Journal of Research in Biology Volume 3 Issue 3

Journal of Research in Biology

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An International Scientific Research Journal for Biology Volume 3 Issue 3

ISSN: 2231 –6280 EISSN: 2231- 6299

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List of Editors of Editors in the Journal of Research in Biology

Managing and Executive Editor:

Abiya Chelliah [Molecular Biology]

Publisher, Journal of Research in Biology.

Editorial Board Members:

Ciccarese [Molecular Biology] Universita di Bari, Italy.

Sathishkumar [Plant Biotechnologist]

Bharathiar University.

SUGANTHY [Entomologist]

TNAU, Coimbatore.

Elanchezhyan [Agriculture, Entomology]

TNAU, Tirunelveli.

Syed Mohsen Hosseini [Forestry & Ecology]

Tarbiat Modares University (TMU), Iran.

Dr. Ramesh. C. K [Plant Tissue Culture] Sahyadri Science College, Karnataka.

Kamal Prasad Acharya [Conservation Biology]

Norwegian University of Science and Technology (NTNU), Norway.

Dr. Ajay Singh [Zoology]

Gorakhpur University, Gorakhpur

Dr. T. P. Mall [Ethnobotany and Plant pathoilogy]

Kisan PG College, BAHRAICH

Ramesh Chandra [Hydrobiology, Zoology]

S.S.(P.G.)College, Shahjahanpur, India.

Adarsh Pandey [Mycology and Plant Pathology]

SS P.G.College, Shahjahanpur, India

Hanan El-Sayed Mohamed Abd El-All Osman [Plant Ecology]

Al-Azhar university, Egypt

Ganga suresh [Microbiology]

Sri Ram Nallamani Yadava College of Arts & Sciences, Tenkasi, India.

T.P. Mall [Ethnobotany, Plant pathology]

Kisan PG College,BAHRAICH, India.

Mirza Hasanuzzaman [Agronomy, Weeds, Plant]

Sher-e-Bangla Agricultural University, Bangladesh

Mukesh Kumar Chaubey [Immunology, Zoology]

Mahatma Gandhi Post Graduate College, Gorakhpur, India.

N.K. Patel [Plant physiology & Ethno Botany]

Sheth M.N.Science College, Patan, India.

Kumudben Babulal Patel [Bird, Ecology]

Gujarat, India.

CHANDRAMOHAN [Biochemist]

College of Applied Medical Sciences, King Saud University.

B.C. Behera [Natural product and their Bioprospecting]

Agharkar Research Institute, Pune, INDIA.

Kuvalekar Aniket Arun [Biotechnology]

Lecturer, Pune.

Mohd. Kamil Usmani [Entomology, Insect taxonomy]

Aligarh Muslim university, Aligarh, india.

Dr. Lachhman Das Singla [Veterinary Parasitology]

Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India.

Vaclav Vetvicka [Immunomodulators and Breast Cancer]

University of Louisville, Kentucky.

José F. González-Maya [Conservation Biology]

Laboratorio de ecología y conservación de fauna Silvestre,

Instituto de Ecología, UNAM, México.

Dr. Afreenish Hassan [Microbiology]

Department of Pathology, Army Medical College, Rawalpindi, Pakistan.

Gurjit Singh [Soil Science]

Krishi Vigyan Kendra, Amritsar, Punjab, India.

Dr. Marcela Pagano [Mycology]

Universidade Federal de São João del-Rei, Brazil.

Dr.Amit Baran Sharangi [Horticulture]

BCKV (Agri University), West Bengal, INDIA.

Dr. Bhargava [Melittopalynology]

School of Chemical & Biotechnology, Sastra University, Tamilnadu, INDIA.

Dr. Sri Lakshmi Sunitha Merla [Plant Biotechnology]

Jawaharlal Technological University, Hyderabad.

Dr. Mrs. Kaiser Jamil [Biotechnology]

Bhagwan Mahavir Medical Research Centre, Hyderabad, India.

Ahmed Mohammed El Naim [Agronomy]

University of Kordofan, Elobeid-SUDAN.

Dr. Zohair Rahemo [Parasitology]

University of Mosul, Mosul,Iraq.

Dr. Birendra Kumar [Breeding and Genetic improvement]

Central Institute of Medicinal and Aromatic Plants, Lucknow, India.

Dr. Sanjay M. Dave [Ornithology and Ecology]

Hem. North Gujarat University, Patan.

Dr. Nand Lal [Micropropagation Technology Development]

C.S.J.M. University, India.

Fábio M. da Costa [Biotechnology: Integrated pest control, genetics]

Federal University of Rondônia, Brazil.

Marcel Avramiuc [Biologist]

Stefan cel Mare University of Suceava, Romania.

Dr. Meera Srivastava [Hematology , Entomology] Govt. Dungar College, Bikaner.

P. Gurusaravanan [Plant Biology ,Plant Biotechnology and Plant Science]

School of Life Sciences, Bharathidasan University, India.

Dr. Mrs Kavita Sharma [Botany]

Arts and commerce girl’s college Raipur (C.G.), India.

Suwattana Pruksasri [Enzyme technology, Biochemical Engineering]

Silpakorn University, Thailand.

Dr.Vishwas Balasaheb Sakhare [Reservoir Fisheries]

Yogeshwari Mahavidyalaya, Ambajogai, India.

Dr. Pankaj Sah [Environmental Science, Plant Ecology]

Higher College of Technology (HCT), Al-Khuwair.

Dr. Erkan Kalipci [Environmental Engineering]

Selcuk University, Turkey.

Dr Gajendra Pandurang Jagtap [Plant Pathology]

College of Agriculture, India.

Dr. Arun M. Chilke [Biochemistry, Enzymology, Histochemistry]

Shree Shivaji Arts, Commerce & Science College, India.

Dr. AC. Tangavelou [Biodiversity, Plant Taxonomy]

Bio-Science Research Foundation, India.

Nasroallah Moradi Kor [Animal Science]

Razi University of Agricultural Sciences and Natural Resources, Iran

T. Badal Singh [plant tissue culture]

Panjab University, India

Dr. Kalyan Chakraborti [Agriculture, Pomology, horticulture]

AICRP on Sub-Tropical Fruits, Bidhan Chandra Krishi Viswavidyalaya,

Kalyani, Nadia, West Bengal, India.

Dr. Monanjali Bandyopadhyay [Farmlore, Traditional and indigenous

practices, Ethno botany]

V. C., Vidyasagar University, Midnapore.

M.Sugumaran [Phytochemistry]

Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram District.

Prashanth N S [Public health, Medicine]

Institute of Public Health, Bangalore.

Tariq Aftab

Department of Botany, Aligarh Muslim University, Aligarh, India.

Manzoor Ahmad Shah

Department of Botany, University of Kashmir, Srinagar, India.

Syampungani Stephen

School of Natural Resources, Copperbelt University, Kitwe, Zambia.

Iheanyi Omezuruike OKONKO

Department of Biochemistry & Microbiology, Lead City University,

Ibadan, Nigeria.

Sharangouda Patil

Toxicology Laboratory, Bioenergetics & Environmental Sciences Division,

National Institue of Animal Nutrition

and Physiology (NIANP, ICAR), Adugodi, Bangalore.

Jayapal

Nandyal, Kurnool, Andrapradesh, India.

T.S. Pathan [Aquatic toxicology and Fish biology]

Department of Zoology, Kalikadevi Senior College, Shirur, India.

Aparna Sarkar [Physiology and biochemistry] Amity Institute of Physiotherapy, Amity campus, Noida, INDIA.

Dr. Amit Bandyopadhyay [Sports & Exercise Physiology]

Department of Physiology, University of Calcutta, Kolkata, INDIA .

Maruthi [Plant Biotechnology]

Dept of Biotechnology, SDM College (Autonomous),

Ujire Dakshina Kannada, India.

Veeranna [Biotechnology]

Dept of Biotechnology, SDM College (Autonomous),

Ujire Dakshina Kannada, India.

RAVI [Biotechnology & Bioinformatics]

Department of Botany, Government Arts College, Coimbatore, India.

Sadanand Mallappa Yamakanamardi [Zoology]

Department of Zoology, University of Mysore, Mysore, India.

Anoop Das [Ornithologist]

Research Department of Zoology, MES Mampad College, Kerala, India.

Dr. Satish Ambadas Bhalerao [Environmental Botany]

Wilson College, Mumbai

Rafael Gomez Kosky [Plant Biotechnology]

Instituto de Biotecnología de las Plantas, Universidad Central de Las Villas

Eudriano Costa [Aquatic Bioecology]

IOUSP - Instituto Oceanográfico da Universidade de São Paulo, Brasil

M. Bubesh Guptha [Wildlife Biologist] Wildlife Management Circle (WLMC), India

Rajib Roychowdhury [Plant science]

Centre for biotechnology visva-bharati, India.

Dr. S.M.Gopinath [Environmental Biotechnology]

Acharya Institute of Technology, Bangalore.

Dr. U.S. Mahadeva Rao [Bio Chemistry]

Universiti Sultan Zainal Abidin, Malaysia.

Hérida Regina Nunes Salgado [Pharmacist]

Unesp - Universidade Estadual Paulista, Brazil

Mandava Venkata Basaveswara Rao [Chemistry]

Krishna University, India.

Dr. Mostafa Mohamed Rady [Agricultural Sciences]

Fayoum University, Egypt.

Dr. Hazim Jabbar Shah Ali [Poultry Science]

College of Agriculture, University of Baghdad , Iraq.

Danial Kahrizi [Plant Biotechnology, Plant Breeding,Genetics]

Agronomy and Plant Breeding Dept., Razi University, Iran

Dr. Houhun LI [Systematics of Microlepidoptera, Zoogeography, Coevolution,

Forest protection]

College of Life Sciences, Nankai University, China.

María de la Concepción García Aguilar [Biology] Center for Scientific Research and Higher Education of Ensenada, B. C., Mexico

Fernando Reboredo [Archaeobotany, Forestry, Ecophysiology]

New University of Lisbon, Caparica, Portugal

Dr. Pritam Chattopadhyay [Agricultural Biotech, Food Biotech, Plant Biotech]

Visva-Bharati (a Central University), India

Table of Contents (Volume 3 - Issue 3)

Serial No Accession No Title of the article Page No

1 RA0328 An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India.

Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and Basant Kumar Agarwala.

852-860

2 RA0327 Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms.

Ujowundu CO, Nwaogu LA, Igwe KO, Ujowundu FN, Belonwu DC

861-869

3 RA0167

Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats.

Akpa GN, Alphonsus C and Usman N.

870-875

4 RA0245 Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India.

Samit Roy Choudhury and Basant Kumar Agarwala.

876-885

5 RA0329 Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema.

Renju GL and Muraleedhara Kurup G.

886-894

6 RA0332 Identification of animal Pasteurellosis by PCR assay.

Venkatesan PS, Deecaraman M and Vijayalakshmi.

895-899

7 RA0345 Source of light emission in a luminous mycelium of the fungus Panellus stipticus.

Puzyr Alexey, Burov Andrey and Bondar Vladimir.

900-905

8 RA0274 Local people’s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India.

Lakshmi Kodoth and Ramamoorthy D.

906-910

9 RA0318 Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria.

Nwanyanwu CE and Abu GO.

911-921

10 RA0317 Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria.

Nwanyanwu CE, Nweke CO, Orji JC and Opurum CC.

922-931

11 RA0337 Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats.

Nwankpa P, Ezekwe AS, Ibegbulem CO and Egwurugwu JN.

932-939

http://jresearchbiology.com/documents/RA0328.pdf








jresearchbiology.com/documents/RA0318.pdf



Jou

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esearch

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Biology

An ornithological survey in the vicinity of Agartala city of Tripura state,

north-eastern India

Keywords: Avifauna, biodiversity hotspot, Agartala, Tripura, north-east India .

ABSTRACT:

North-east India is a part of Indo-Burma hotspot and among the richest bird zones in India. Tripura lies in the border of Indo-Burma global biodiversity hotspot area but is poorly covered by ornithological works. Avifauna of Tripura state is known by 277 species but there is lack of information about their distribution, particularly in and around Agartala city, which is the capital of Tripura state and is a tourist destination along the border of Bangladesh for its natural landscapes, inland water species, and strong presence of green flora. With a view to enhance its value for tourist attraction and naturalists, a study was conducted to record the species of birds that occur in and around the City. In the present study 73 bird species were recorded from Agartala city and its adjacent areas belonging to 41 families and 14 orders.

852-860 | JRB | 2013 | Vol 3 | No 3

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.



An International Scientific

Research Journal

Authors: Partha Pratim Bhattacharjee,

Rahul Lodh, Dipten Laskar,

Joydeb Majumder and

Basant Kumar Agarwala.

Institution:

Ecology & Biodiversity

Laboratories, Department of

Zoology, Tripura University,

Suryamaninagar-799 022,

Tripura, India.

Corresponding author: Basant Kumar Agarwala.

Email: [email protected]

Web Address: http://jresearchbiology.com/documents/RA0328.pdf.

Dates: Received: 28 Jan 2013 Accepted: 15 Feb 2013 Published: 10 Apr 2013

Article Citation: Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and Basant Kumar Agarwala. An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India. Journal of Research in Biology (2013) 3(3): 852-860

Journal of Research in Biology An International Scientific Research Journal

Original Research

INTRODUCTION

Avifauna contributes most significantly to the

diversity of terrestrial vertebrates, which have a special

role in conservation of biodiversity of a particular area

(Daniels, 1994). Birds are very good indicator

of environmental changes as they respond in the minute

change in habitat structure and composition

(Robert et al., 2001). Indian subcontinent harbour nearly

1300 species of birds, which is more than 13% of total

bird species of the world (Grimmet et al., 2004), and

more than 60% of Indian birds are found in north-east

India (Choudhury, 2010). North-east India is one of the

most significant biodiversity hotspots of the world and

among the richest bird zones in India because of

convergence of the Indo-Malayan, Indo-Chinese and

Indian biogeographical realms. As a result, it is unique in

providing an abundance of habitats that harbour diverse

biota with a high degree of endemism (Chatterjee et al.,

2006; Narwade et al., 2011). Tripura (22°56´- 24°32´ N

and 91°10´- 92°21´ E, with an area of 10,490 km2) is a

small state of north-east India bounded by Bangladesh

on three sides and with Assam and Mizoram on the other

side. It lies in the border of Indo-Burma global

biodiversity hotspot area (Myers et al., 2000) but

very poorly covered by ornithological works

(Choudhury, 2010). Although avifaunal checklist for

Tripura state listed 277 species (Choudhury, 2010) but

little is known regarding the bird species found in the

vicinity of Agartala city, situated by international

boundary of Bangladesh.

STUDY SITES

Agartala city is situated in the western region of

Tripura state with the latitude of 23°45' North and

longitude of 91°45' East and an average elevation of

20.36 m above sea level. It is the capital town of Tripura

with a mix of urban and semi urban complex and a rich

green cover. Forests and farms adjoin the town on three

sides, and therefore, it is also called ‘Green City’. The

total city area is 62.02 km2 and is delimited on the west

side by international boundary with Bangladesh.

Climatic condition is of tropical monsoon type with an

average annual rainfall of 220 cm. Average minimum

and maximum temperature recorded in the region are

6.8°C in January and 37.70°C in June, respectively.

Present study was carried out in eleven

different sites (viz., College Tilla lake area, Golbazar,

Pratapgarh, Dashamighat, Arundhutinagar, Shanmura,

Bhubanban, Barjala, Jagannath Bari lake area, G B Bazar

and Nandannagar) (Table 1, Figure 1) covering different

sides of Agartala city and its adjacent areas.

METHODOLOGY

The study sites were visited fortnightly

throughout the study period from 2009-2011. Data on

Bhattacharjee et al., 2013

853 Journal of Research in Biology (2013) 3(3): 852-860

Sl. No Sites Coordinates Altitude (m)

1. College Tilla lake area 23°49'35.45" N; 91°17'42.28" E 17

2. Golbazar 23°49'38.30" N ; 91°16'57.15" E 16

3. Pratapgarh 23°49'08.98" N ; 91°17'17.10" E 16

4. Dashamighat 23°49'46.34" N ; 91°15'51.45" E 16

5. Arundhutinagar 23°49'01.44" N ; 91°16'21.68" E 31

6. Shanmura 23°50'51.98" N ; 91°16'07.20" E 15

7. Bhubanban 23°51'56.50" N; 91°15'73.70" E 21

8. Barjala 23°52'05.05" N ; 91°16'32.13" E 23

9. Jagannath Bari lake area 23°50'05.43" N; 91°16'53.70" E 14

10. G B Bazar 23°51'33.74" N ; 91°17'33.97" E 27

11. Nandannagar 23°51'43.68" N ; 91°17'57.00" E 28

Table 1: Geo-coordinate details of the study sites

present bird species were collected by direct observations

with the help of binoculars (VISTA LE 8 X 40). Almost

all the species mentioned in the checklist were

photographed. For this purpose, digital cameras of Canon

Power shot SX 200 IS (12 X Digital zoom), Cannon SRL

EOS 50D and Panasonic Lumix DMC FZ 40 were used.

In lake areas birds were observed from the bank,

peripheral areas, and urban areas were surveyed on foot

regularly. Farm and forested areas in the vicinity of the

city were surveyed to record the assemblages of different

bird species. Most of visits were made in morning and

afternoon time when birds are most active. Identification

of birds was based on the field guides produced by Ali

and Ripley (1995), Ali (1996 and 2002) and Grimmett et

al., (2003).

Status of the birds was classified as C-Common,

MC-Most common, NC-Not common, S-Singleton,

W- Winter visitor.

RESULTS AND DISCUSSION

In the present study 73 bird species were

recorded from Agartala city and its adjacent areas

belonging to 41 families and 14 orders (Table 2, Plate 1

and 2). There is no authentic information about the

avifauna of Tripura except that by Blyth (1845, 1846),

Ali and Ripley (1968-74) and International Waterfowl

and Wetlands Research Bureau on Asian Waterfowl

Census, 1989 (Scott and Rose 1989). Majumdar et al.,

(2002) recorded 259 species of birds, belonging to

56 families and 16 orders. Recently Choudhury (2010)

recorded 277 species of birds, in the annotated checklist

from Tripura, but avifaunal diversity of Agartala city is

not yet available. Out of 14 orders. Passeriformes was

found dominant with 22 families followed by

Coraciiformes with 3 families and Pelecaniformes and

Piciformes with 2 families each. Dominance of

Passeriformes was also recorded by Choudhury (2010)

and Majumdar et al., (2002) from the state and from

Nagpur district of central India (Chinchkhede and Kedar,

2012). The resident birds such as Pond heron, Cattle

egret, Lapwing, Blue rock pigeon, Spotted dove,

Parakeets, Asian koel, Kingfisher, Bee eater, Lineated

barbet, Woodpecker, Bush lark, Bulbul, Shrike, Robin,

Tailorbird, Cinereous tit, Sunbird, Sparrow, Starling,

Myna, Oriole, Black drongo and Crow etc were found

regularly throughout the study period. Little Cormorant,

Asian Openbill-Stork, Black headed Ibis, Lesser

Whistling Duck, Crested Serpent Eagle, Red Junglefowl,

Red Collared Dove, Yellow-footed Green Pigeon, Brown

Fish Owl, Asian Palm Swift, Indian Roller, Coppersmith

Barbet, Orange headed Thrush, Blue Rock Thrush,

White-rumped Shama, Scarlet-backed Flower pecker,

Tricoloured Munia etc were found less common in this

study. Common Sandpiper and Black headed Ibis were

observed during the winter season only in the paddy

fields of peripheral areas of the city. Common Hoopoe


Journal of Research in Biology (2013) 3(3): 852-860 854

Figure 1. Showing the study sites in and around Agartala City.



Sl.

No. Common name Scientific name

Status

IUCN Abundance

Cormorants [Phalacrocoracidae]

1. Little Cormorant Phalacrocorax niger Vieillot, 1817 LC NC

Herons & Egrets [Ardeidae]

2. Indian Pond Heron Ardeola grayii (Sykes, 1832) LC MC

3. Cattle Egret Bubulcus ibis (Linnaeus, 1758) LC MC

4. Median Egret Mesophoyx intermedia (Wagler, 1827) LC C

Storks [Ciconiidae]

5. Asian Openbill-Stork Anastomus oscitans Boddaert, 1783 LC NC

6. Black headed Ibis Threskiornis melanocephalus (Latham, 1790) NT W, NC

Ducks [Anatidae]

7. Lesser Whistling Duck Dendrocygna javanica (Horsfield, 1821) LC NC

Hawks & Eagles [Accipitridae]

8. Crested Serpent Eagle Spilornis cheela Latham, 1790 LC NC

9. Black Kite Milvus migrans (Boddaert, 1783) LC C

Pheasants [Phasianidae]

10. Red Junglefowl Gallus gallus (Linnaeus, 1758) LC NC

Rails & Coots [Rallidae]

11. White-breasted Waterhen Amaurornis phoenicurus Pennant, 1769 LC C

Lapwings [Charadriidae]

12. Red-wattled Lapwing Vanellus indicus (Boddaert, 1783) LC MC

Sandpipers [Scolopacidae]

13. Common Sandpiper Actitis hypoleucos (Linnaeus, 1758) LC W, C

Pigeons & Doves [Columbidae]

14. Blue Rock Pigeon Columba livia Gmelin, 1789 LC MC

15. Spotted Dove Streptopelia chinensis (Scopoli, 1768) LC MC

16. Red Collared Dove Streptopelia tranquebarica (Hermann, 1804) LC NC

17. Orange-breasted Green

Pigeon Treron bicinctus (Jerdon, 1840) LC C

18. Yellow-footed Green Pigeon Treron phoenicoptera (Latham, 1790) LC NC

Parakeets [Psittacidae]

19. Rose-ringed Parakeet Psittacula krameri (Scopoli, 1769) LC C

20. Red-breasted Parakeet Psittacula alexandri (Linnaeus, 1758) LC C

Cuckoos & Coucals [Cuculidae]

21. Asian Koel Eudynamys scolopaceus (Linnaeus, 1758) LC MC

22. Greater Coucal Centropus sinensis (Stephens, 1815) LC C

Owls [Strigidae]

23. Collared Scops Owl Otus lettia Hodgson, 1836 LC S

24. Spotted Owlet Athene brama (Temminck, 1821) LC C

25. Brown Fish Owl Bubo zeylonensis (Gmelin, 1788) LC NC

Table 2: List of birds in and around Agartala city during 2009-2011



Swifts [Apodidae]

26. Asian Palm Swift Cypsiurus balasiensis Gray, 1829 LC NC

27. House Swift Apus affinis (J E Gray, 1830) LC C

Kingfishers [Alcedinidae]

28. Common Kingfisher Alcedo atthis (Linnaeus, 1758) LC MC

29. Stork-billed Kingfisher Halcyon capensis (Linnaeus, 1766) LC C

30. White-throated Kingfisher Halcyon smyrnensis (Linnaeus, 1758) LC MC

Bee-eaters [Meropidae]

31. Little Green Bee-eater Merops orientalis Latham, 1802 LC MC

Rollers [Coraciidae]

32. Indian Roller Coracias benghalensis (Linnaeus, 1758) LC NC

Hoopoe [Upupidae]

33. Common Hoopoe Upupa epops Linnaeus, 1758 LC S

Barbets [Capitonidae]

34. Lineated Barbet Megalaima lineata (Vieillot, 1816) LC MC

35. Coppersmith Barbet Megalaima haemacephala Muller, 1776 LC NC

Woodpeckers [Picidae]

36. Rufous Woodpecker Celeus brachyurus (Vieillot, 1818) LC C

37. Greater Flameback Chrysocolaptes lucidus (Scopoli, 1786) LC C

38. Fulvous-breasted Woodpecker Dendrocopos macei (Vieillot, 1818) LC C

Larks [Alaudidae]

39. Singing bush lark Mirafra cantillans Blyth, 1844 LC C

Pipits & Wagtails [Motacillidae]

40. Paddy field Pipit Anthus rufulus Vieillot, 1818 LC C

41. White Wagtail Motacilla alba Linnaeus, 1758 LC W,C

Bulbuls [Pycnonotidae]

42. Red-whiskered Bulbul Pycnonotus jocosus (Linnaeus, 1758) LC C

43. Red-vented Bulbul Pycnonotus cafer (Linnaeus, 1766) LC MC

Loras [Irenidae]

44. Common Lora Aegithina tiphia (Linnaeus, 1758) LC C

Shrikes [Laniidae]

45. Brown Shrike Lanius cristatus Linnaeus, 1758 LC W, MC

46. Grey-backed Shrike Lanius tephronotus (Vigors, 1831) LC W, C

Thrushes [Turdidae]

47. Orange headed Thrush Zoothera citrina (Latham, 1790) LC NC

Flycatchers [Muscicapidae]

48. Blue Rock Thrush Monticola solitarius (Linnaeus, 1758) LC NC

49. White-rumped Shama Copsychus malabaricus (Scopoli, 1786) LC NC

50. Oriental Magpie Robin Copsychus saularis (Linnaeus, 1758) LC MC

http://en.wikipedia.org/wiki/Muscicapidae



Babblers [Timaliidae]

51. Rufous-necked Laughing-

thrush

Garrulax ruficollis (Jardine & Selby, 1838) LC C

Warblers [Sylviidae]

52. Common Tailorbird Orthotomus sutorius (Pennant, 1769) LC MC

Flycatchers [Stenostiridae]

53. Grey-headed Canary-

flycatcher

Culicicapa ceylonensis (Swainson, 1820) LC W, C

Tits [Paridae]

54. Cinereous Tit Parus cinereus Vieillot, 1818 LC MC

Flowerpeckers [Dicaeidae]

55. Scarlet-backed Flowerpecker Dicaeum cruentatum (Linnaeus, 1758) LC NC

Sunbirds [Nectariniidae]

56. Ruby-cheeked Sunbird Anthreptes singalensis (Gmelin, 1788) LC C

57. Purple-rumped Sunbird Nectarinia zeylonica (Linnaeus, 1766) LC C

58. Purple Sunbird Cinnyris asiaticus Latham, 1790 LC MC

White-eyes [Zosteropidae]

59. Oriental White-eye Zosterops palpebrosus (Temminck, 1824) LC C

Munias [Estrildidae]

60. Scaly-breasted Munia Lonchura punctulata (Linnaeus, 1758) LC C

61. Tricoloured Munia Lonchura malacca (Linnaeus, 1766) LC NC

Sparrows [Passerinae]

62. House Sparrow Passer domesticus (Linnaeus, 1758) LC MC

Weavers [Ploceidae]

63. Baya Weaver Ploceus philippinus (Linnaeus, 1766) LC C

Starlings & Mynas [Sturnidae]

64. Chestnut-tailed Starling Sturnus malabaricus (Gmelin, 1789) LC C

65. Asian Pied Starling Gracupica contra (Linnaeus, 1758) LC MC

66. Common Myna Acridotheres tristis (Linnaeus, 1766) LC MC

67. Jungle Myna Acridotheres fuscus (Wagler, 1827) LC MC

Orioles [Oriolidae]

68. Black-hooded Oriole Oriolus xanthornus (Linnaeus, 1758) LC MC

Drongos [Dicruridae]

69. Black Drongo Dicrurus macrocercus (Vieillot, 1817) LC MC

70. Greater racket-tailed Drongo Dicrurus paradiseus Linnaeus, 1766 LC NC

Crows & Treepie [Corvidae]

71. Rufous Treepie Dendrocitta vagabunda (Latham, 1790) LC C

72. House Crow Corvus splendens Vieillot, 1817 LC MC

73. Jungle Crow Corvus macrorhynchos Wagler, 1827 LC MC

Abbreviations:

Status: LC = least concern; NT = near threatened; C = common; MC = most common; NC = not common;

S = singleton; W = winter visitor.

http://en.wikipedia.org/wiki/Johann_Georg_Wagler



Plate 1. A-Lineated barbet, B-Cattle egret, C-Red-wattled Lapwing, D-Common Hoope, E-Little cormorant,

F-Stripe-breasted woodpecker, G-Rufous woodpecker, H-White throated kingfisher, I-Yellow footed green

pegion, J-Asian open bill stork, K-Chestnut-tailed starling, L-Collared scops owl.

Plate 2: M-Asian Koel, N-Crested serpent eagle, O-Common Tailorbird, P-Cinereous Tit, Q-Emerald dove,

R-Little green bee-eater, S-Grey-baked shrike, T-Indian pond heron, U-Red collared dove, V-White-rumped

shama, W-Singing bush lark, X-Black headed ibis.

and Collared Scops Owl were sighted only once in the

two years study. Brown Shrike, Grey-backed Shrike,

Grey-headed Canary-flycatcher were observed in the

winter season only (Table 2), which corroborates with

the findings of Choudhury (2010) and Majumdar et al.,

(2002).

CONCLUSION

The present avifaunal survey of Agartala city and

its adjacent areas revealed 73 bird species which is very

important as it is the first ornithological record of the city

and will give a baseline data for future study. Rich bird

diversity is influenced by the topographical location of

the city and adjacent areas of Bangladesh.

Expansion of the city by construction activities,

reducing forest and farm areas with population pressure,

filling of pond and lake areas, dumping of wastes and

garbage in the low lands, use of chemical pesticides in

agricultural fields and hunting of birds are the major

threats to the avifaunal diversity here which needs proper

conservation management practices.

ACKNOWLEDGEMENT

Authors are thankful to Mr. Dipankar Kishore

Sinha for his constant services, tireless field assistance

and in capturing photographs during the study.

REFERENCES

Ali S. 1996. The book of Indian birds, Twelfth Revised

Edition, Bombay Natural History Society Oxford

University Press, Mumbai.

Ali S. 2002. The book of Indian birds, Thirteenth

Revised Edition, Bombay Natural History Society

Oxford University Press, Mumbai.

Ali S and Ripley SD. 1968-74. Pakistan 10 vols.,

Oxford University Press, Bombay.

Ali S and Ripley SD. 1995. A pictorial guide to the

birds of Indian Subcontinent. Bombay Natural History

Society, Mumbai.

BirdLife International 2009: IUCN 2011. IUCN Red

List of Threatened Species. Version 2011.2.

<www.iucnredlist.org>. Downloaded on 18 June 2012.

Blyth E. 1845. Notices and descriptions of various new

or little known species of birds, Journal of the Asiatic

Society of Bengal, 14: 546-602.

Blyth E. 1846. Notices and descriptions of various new

or little known species of birds, Journal of the Asiatic

Society of Bengal, 15: 1-54.

Chatterjee S, Saikia A, Dutta P, Ghosh D, Pangging

G and Goswami AK. 2006. Biodiversity significance of

north east India: WWF-India. New Delhi. pp-71.

Chinchkhede KH and Kedar GT. 2012. Avifaunal

diversity of Koradi lake in Nagpur district of central

India. Journal of Research in Biology, 2: 70-76.

Choudhury A. 2010. Recent ornithological records from

Tripura, north-eastern India, with an annotated checklist.

Indian BIRDS 6(3): 66-74.

Daniels RJR. 1994. A landscape approach to

conservation of birds. Journal of Bioscience 19(4): 503-

509.

Grimmet R, Inskip T and Islam MZ. 2004. Birds of

Northern India. Christopher Helm A and C Bleak

Publishers Ltd. London.

Grimmett R, Inskipp C and Inskipp T. 2003. Pocket

Guide to the Birds of the Indian Subcontinent, Oxford

University Press, New Delhi.

Majumdar N, Ray CS and Datta BK. 2002. Aves. In:

Fauna of Tripura (Part 1) (Vertebrates). State Fauna

Series 7, pp. 47-158 (Ed.: Director 2002). Kolkata:

Zoological Survey of India.



Submit your articles online at www.jresearchbiology.com

Advantages

Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright

[email protected]

www.jresearchbiology.com/Submit.php.

Myers N, Russell A, Mittermelert C, Mittermelert G,

Gustavo AB and Fonseca KJ. 2000. Biodiversity

hotspots for conservation priorities. Nature 403: 853-

858.

Narwade S, Kalra M, Jagdish R, Varier D, Satpute S,

Khan N, Talukdar G, Mathur VB, Vasudevan K,

Pundir DS, Chavan V and Sood R. 2011. Literature

based species occurrence data of birds of North-East

India. In: Smith V, Penev L (Eds) e-Infrastructures for

data publishing in biodiversity science. ZooKeys 150:

407-417.

Robert A Fimbel, John AG and Robinson G. 2001.

The Cutting Edge: Conserving Wildlife in Logged

Tropical Forest.

Scott DA and Rose PM. 1989. Asian Waterfowl

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Jou

rn

al of R

esearch

in

Biology

Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of

Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms.

Keywords: Oil and gas, Pollution, Phytochemicals, Vitamins, Oha, Okra.

ABSTRACT: The phytochemical, proximate, mineral and vitamin contents of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms were investigated. Plant samples were harvested from Polluted Environment (PE) at Izombe in Oguta Local Government Area- an oil drilling and gas flaring environment. The results obtained were compared to identical vegetables harvested from Eziobodo in Owerri West Local Government Area, designated as Unpolluted Environment (UPE). Our result showed that A. esculentus and P. mildbraedii have excellent nutritional value, which can confer biochemical and physiological advantage to humans. The quantitative proximate composition showed that the carbohydrate and ash contents of samples harvested from PE differed significantly (P<0.05) from samples obtained from unpolluted environment. The protein, crude fibre, moisture and total fat contents of samples from PE differed non significantly (P<0.05) when compared with samples obtained from UPE. The phytochemical contents of A. esculentus and P. mildbraedii were significantly higher in samples from UPE than in samples from PE. The mineral and vitamin contents were also determined. The concentration of nutritionally important macro and micro elements indicates that the two vegetable samples studied are rich sources of minerals and, therefore, can be used to improve the diet of both humans and livestock. This study also showed that environmental pollutants emanating from the activities of oil and gas industries can impact negatively on some important chemical and nutritive compositions of edible vegetables.

861-869 | JRB | 2013 | Vol 3 | No 3






Research Journal

Authors:

Ujowundu CO1,

Nwaogu LA1, Igwe KO1,

Ujowundu FN1,

Belonwu DC2.

Institution:

1. Department of

Biochemistry, Federal

University Technology

Owerri, Nigeria.

2. Departmentof

Biochemistry, University of

Portharcourt, Nigeria.

Corresponding author:

Ujowundu CO.



Dates: Received: 16 Jan 2013 Accepted: 09 Feb 2013 Published: 11 Apr 2013

Article Citation: Ujowundu CO, Nwaogu LA, Igwe KO, Ujowundu FN, Belonwu DC. Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms. Journal of Research in Biology (2013) 3(3): 861-869


Original Research

INTRODUCTION

Nigeria, a major producer of crude oil, benefits

as well as suffers from the positive and negative effects

of crude oil drilling and gas flaring (Adeniye et al.,

1983). Gas flaring is the unscientific burning of excess

hydrocarbons gathered in an oil/gas production flow

station. Gas flaring is a major source of pollution in

Nigeria's Niger Delta because it is the preferred means of

disposing waste gas associated with oil exploitation in

that region by many multinational oil companies that

operate its fields. Gas flaring releases carbon monoxide,

oxides of sulphur and nitrogen, hydrocarbons, soot and

heavy metals (Coker, 2007; Ikoro, 2003). These

pollutants actually interfere with growth and survival of

living organisms in such environment especially in

plants. It pollutes seedlings and fruits of plants which in

turn have a devastating effect on humans who consume

them. Such effects include respiratory or cardiovascular

diseases. This study used Abelmoschus esculentus

Moench and leaves of Pterocarpus mildbraedii Harms,

two commonly consumed indigenous vegetables to

evaluate the biochemical effects of these environmental

pollutants.

Abelmoschus esculentus Moench (local name,

Okra) and leaves of Pterocarpus mildbraedii Harms

(local name, Oha) are vegetables commonly consumed

as a source of food and medication for their high content

of nutrients and phytochemicals and mainly used for

soup preparation. Consumption of vegetables provide

taste, palatability, increases appetite and provides fiber

for digestion and to prevent constipation. They play key

roles in neutralizing acids produced during digestion of

proteins and fatty foods and also provide valuable

roughages which helps in movement of food in the

intestine. Some of these vegetables possess the ability to

reduce or reverse so many disease conditions and

disorders such as those which require a reduced intake of

glucose (diabetes) (Mcdowell, 2001: Ogbonnia et al.,

2008). These vegetables can be deeply affected by

pollution.

The most glaring sight in gas production flow

station is the ten-meter-high flame that burns

continuously from vertical pipes at many facilities owned

by oil companies. These vertical pipes are fed with gas

given off during production. Gas flaring, for about four

decades has contributed to the high pollution level, and

the ecosystem of Izombe may have been impacted

negatively. A good example of such negative effect is

high soil acidity that creates chemical and biological

conditions which may be harmful to the soil and plants

(Nwaugo et al., 2006). One of these conditions is the

reduction in the capacity of plants to absorb cations

(Wild et al., 2005). The higher acidic nature of soil is

attributable to high concentrations of sulphur dioxide and

particulates from gases flared into the atmosphere which

is washed back to the soil as acid rain.

This study has two objectives, first to contribute

to the knowledge of nutritional and antinutritional

composition of A. esculentus Moench and P. mildbraedii

Harms. Secondly, to evaluate the effect of environmental

pollution resulting from crude oil exploration and

exploitation and other industrial processes within the

area of study. The arable nature and vast land mass of

Izombe, confers it the status of food basket of Imo State,

Nigeria.

MATERIALS AND METHODS

Collection and preparation of plant samples

Samples of A. esculentus Moench and

P. mildbraedii Harms were obtained at Izombe, in Oguta

Local Government Area and at Eziobodo, in Owerri

West Local Government Area both in Imo state, Nigeria

and identified by a plant taxonomist in the Federal

University of Technology, Owerri (FUTO). Izombe is a

rainforest ecosystem, which hosts multinational

industries specialized in crude oil exploration and

exploitation. Flaring of gases constitutes the major

method of waste gas disposal at these oil fields. Situate

Ujowundu et al., 2013


to these oil fields are communities of indigenous

inhabitants whose occupation are subsistence and semi-

commercial farming. Eziobodo is also within the

rainforest region of Nigeria, occupied by indigenous and

FUTO students population. It has no known industrial

activities, except few automobiles that convey

inhabitants in and out of the village. Samples were sorted

by removing extraneous materials, spoilt and unhealthy

ones. After washing, okro samples were carefully sliced.

The samples were oven dried, macerated, sieved and

properly stored.

Evaluation of proximate composition

The method described by James (1995) and

Onwuka (2005) were used to determine crude fiber. Fat

content was determined by the method of Min and Boft

(2003). Moisture content was determined by the method

of AOAC (1990). The sample’s total protein content was

determined by microkjeldhal method described by

James (1995). Protein concentration was obtained by

determining total nitrogen and multiplied by the

factor- 6.25. Carbohydrate contents was calculated using

the arithmetical difference method described by Pearson

(1976) and James (1995).

Evaluation of phytochemical content

Tannin content of samples were determined by

Folin-Denis colorimetric method (Kirk and Sawyer,

1998). Saponin, alkaloid and flavonoid were done by

method described by Harborne, (1973). The

spectrophotometric method as described by Griffiths and

Thomas (1981) was used for determining phytate

content. Determinations were done in triplicates and

results were expressed as averages of percent values on

dry weight basis.

Evaluation of vitamins content

Retinol, ascorbic acid and α-tocopherol contents

in the samples were determined using the method of

Association of Vitamin Chemist as described by Kirk

and Sawyer (1998).

Evaluation of mineral content

Some mineral contents were determined by

atomic absorption spectrophotometer (James, 1995). The

dry samples were burnt to ashes to remove all organic

materials leaving inorganic ash. The resulting ash was

dissolved in 10 ml of 2 M HCl solution and diluted to

100 ml with distilled water in a volumetric flask. The

mixture was filtered and the resulting extract was used

for the specific evaluation of copper, zinc and iron.

Sodium, potassium, calcium and magnesium were

determined with the aid of Jaway digital flame

photometer. Phosphorus was determined as phosphate by

the vanadomolybdate colorimetric method (Pearson,

1976)



Environment of

sampling Carbo- hydrates

Crude

Protein Ash Crude Fibre Moisture Total Fat

Polluted 32.47±2.22a 14.28±0.30a 13.78±0.40a 22.13±1.40a 10.52±0.89a 6.82±0.90a

Unpolluted 37.94±1.78b 12.67±0.07a 7.99±0.16b 21.83±0.23a 12.71±1.71a 6.85±0.03a

Table 1: Proximate composition (%) of Abelmoschus esculentus Moench

Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Environment of

sampling Carbo- hydrates

Crude

Protein Ash Crude Fibre Moisture Total Fat

Polluted 29.38±1.24c 8.06±1.43c 19.40±0.57c 17.65±0.79c 22.37±1.18c 3.14±0.33c

Unpolluted 34.82±0.30d 9.67±0.07c 12.05±0.18d 16.58±0.21c 23.67±0.29c 3.21±0.06c

Table 2: Proximate Composition (%) of Pterocarpus mildbraedii Harms


Statistical analysis

Data obtained were expressed as

means± standard deviation. Statistical Package for the

Social Sciences (SPSS) was used for the Analysis of

Variance (ANOVA) for the test of significant difference

between means (P<0.05).

RESULTS

The proximate contents of A. esculentus and

P. mildbraedii are presented in Tables 1 and 2

respectively. Results obtained from unpolluted

environment (UPE) showed that A. esculentus have

higher content of carbohydrate, protein, fibre and total

fat compared to P. mildbraedii. However, higher ash

and moisture content were observed in P. mildbraedii

when compared to A. esculentus from UPE.

Carbohydrate content in samples obtained from polluted

environment (PE) were significantly (P<0.05) lower than

the UPE. But the ash contents were significantly higher

in samples from PE. The protein, crude fiber, moisture

and total fat contents of the samples showed no

significant difference.

Results of phytochemical analysis are presented

in tables 3 and 4. The concentrations of phytochemicals

were significantly higher (P<0.05) in samples from PE.

Also, the phytochemical contents of P. mildbraedii from

PE were higher than that of A. esculentus from PE. The

highest concentrations of phytochemicals were observed

in flavonoids (0.54±0.02%) and tannin (1.83±0.01%)

from A. esculentus and P. mildbraedii respectively from

PE. However, alkaloids and tannins contents were

highest in A. esculentus and P. mildbraedii respectively

from UPE.

Vitamin contents were presented in tables 5 and

6. Vitamin A concentration in A. esculentus and

P. mildbraedii were 627.59±0.47 mg/100g and

375.48±0.18 mg/100g respectively, indicating the

highest vitamin content in the samples. Also, vitamin C

and B5 contents in samples from UPE were significantly

higher (P<0.05) when compared to samples from PE.

Similarly P. mildbraedii have significantly higher value

of vitamin B5 (189.33±2.31 mg/100g) compared to

A. esculentus from UPE. In A. esculentus (table 5), all

the vitamins determined were significantly (P<0.05)

lower except vitamin B5, vitamin B9 and vitamin E in

samples from PE. Also, samples of P. mildbraedii from

PE when compared with samples from UPE showed

significantly lower content in all the vitamins (table 6)

except in vitamin B2, vitamin B3 and vitamin E.

The mineral contents are shown in tables 7 and 8.

The concentrations of copper, iron, zinc and lead in

A. esculentus from PE were significantly higher (P<0.05)



Environment of

sampling Saponins Tannins Phytates Alkaloids Phenols Flavonoids

Polluted 0.33±0.03a 0.37±0.01a 0.23±0.01a 0.43±0.01a 0.26±0.04a 0.54±0.02a

Unpolluted 0.12±0.05b 0.20±0.01b 0.09±0.01b 0.27±0.09b 0.11±0.00b 0.24±0.02b

Table 3: Phytochemical Composition (%) of Abelmoschus esculentus Moench


Environment

of sampling Saponins Tannins Phytates Alkaloids Phenols Flavonoids

Polluted 0.41±0.02c 1.83±0.00c 0.37±0.01c 0.57±0.01c 0.44±0.02c 0.63±0.01c

Unpolluted 0.23±0.08d 1.56±0.14d 0.15±0.01d 0.28±0.12d 0.35±0.00d 0.45±0.08d

Table 4: Phytochemical Composition (%) of Pterocarpus mildbraedii Harms Vegetables

Values (mean ± SD of triplicate determinations) with different superscripts per column are significantly

(P<0.05) different.



En

vir

on

men

t

of

sam

pli

ng

V

ita

min

A

Vit

am

in B

1

Vit

am

in B

2

Vit

am

in B

3

Vit

am

in B

5

Vit

am

in B

6

Vit

am

in B

9

Vit

am

in C

V

ita

min

E

Poll

ute

d

59

2.7

8±

19

.69

a 0

.02

±0.0

2a

0.0

5±

0.0

1a

0.8

3±

0.1

0a

21

.00

±2

.52

a 0

.58

±0.0

2a

0.6

7±

0.1

1a

68

.41

±2

.31

a 1

.52

±0.0

2a

Un

poll

ute

d

62

7.5

9±

0.4

7b

0.0

8±

0.0

1b

0.0

8±

0.0

1b

1.1

2±

0.0

1b

23

.33

±2

.31

a 0

.81

±0.1

0b

0.7

3±

0.0

8a

78

.03

±1

.02

b

1.8

8±

0.1

7a

Tab

le 5

: V

ita

min

Con

ten

t (m

g/1

00g

) of

Abelm

osc

hu

s esc

ule

ntu

s M

oen

ch

Va

lues

(mea

n +

SD

of

trip

lica

te d

ete

rm

ina

tion

s) w

ith

dif

fere

nt

sup

ersc

rip

ts p

er c

olu

mn

are s

ign

ific

an

tly (

P<

0.0

5)

dif

feren

t.

En

vir

on

men

t

of

sam

pli

ng

V

ita

min

A

Vit

am

in B

1

Vit

am

in B

2

Vit

am

in B

3

Vit

am

in B

5

Vit

am

in B

6

Vit

am

in B

9

Vit

am

in C

V

ita

min

E

Poll

ute

d

30

2.5

7±

7.0

1c

0.0

7±

0.0

2c

0.0

4±

0.0

1c

0.6

5±

0.0

9c

17

5.2

2±

6.9

7c

0.2

5±

0.0

8c

0.5

3±

0.1

7c

85

.29

±1

.79

c 1

.97

±0.1

8c

Un

poll

ute

d

37

5.4

8±

0.1

8d

0.1

2±

0.0

0d

0.0

6±

0.0

1c

0.5

7±

0.0

2c

18

9.3

3±

2.3

1d

0.5

9±

0.0

5d

0.8

9±

0.0

2d

99

.15

±2

.69

d

2.2

7±

0.1

7c

Tab

le 6

: V

ita

min

Con

ten

t (m

g/1

00g

) of

Pte

roca

rpu

s m

ild

bra

ed

ii H

arm

s V

egeta

ble

s

Va

lues

(mea

n +

SD

of

trip

lica

te d

ete

rm

ina

tion

s) w

ith

dif

fere

nt

sup

ersc

rip

ts p

er c

olu

mn

are s

ign

ific

an

tly (

P<

0.0

5)

dif

feren

t.

En

vir

on

men

t

of

sam

pli

ng

M

ag

nesi

um

C

alc

ium

P

ho

sph

oru

s S

od

ium

P

ota

ssiu

m

Cop

per

Iron

Z

inc

Lea

d

Poll

ute

d

22

.17

±1

.39

a 7

0.2

3±

2.0

2a

57

.35

±0

.59

a 6

.34

±0.4

6a

10

7.6

3±

4.4

5a

0.4

2±

0.1

5a

0.9

5±

0.2

6a

0.4

8±

0.1

4a

0.6

5±

0.0

7a

Un

poll

ute

d

39

.60

±1

.39

b

82

.83

±2

.32

b

68

.28

±0

.46

a 7

.47

±0.2

3a

13

0.4

0±

6.5

5b

0.0

6±

0.0

1b

0.7

4±

0.2

5b

0.3

2±

0.0

7b

0.3

5±

0.1

0b

Tab

le 7

: M

iner

al

Con

ten

t (m

g/1

00g

) of

Ab

elm

osc

hu

s esc

ule

ntu

s M

oen

ch

.

Va

lues

(mea

n +

SD

of

trip

lica

te d

ete

rm

ina

tion

s) w

ith

dif

fere

nt

sup

ersc

rip

ts p

er c

olu

mn

are s

ign

ific

an

tly (

P<

0.0

5)

dif

feren

t.

En

vir

on

men

t

of

sam

pli

ng

M

ag

nesi

um

C

alc

ium

P

ho

sph

oru

s S

od

ium

P

ota

ssiu

m

Cop

per

Iron

Z

inc

Lea

d

Poll

ute

d

48

.67

±4

.38

c 6

4.3

8±

3.9

5c

40

9.8

9±

0.4

3c

17

.16

±1

.71

c 2

50.7

3±

4.2

9c

0.3

4±

0.1

3c

0.7

7±

0.1

8c

0.3

4±

0.0

6c

0.5

0±

0.2

2c

Un

poll

ute

d

52

.80

±2

.40

c 7

7.4

8±

2.3

2d

41

3.8

9±

12

.48

c 2

1.1

3±

0.1

2d

28

4.2

7±

2.4

4d

0.0

4±

0.0

0d

0.5

8±

0.0

9d

0.1

6±

0.0

2d

0.1

9±

0.1

3d

Tab

le 8

: M

iner

al

Con

ten

t (m

g/1

00g

) of

Pte

roca

rpu

s m

ild

bra

ed

ii H

arm

s V

egeta

ble

s.

Va

lues

(mea

n +

SD

of

trip

lica

te d

ete

rm

ina

tion

s) w

ith

dif

fere

nt

sup

ersc

rip

ts p

er c

olu

mn

are s

ign

ific

an

tly (

P<

0.0

5)

dif

feren

t.

when compared to samples from UPE. Results presented

in table 8 showed that P. mildbraedii from UPE are

excellent source of phosphorus (413.89±12.48),

potassium (284.27±2.44), calcium (77.48±2.32) and

magnesium (52.80±2.40). Also, the concentration of

minerals in P. mildbraedii from PE and UPE were

significantly different in all except in magnesium and

phosphorus.

DISCUSSION

Gas flaring and other oil and gas activities for

about four decades have contributed to pollution in

Oguta, which have impacted on the ecosystem. Soots

were seen on vegetation within the communities around

the flaring site. Plants growing in such environment have

over the years taken in varying doses of pollutants which

invariably may affect the nutritional and chemical

contents.

Our result showed that A. esculentus had better

nutritional value than P. mildbraedii with respect to

protein and carbohydrate contents. Also, A. esculentus

and P. mildbraedii showed higher values in proximate

contents (except in protein) than A. hybridus as reported

by Nwaogu et al., (2006). Carbohydrates provide energy

to cells in the body, particularly to the brain, a

carbohydrate dependent organ in the body. (Nelson and

Cox, 2005). These vegetables can supplement the daily

energy intake of humans (Bingham, 1998; Effiong et al.,

2009). The crude fibre content, indicates that the

vegetables are good sources of fibre, thus making them

veritable source of roughage. The concentrations of

carbohydrate were significantly reduced while ash

contents were increased in plants from polluted

environment when compared to plants from unpolluted

environment (UPE). The reduced carbohydrate can be

attributed to the effect of air pollutants as reported by

Farzana (2005), in which he affirmed that it reduces

photosynthesis in chloroplasts. The contents of protein,

crude fiber and fat in samples from PE were lower than

those from UPE but were not significantly different,

which indicates that they were not adversely affected by

the pollution.

The phytochemical results indicate that

A. esculentus and P. mildbraedii are good sources of

these beneficial chemicals. They have antioxidative,

hypocholesterolemic, chemoprotective and antibacterial

properties (Price et al., 1987; Enechi and Odonwodo,

2003; Okwu, 2004). Both vegetables are rich in

alkaloids, flavonoids and tannins which indicates that

they have diuretic, antispasmodic, anti-inflammatory and

analgesic effects (Owoyele et al., 2002; Nobre-Junior,

2007 ; Alisi et al., 2011). Comparatively, P. mildbraedii

had higher content of the phytochemicals studied. Also,

significantly higher amount of phytochemicals were

observed in vegetables obtained from PE. The increase

can be linked to their role in oxidative stress in plants.

Phytochemicals are secondary metabolite of plants,

known to exhibit diverse pharmacological and

biochemical effects on living organisms. It has been

reported that certain phytochemicals play important role

in antioxidant defense systems of vegetative plants

(Ugochukwu and Babady, 2003). Pollution by gas flaring

is taught to generate free radicals in surrounding

environment. Thus, it is expected that plants may

increase synthesis of antioxidant defense compounds.

These vegetables showed significantly high

amount of vitamins especially vitamins A, B1, B2, B5, B6

and C in samples from UPE when compared

to samples from PE. These vitamins are involved in

intermediary metabolism of both plants and animals

acting as part or whole coenzyme to some specific

enzyme system and playing important role in both

enzyme and non enzyme oxidative stress defense

systems. The high concentrations of vitamins A and C

will contribute significantly to the daily requirements in

view of the reports of Murray (1998). Vitamin C

maintains blood vessel flexibility and improves

circulation in the arteries of smokers. The most important



benefit of vitamins A and C is their involvement in free

radical scavenging processes (Trumbo et al., 2004;

Nwaogu et al., 2011). These chemically active radicals

are byproducts of many normal biochemical processes.

Their numbers are increased by environmental assaults

such as chemicals and toxins. The lower concentrations

of these vitamins in samples from PE suggest an inability

of the plants to synthesize these vitamins in sufficiently

large amount for their metabolic functions. Oxidative

stress caused by gas flaring in Oguta community can

interfere with the synthetic mechanisms of the plants in

the environment (Farzana, 2005).

Some of the mineral contents of

A. esculentus Moench and P. mildbraedii Harms are

comparable or higher than that reported for

Amaranthus hybridus (Nwaogu et al., 2006)

Mucuna utilis (Ujowundu et al., 2010),

Commelina nudiflora and Boerhavia diffusa (Ujowundu

et al., 2008). The values obtained for the minerals

indicates that the samples are good sources of mineral

and are of great nutritional importance. In animals,

potassium and sodium are important electrolytes.

Potassium is a major intracellular cation. Sodium is

involved in the regulation of acid-base equilibrium,

protection against dehydration and maintenance of

osmotic pressure in living system. It plays a role in the

normal irritability of muscles and cell permeability

(Schwart, 1975). Copper (Cu) is essential for

haemoglobin synthesis, normal bone formation and the

maintenance of myelin within the nervous system

(Passmore and Eastwood, 1986). In animals, the

manifestations of copper deficiency include; anaemia,

hypo-pigmentation, defective wool keritinization,

abnormal bone formation with spontaneous reproductive

and heart failure (Williams, 1982). In humans, it has

been established that occurrence of Cu absorption

disorder in after partial gastetomycin leads to severe

malnutrition just as when protein is severely deficient in

the diet; as in kwashiorkor (Davies, 1972). Calcium and

phosphorus are important and indispensable for the

synthesis of strong bones and teeth, kidney function and

cell growth (Uddoh, 1988; Brody, 1994). Phosphorus

and magnesium are also important in the regulation of

acid-alkaline balance in the body (Fallon, 2001).

The mineral contents, like Mg, Ca, P, S and K in

vegetables from PE have significantly (P<0.05) reduced

value compared to vegetables obtained from UPE. The

release of pollutants such as oxides of sulphur and

nitrogen, hydrocarbons and other volatile organic

carbons can create chemical and biological conditions

which may be harmful to plants and soil microorganisms.

One of such conditions is the reduction in the capacity of

plants to absorb cations (Wild et al., 2000). Crops grown

in soil with low mineral contents exhibit various forms of

mineral deficiency. In plants, potassium is an essential

nutrient and has an important role in the synthesis of

amino acids and proteins (Malik, 1982). Ca and Mg play

significant role in photosynthesis, carbohydrate and

nucleic acids metabolism (Russel, 1973). The reduced

content of these minerals will definitely affect these

important plant processes. Lead is yet to record any

physiological role in the biological system and are

known to be extremely toxic even at the slightest

concentration. Their presence in the samples calls for

serious concern

This study has shown that A. esculentus Moench

and P. mildbraedii Harms are good sources of nutrients

and their consumption should be encouraged. Improved

information on these plants will contribute to the

awareness of their nutritive value, especially in this time

of increased food insecurity. Also, gas flaring showed

negative effects on these plants, which could affect

animals that consume them. Similarly, the adverse health

consequences on the inhabitants around the gas flare

site are of great concern. Communities around such

environment should be enlightened on the inherent

dangers. Oil and gas industries should be compelled to

upgrade their waste disposal technologies, with emphasis



in gas disposal. This will reduce the detrimental effects

on the health and well-being of inhabitants of Izombe in

Oguta Local Government Area of Imo State.

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Jou

rn

al of R

esearch

in

Biology

Effect of age, sex and hemoglobin type on adaptive and blood biochemical

characteristics in Red Sokoto Goats

Keywords: Adaptive coefficient, heart rate, rectal temperature, blood biochemical characteristics.

ABSTRACT: This study was conducted to evaluate the effect of haemoglobin (Hb) types, sex and age on adaptive and blood biochemical characteristics of Red Sokoto goats. Ninety four (94) goats were sampled from two locations: Dei-dei and Gwagwalada grazing reserved, Abuja. Data were collected on adaptive characteristics {heart rate(HR) and rectal temperature(RT) and adaptive coefficient (AC) was calculated from the HR and RT} and blood biochemical characteristics{ haemoglobin (Hb) types, Hb-concentration (Hb-conc), Potassium concentration (K-conc) and albumin concentration (alb-conc)}. The effects of haemoglobin type, sex and age on the adaptive and blood biochemical characteristics of the goats was analyzed by general linear model (GLM) procedure of SAS. The results showed that the mean RT of the sampled goats was 38.9°C with very minimal variations (CV=0.5). The mean HR of the goats was 76.1bpm, with min and max HR of 70 and 80bpm. The mean albumin, Hb and K concentration were 38.4g/l, 8.9g/dl and 4.0Mmol/l, respectively. The variation of Hb type with adaptive and blood biochemical characteristics was significant (P<0.05) except Hb concentration. Higher HR was observed in goats with Hb AA and AB. Age and sex had significant effect (P<0.05; P<0.01) on HR, AC and albumin concentration of the goats. Although there was no trend in the variation of HR and AC with age, but HR and AC were higher in the older goats than the younger, however the albumin concentration significantly decreased with progressive increase in age of the goats.

870-875 | JRB | 2013 | Vol 3 | No 3




Authors:

Akpa GN, Alphonsus C

and Usman N.

Institution:

Animal Science

Department, Ahmadu Bello

University, Zaria, Nigeria.


Alphonsus C.

Email:

[email protected]


Dates: Received: 15 Dec 2011 Accepted: 01 Jan 2012 Published: 16 Apr 2013

Article Citation: Akpa GN, Alphonsus C and Usman N. Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats Journal of Research in Biology (2013) 3(3): 870-875


Original Research



Research Journal

mailto:[email protected]

INTRODUCTION

In recent years, advances in the field of

biotechnology have opened up a completely new area at

molecular levels with the introduction of techniques such

as routine electrophoresis employed for detection of

polymorphism at protein and enzyme loci as well as

other serological and immunogenetic procedures for the

measurement of variations (Salako et al., 2007). Data

obtain from this type of study could be useful as genetic

markers for important economic characteristics and

could aid significantly in selection of superior animals

for breeding purposes.

Haemoglobin typing is very important as

different Hb types may have selective advantage in

different geographical regions (Ndamukong, 1995).

Economic pressures of various kinds are forcing the

production of livestock into climatic environments that

are increasingly more remote from the considered ideal

for optimal production and feed utilization.

Thermal stress, which is one of the major factors

that affect the productivity of many farm animals can be

reflected in an easily observable changes in pulse rate,

respiration rate, and rectal temperature, although the

whole body reacts to thermal stress by an elaborate series

of chain reactions (Ahmed, 2004). The most obvious

index of thermal stress is body temperature response.

Deviation from normal rectal temperature indicates that

the animal is under stress, that its homeothermic

mechanisms are overtaxed Ahmed, 2004).

Adaptive characteristics of animals serve as a

key to managing any livestock operation. Adaptive traits

such as rectal temperature, heart rate, and flank

movement have been documented to have some

significant effect on genetic variations. Every normal

animal has a range of individual adaptive traits in

relation to a specific physiological pattern.

Because study of environmental physiology

involves so many variables and scientific discipline,

much is being published on this subject especially as

related to farm animals. Comprehensive reviews have

appeared under the authorship of Alderson, 1992,

Derman and Noakes, 1994, Tambuwal et al., (2002),

Otoikhian et al., 2009, chukwuka et al., 2010,

Opara et al., 2010, Gurcan et al., 2010 and a lot of

others. Research results reported in this paper is intended

to supplement data reviewed by authors listed.

Eventually, accumulated data will permit specific

recommendation on breeding, feeding and management

of farm animals.

This study therefore aimed at studying the effects

of Haemoglobin type, sex and age on adaptive and blood

biochemical characteristics of Red Sokoto goats.


Location

The study was conducted at the Federal Capital

Territory Abuja, located within the Northern guinea

Savanna zone of Nigeria. It is laying between latitudes

8.25° and 9.0° N of the equator and Longitude 6.45° and

7.39° E of the Greenwich Meridian. (Presentation

Copyright@ falling Rain Genomics, 1996-2010).

Data collection

Ninety four (94) goats were sampled from two

locations: Dei-dei and Gwagwalada grazing reserve,

Abuja. Data were collected on the adaptive and blood

biochemical traits. The adaptive traits were Heart rate

(HR), Rectal temperature(RT) and Adaptive coefficient

(AC) while the blood biochemical characteristics were

haemoglobin (Hb) types, Hb-concentration (Hb-conc),

Potassium concentration (K-conc) and albumin

concentration (alb-conc).

METHODS OF MEASUREMENTS

Adaptive Traits

Heart Rate (HR)

Heart rate was taken by placing stethoscope on

femoral artery of the hind limb of the goat to count the

number of beat per minute.


Akpa et al.,2013

Rectal Temperature (RT)

This was taken using clinical thermometer which

was inserted into the rectum of the goat and left for

45-50 seconds. It was then removed and the temperature

level was read. The values read were recorded, and the

process was repeated for the other goats.

Blood Biochemical Characteristics

Blood samples were taken from each of the

experimental goats through the jugular vein. 5 mls of the

blood was taken from each goat, from which 2 mls was

put into heparinized vacutainer tubes containing

anticoagulant ethylene diamine tetra acetic acid (EDTA).

The remaining 3 mls of the blood was put into sterile

vacutainer tubes (without anticoagulant). The samples

were labeled accordingly. The blood samples in the

sterile vacutainer tube were centrifuged in order to have

a clear layer of serum. This serum was pipetted into

another sterile bottle and store in a refrigerator. The

blood samples were taken to the Haematological

laboratory of Ahmadu Bello Teaching Hospital from

where the analysis of blood biochemical characteristics

was carried out.

A spectrometer with wavelength capability of

600-650 nm (Zenway 5041 colorimeter) was used

to analyzed for the albumin concentration, while the

K concentration was analysed using Corning

flame photometer 410. Electrophoresis and

Cyanmethaemoglobin method was used to analyzed for

Hb-types and Hb-conc, respectively.

Data Analysis

The adaptability of the goats were measured by

determining the adaptive coefficient from the values of

the Rectal Temperature (RT) and Heart Rate (HR) as

thus

Adaptive coefficient (AC) = (RT/38.33) + (HR/23.00)

The effects of haemoglobin types, sex and age on

the adaptive and blood biochemical characteristics of the

goats were determined by general linear model (GLM)

procedure of SAS, (2005).


Rectal Temperature (RT) is directly affected by

the surrounding and ambient temperature, and high

ambient temperature has a negative effect on

productivity of the animal. Chukwuka et al., (2010)

reported that negative effect of high ambient temperature

is direct in the form of stress suffered by the animal and

the diversion of energy from the purpose of production to

regulation of body temperature and indirectly by

affecting the availability of feed resources upon which

production is dependent. In this study, the mean RT of

the goats was 38.9°C with minimum and maximum body

temperature of 38.1 and 39.4 °C (Table 1). These values

were within the reference range of previous study

of goats in thermal neutral condition (Otoikhian et al.,

2009) and this indicate that the goats used for this

research showed no clinical signs of stress during the

research period. The body temperature of the goats

exhibited minimal variations (CV=0.5%), thus implying

that goats are homoeothermic animals, they can maintain

near constant body temperature under wide range of

environmental conditions.

The Heart Rate (HR) is the pulse that helps to

know the beating rate of the heart which is measured


Akpa et al.,2013

Characteristics N Mean±SE CV(%) Min Max

Rectal Temperature (°C) 94 38.9±0.02 0.5 38.1 39.4

Heart Rate (bpm) 94 76.1±0.39 3.8 70.0 81.0

Adaptive coefficient 94 4.3±0.02 5.0 4.1 4.6

Albumin concentration (g/l) 94 38.4±0.34 8.5 31.0 48.0

Hemoglobin concentration (g/dl) 94 8.9±0.16 17.1 4.0 12.7

Potassium concentration (Mmol/l) 94 4.0±0.06 14.7 3.0 5.8

Table 1: Summary Statistics of the measured characteristics in Red Sokoto Goats

in beats per minute (bpm) using stethoscope

(Otoikhian et al., 2009). The mean HR of the goats used

in this study was 76.1bpm, with the min and max HR of

70 and 80bpm. This is slightly higher than the range of

70-75bpm reported by Derman and Noaks (1994) in

goats. The minor difference observed in the values of the

HR may be explained by differences in geographical

conditions, season or climate and physiological

conditions of the sample goats.

Rectal temperature and heart rate have been

shown to be good indicators of the thermal stress and

may be used to assess thermal adversity of the

environment (Al- Haidary, 2004).

The Adaptive Coefficient (AC) (which is the

function of RT and HR) signifies the level of adaptability

of the goats to the environments varied significantly

(P<0.05) with Hb types, sex and age of the goats. The

goats with Hb AA and AB had higher AC than those

with BB and AC; likewise the bucks had higher AC than

the does.

Potassium is one of the intracellular elements

that regulate the intracellular density of the cell. The

amount of K-concentration is fairly high at intracellular

membranes (Gurcan et al., 2010). The values of

K-concentration reported by Opara et al., (2010) for

WAD bucks and does were 17.8 and 6.9mmol/l,

respectively. These values were higher than the mean

value of 4.0mmol/l observed in this study; this is

probably due to differences in breed and physiological

conditions of the sampled animals. Researchers had

identified the existence of different type of K in different

species of animals, and that in sheep for instance, there

are two types of K which is high and low K with the

low K type dominant over the high K type

(Soysal et al., 2003). Also Gurcan et al., (2010) reported

a range of 4.23 to 11.69mmol/l for low K type in

animals.

The concentration of albumin in this study

(38.4g/dl) was slightly higher than the 34.5g/dl reported

by Opara et al., (2010).

The variation of Hb type with adaptive and blood

biochemical characteristics was significant (P<0.05)

except Hb-concentration (Table 2). The relationship

between Hb types and HR can be linked to the different

Akpa et al.,2013

Characteristics Hemoglobin Type

AA BB AB AC SEM LOS

Rectal Temperature (°C) 39.0a 39.0a 39.0a 38.9b 0.02 *

Heart Rate (bpm) 76.6a 75.3b 76.2a 75.1b 0.40 *

Adaptive coefficient 4.4a 4.3b 4.4a 4.3b 0.02 *

Albumin concentration (g/l) 37.6b 39.2a 38.6a 38.8a 0.34 *

Hemoglobin concentration (g/dl) 9.1 8.6 8.8 8.6 0.16 ns

Potassium concentration (Mmol/l) 3.8b 3.9a 4.0ab 4.4a 0.06 *

Number of observations 30 11 41 12 94 ab: means within the same row with different superscripts differ significantly(P<0.05); ns:not significant;

Table 2: Effect of hemoglobin type on adaptive and blood biochemical characteristics

Characteristics Sex

Buck Doe SEM LOS

Rectal Temperature (°C) 39.0 39.0 0.03 ns

Heart Rate (bpm) 78.5a 75.0b 0.49 **

Adaptive coefficient 4.4a 4.3b 0.02 **

Albumin concentration (g/l) 39.8a 37.7b 0.49 **

Hemoglobin concentration (g/dl) 9.1 8.7 0.26 ns

Potassium concentration (Mmol/l) 3.9 4.0 0.08 ns

Number of observations 30 64 94 ab: means within the same row with different superscripts differ significantly(P<0.01); ns:not significant;

Table 3: Effect of Sex on adaptive and blood biochemical characteristics


levels of oxygen carrying capacity of the different

Hb types. In this study, higher HR was observed in goats

with Hb AA and AB, and Hb A is known to be the

haemoglobin allele with highest affinity for oxygen. This

is in line with the earlier report of Huisman et al., (1959)

who relates the preponderance of Hb A to it greater

affinity to oxygen. This could also explain the high

adaptive coefficient observed on goats with Hb types AA

and AB since adaptive coefficient is a function of HR

and RT.

The variation of HR, AC and Albumin

concentration with sex was highly significant (P<0.01;

Table 3). The RT of the buck and does were similar

however, the HR was higher in bucks (78.5bpm) than the

does (75.0bpm) this is probably due to the high sexual

activity of the bucks. There was no significant (P>0.05)

difference between the bucks and does in Hb and

K concentration. This is contrary to the study of

opera et al., (2010) who reported significant differences

between WAD bucks and does in there Hb and

K concentration. This is probably due to differences in

breeds and location of the animal, Hb type had been

reported to vary with breed and location (Ndamukong,

1995, Abdussamad et al., 2004 Essien et al., 2011)

Age significantly (P<0.05) influence HR, AC

and albumin concentration but had no significant

influence on the RT, Hb and K concentration (Table 4).

Although there was no trend in the variation of HR and

AC with age, but it was observed that the HR AC was

higher in the older goats than the younger, however the

albumin concentration significantly decreased with

progressive increase in age of the goats. The observed

significant influence of age on albumin concentration

is at variance with the earlier studies of

Piccione et al., (2009) and Opara et al., (2010) who

reported non-significant effect of age on albumin

concentration of WAD goats.

CONCLUSION

The mean body temperature (38.9°C) of the

goats used was within the reference normal range for

goats in thermal neutral condition and this indicates that

the goats showed no clinical signs of stress during the

research period.

The albumin concentration, heart rate and

adaptive coefficient of the goats had clear variation

based on differences in haemoglobin type, sex and age of

the animals.

REFERENCES

Abdussamad AM, Esievo KAN, Akpa GN. 2004.

Haemoglobin types in the Nigerian Zebu and their

crosses in Zaria. Proceedings of the Nigerian Society for

Animal Production (NSAP). 29-31.

Alderson GLH. 1992. genetic conservation of domestic

livestock. CAD International, Wallingford, UK. 242.

AL-Haidary. 2004. Physiological Responses of Naimey

Sheep to Heat Stress Challenge under Semi-Arid

Environments. International Journal of Agriculture

&Biology 06(2):307-309.


Akpa et al.,2013

Characteristics Age of goat

12mon 18mon 24mon 30mon SEM LOS

Rectal Temperature (oC) 39.0 39.0 39.0 39.0 0.02 ns

Heart Rate (bpm) 77.1b 74.4c 74.4c 78.5a 0.37 *

Adaptive coefficient 4.4b 4.3c 4.3c 4.5a 0.02 *

Albumin concentration (g/l) 39.2a 37.4b 36.7c 36.5c 0.32 *

Hemoglobin concentration (g/dl) 9.1 8.7 8.3 8.5 0.16 ns

Potassium concentration (Mmol/l) 4.0 4.0 4.0 4.0 0.06 ns

Number of observations 55 25 12 2 94 ab: means within the same row with different superscripts differ significantly(P<0.05); ns:not significant;

Table 4: Effect of Age on adaptive and blood biochemical characteristics

Chukwuka OK, Okoli IC, Okeudo NJ, Opara MN,

Herbert U, Obguewu IP and Ekenyem BU. 2010.

Reproductive potentials of West African Dwarf sheep

and goats. A Review, Research Journal of Vetrenary

Sciences 3(2):86-10.

Derman KD and Noakes TD. 1994. Comparative aspect

of exercise physiology. In: Hodgson, D.R., Rose, R.J

(Eds). The Athletic horse: Principle and practice of

Equine Sports Medicine. Philadelphia. W.B. Saunders.

13-25.

Essien IC, Akpa GN, Barje PP, Alphonsus C. 2011.

Haemoglobin types in Bunaji cattle and their Friesian

crosses in Shika, Zaria-Nigeria. Afri. J. Anim. Biomed.

Sci., 6(1):112-116.

Gurcan EK, Erbas C and Ozden C. 2010.

Biochemical polymorphism of erythrocyte, Potassium

and glutathione protein: the relationship with some blood

parameters in Kivircik sheep breed. African Journal of

Agricultural Research 2(10):1022-1027.

Huisman THJ, Van Der Helm HJ, Visser HKA and

Van Vilet G. 1959. Symposium on abnormal

Haemoglobin. Blackwell Scientific Publication. 181.

Ndamukong. 1995. Haemoglobin polymorphism in

Grassland dwarf sheep and goats of the North West

province of Cameroon. Bulletin of Animal Health and

Production in Africa. 43:53-56.

Opara MN, Udeyi N and Okoli IC. 2010.

Haematological parameters and blood chemistry of

apparently healthy West African Dwarf (WAD) goats in

Owerri, South-eastern Nigeria. Tropical Animal Health

and Welfare Research group. New York Science Journal

3(8).

Otoikhian CSO, Orheruata MA, Imaseuen JA and

Akporhuarho OP. 2009. Physiological response of local

(West African Dwarf) and adapted Switzerland

(White Bornu) goat breed to varied climatic conditions in

South-south Nigeria. African Journal of General

Agriculture. 5(1):1-6.

Piccione G, Casella S, Lutri L, Vazzana I, Ferrantelli

V, Caola G. 2009. Reference values of some

Haematological, Haematochemical and electrophoretic

parameters in the Girgentana goats. Faculty of Vetrinary

Medicine, University of Messina, Italy.

Presentation Copyright @Falling Rain Genomics, Inc

1996-2010.

SAS. 2005. user guide for personal computers, statistical

programme 9.01 windows version.(SAS Institute Inc.

Cary, NC).

Salako AE, Ijadunola TO and Agbesola YO. 2007.

Haemoglobin polymorphism in Nigerian indigenous

Small Ruminant population-preliminary investigation.

African Journal of Biotechnology 6(22):2636-2638.

Soysal ML, Gurcan EK, Ozkan E. 2003. Turkiye de

yetistirilen cesitli koyun Irklrinda trim kan potasyum

konsan trasyonu polimorfizmi vizerine arastirmalar.

GAP III Tarim Kongresi, sanliurfa.

Tambuwal FM, Agala BM and Bangana A. 2002.

Haematological and Biochemical values of apparently

healthy Red Sokoto goats. Proceeding of 27th Annual

Conference, Nigerian Society of Animal Production

(NSAP).FUTA, Akure, Nigeria. 50-53.


Akpa et al.,2013

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Biology

Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with

special reference to its life table attributes in Tripura, India

Keywords: Catopsilia pomona butterfly, Pieridae, eco-biology, life table, Tripura, north east India.

ABSTRACT: Butterflies of the family Pieridae are common in tropical parts of the world. They are considered as major pollinators as well as pests of economically important plants. Catopsilia pomona is a dominant pierid butterfly found in association with wild plants of Tripura, northeast India. It is abundant throughout the year. Present study was conducted to document the eco-biology of Catopsilia pomona with special reference to its life table attributes in the state of Tripura. Survival rates of life cycle stages in the semi-natural as well as in the field were the maximum during the wet and hot season. Mortality (k value) of different life cycle stages as a proportion of individuals dying during development varied from 0.16 to 0.46 in different seasons. Results suggested that abiotic factors and mortality factors of egg significantly influenced the survival rate of C. pomona population. This butterfly depends on three species of Cassia plants, all shrubs, for their oviposition and larval development in the environment of Tripura.

876-885 | JRB | 2013 | Vol 3 | No 3



www.jresearchbiology.com Journal of Research in biology


Research Journal

Authors:

Samit Roy Choudhury and

Basant Kumar Agarwala*

Institution:

Ecology & Biodiversity

Laboratories, Department of

Zoology, Tripura University,

Suryamaninagar- 799022,

Tripura, India.


Basant Kumar Agarwala

Email:

[email protected]

Phone No:

0091 381 237 9083/9123


Dates: Received: 22 May 2012 Accepted: 28 May 2012 Published: 17 Apr 2013

Article Citation: Samit Roy Choudhury and Basant Kumar Agarwala. Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India. Journal of Research in Biology (2013) 3(3): 876-885


Original Research

INTRODUCTION

Host selection for survival, development and

reproduction in majority of insects often vary in space

and time (van Nouhuys et al., 2003; Nylin et al., 2009)

which, in turn, depends on the availability (minimum

density per unit area) of closely related host plant species

(Thorsteinson, 1960), and trade off between host

preference by females for oviposition and larval

performance of insects (van Nouhuys et al., 2003).

However, adult butterflies and their caterpillars show

preference for certain host plants for tender shoots,

pollen and nectar as food source. Thus, butterfly

diversity of a particular habitat generally reflects the

overall plant diversity of that habitat. Butterflies are

essential component of any natural ecosystem. Their

value as indicators of biotope quality is being recognized

because of their sensitivity to minor changes in micro-

habitat, climatic conditions as well as seasonal changes

(Kremen, 1992; Murugesan and Muthusamy, 2011).

They are considered as ideal subject for ecological

studies of terrestrial landscapes (Thomas and Malorie,

1985).

North eastern region of India is blessed with

vegetation rich landscapes that support diverse butterfly

fauna and other insects (Alfred et al., 2002). The state of

Tripura, being a part of this region, also contains large

number of butterfly species which is evident from

infrequent records of these taxa (Mandal et al., 2002;

Agarwala et al., 2010; Majumder et al., 2011; Roy

Choudhury et al., 2011). Butterflies of the family

Pieridae are common in tropical parts of the world and

are considered as major pollinators of crop plants

(Borges et al., 2003), and a few of them are also

considered as pests of economically important plants

(Anonymous, 2007; Capinera, 2008). Despite their

common occurrence, there is a lack of substantial study

on the ecology, seasonal abundance, host preference and

life history of the most common pierid species

Catopsilia pomona F. found in association with wild

plants of north east India, including Tripura. However,

information on life table and host selections are available

on other pierid species that feed and oviposit on crop

plants (Chew, 1995). C. pomona, a dominant pierid

butterfly, is found throughout the year in the state of

Tripura (Agarwala et al., 2010; Majumder et al., 2011;

Roy Choudhury et al., 2011). It prefers green and moist

lands, pasture lands, farms, and edge of drains, moist

deciduous forests, hillocks, and semi-arid areas with high

abundance of grasses, small herbs and shrubs i.e.

secondary type of vegetation (Atluri et al., 2004).

Reported larval host plants of common emigrant

comprise of Cassia fistula L., C. sophera L.,

C. occidentalis L., C. tora L., C. siamea (Lam.) Irwin et

Barneby, Butea frondosa, and Bauhinia racemosa L.

(Kunte, 2000; Atluri et al., 2004). Among these plants

C. fistula, C. tora, C. occidentalis, C. sophera, and

B. racemosa are important as medicinal plants

(Anonymous, 2004; Danish et al., 2011; Harshal et al.,

2011; Singh and Dubey, 2012), and C. siamea is used in

social forestry (Atluri et al., 2004, Borikar et al., 2009).

Hence, it is very important to document the seasonal

occurrence and its host plant preference for oviposition

and larval development of C. Pomona. With this view,

the present study was conducted to know the eco-biology

of Catopsilia pomona with special reference to its life

history attributes in the state of Tripura.

Study site

Present study was conducted in Trishna Wildlife

Sanctuary of south Tripura district (23°26.137’ N,

91°28.184’ E: 51-82 m asl), having an area of about

194.7 sq. km. Study location is characterized by patches

of secondary moist deciduous forests and surrounded by

swamp areas. Forest patches are rich in sal trees, garjan

trees, bamboo bushes, herbs, shrubs and climbers.

Trishna sanctuary is known by 230 tree species, 110

species of shrubs, 400 species of herbs, and 150 species

of climbers (Economic review of Tripura, 2008-2009).

Among the known host plants of C. pomona, the study

Roy Choudhury and Agarwala, 2013


area contains three species of Cassia only viz.

Cassia sophera, C. tora and C. occidentalis which are

considered to be the preferred hosts of larvae. Some part

of the study area is used for rubber cultivation and paddy

cultivation (Figure 1). The area has a tropical climate,

with cold weather from November to February. Average

daily temperature varies from the minimum of 6.8°C in

January to the maximum of 37.7°C in June. The area

receives, on an average, 3353.4 mm rainfall annually.


Field census of eggs, larvae and oviposition

preference of C. pomona

Prior to the study a reconnaissance survey was

made in the Trishna study area to locate the available

host plants distribution of C. pomona. Walk census for

leaves of host plants containing eggs and larvae were

held at an interval of 7-days from March 2007 to

February 2008. For this, two line transects (approx. 1 km

long and 5 m wide) were set up in the study area. Thirty

host plants, 10 plants each of C. sophera, C. tora and

C. occidentalis, were randomly selected for the study

along the length of transects and were marked with

plastic tags. Thus, sixty plants from three species were

selected from transects. Ovipositing females were

followed in the selected host plants for recording number

of eggs laid per female per leaf. Binoculars were used to

observe the females from a distance (about 2 m) without

disturbing them. The same host plant was also observed

for presence of larvae. All the females seen ovipositing

on the selected host plants was recorded during the

transect walk. Two transects were walked in two

consecutive days in a week. Ten apical leaves were

observed within a selected plant for egg and larval counts

which were made between 8.00 AM to 12.00 noon local

time. When a female was found to either laying eggs or

seen perching near a host plant, halt was made for

approx. 8 to10 minutes, and then move to the subsequent

host plants along the transect. Different host plants

selected by females for oviposition were recorded,

photographed, collected and later identified by

comparing with the herbarium deposited in the gallery of

Plant Taxonomy and Biodiversity Laboratories,

Department of Botany, Tripura University.



Figure 1. Geographical map of Trishna and landscape of the Study area.

Larval host range and seasonal variation in

development

Leaves of the host plant species found to contain

freshly laid eggs of C. pomona in field were brought to

the field station (3 km from the study area), and

transferred individually to 10 cm diameter paired Petri

dishes lined with corrugated papers. These were fed with

surplus quantity of tender leaves of respective host plants

from which they were actually collected. Twenty

replicates were used for each host plant species. Food

was changed every 24 hrs intervals. Petri dishes were

cleaned at the time of food change. These were observed

twice in a day at 11 am and again at 5 pm to record the

incubation period of eggs, developmental time of larvae,

and pupae. Mortality in development, if any, was also

recorded. This was simultaneously done on each host

plant, once in five different seasons to record the

seasonal variation, if any. Experiments were set up at the

field station (Temp: 18°C ~ 27°C, RH: 45~75%, and

L: D: 16:8h) i.e. in the controlled environment.

Larval development in field

Selected plants with freshly laid eggs and

subsequent developmental stages were provided with

coloured tags and these were numbered for easy

identification. Individual eggs, larvae and pupae were

followed daily, and the disappearance of individuals or

those that failed to develop in to the next stage at

different life stages were recorded. Larvae were found to

be slightly sluggish and females laid solitary eggs,

usually one on each leaf. The study was repeated once in

different seasons.

Survival rate and K-factor analysis

An age-specific life table was constructed

following the method of Stiling (2002). To prepare the

life table, records were made on the larval durations and

survival rate at each developmental stage i.e. eggs to

emergence of adults from pupae. For this purpose,

409 eggs and 317 eggs of C. pomona were studied in

natural (in field) and in controlled conditions (ambient

condition of field station), respectively. Meteorological

data of Trishna study area were collected from the

records maintained by the Department of Agriculture,

Govt. of Tripura at Arundhuti Nagar, Agartala.

Data analysis

Field data on proportion of host plants used by

C. pomona for laying of eggs and distribution of eggs per

leaf of the different host species during a year were used

to draw population curves. For this purpose, weekly data

were pooled on monthly basis. Developmental time from

egg to the eclosion of pupae on different host plants and

between different seasons was subjected to one-way

analysis of variation (ANOVA). Mean values of

development time on different host plant species and

between different seasons were compared by Tukey’s

multiple comparison test. Differences in development

time recorded in field and in field station were compared

by Students t-test. A significance level of 0.05 was used

to reject the null hypothesis. Field data on distribution of

eggs on different host plant species were subjected to

regression analysis to reveal the relationship between

oviposition preference and host utilization. Based on the

life table data, survival rate and K factor value that

closely mirrors the overall population mortality was



Host plant No. of leaves

observed

No. of larvae

counted

Mean (+SEM) no

of larvae/ leaf

ANOVA No. of eggs

counted

Mean (+SEM)

no of eggs/ leaf

ANOVA

C. sophera 4800 984 0.21 + 0.01 F = 6.909 ,

df = 2,14397,

P = 0.0001

1237 0.26+0.02 F = 5.26,

df = 2,14397,

P = 0.006 C.occidentalis 4800 563 0.12 + 0. 02 899 0.19+0.03

C. tora 4800 647 0.13 + 0.01 816 0.17+0.02

Table 1. Oviposition preference of C. pomona females on different host plants in the study area

determined. At each life stage, number of deaths

(k value) was calculated as under: k = log Nt - log Nt+1,

where Nt is the density of the population before it is

subjected to the mortality and Nt+1 is the density

afterward. Total generational mortality factor K is

determined as the sum of the individual mortality factors

k at egg, larval and pupal stage of the C. pomona species

(Stilling, 2002). For interpretation of colleted data, the

year was divided in to five seasons: spring (March,

April), summer (May, June), rain (July, September),

autumn (October, November), and winter (December-

February). To determine the relationship between

successful development (%) of C. pomona eggs and

climatic factors in the study area regression analysis was

carried out. Origin 7 software (www.originlab.com) was

used for the analysis of data.

RESULTS

Egg abundance and oviposition preference

Females of C. pomona laid solitary eggs at edges

and on undersides of tender or young leaves (one egg/

leaf/female) of C. sophera, C. occidentalis and C. tora

plants throughout the year (Table 1, Figure. 2). In the

year-round census of 10000 m2 (1000 m long x 5 m wide

x 2 transects @ 1 ha) which represents less than 0.5% of

the study area (19.47 ha), 52.54% to 85.07% of

C. sophera plants, 21.31% to 69.47% of C. occidentalis

plants and 23.88% to 56.52% of C. tora plants were

found with one or more eggs. Between the three host

plant species, common emigrant females selected the

highest proportion of C. sophera for oviposition during

hot and wet months, and the maximum was recorded in

the month of August (Figure 2). In comparison,

distribution pattern of eggs on C. occidentalis plants

showed marked difference from the distribution of eggs

on C. sophera. Higher proportion of this host plant

species was recorded during dry and cooler months, and

the maximum was recorded in the month of January

(69.47%) (Figure. 2). In case of C. tora, the trend of egg

distribution was found to be nearly similar to that of

C. sophera but the proportion of host use was found to

be much lower than C. sophera (Figure. 2). Occurrence

of eggs showed that 4800 leaves each of C. sophera,

C. occidentalis and C. tora that were surveyed during the

year, contained 1237, 899 and 816 eggs, respectively

(mean + SEM: C. sophera: 0.26+0.02 eggs per leaf,

C. occidentalis: 0.19+0. 03 eggs per leaf and C. tora:

0.17+0.02 eggs per leaf, ANOVA: F = 5.26, df = 2,

14397, P = 0.006) (Table 1).

Larval host range

Larvae of C. pomona were found to feed on

tender leaves of the three host plant species, viz.

C. sophera, C. occidentalis and C. tora. Higher

proportion of C. sophera plants were used as food and

maximum was recorded in the hot and wet month of

August (26.70%). Incidence of larvae on C. occidentalis



Month N Mean + SEM value (days)

C. sophera C. occidentalis C. tora

March 36 24.50 + 0.26 1 a 24.75 + 0.25 1 a 24.74 + 0.33 1 a

May 36 20.67 + 0.31 2 a 20.92 + 0.42 2 a 20.92 + 0.42 2 a

August 36 18.92 + 0.23 3 a 19.42 + 0.63 3 a 19.00 + 0.28 3 a

October 36 21.17 + 0.24 2 a 21.33 + 0.28 2 a 21.25 + 0.25 2 a

December 36 30.67 + 0.47 4 a 30.83 + 0.41 4 a 31.00 + 0.41 4 a

Dissimilar numbers following means in a column denote significant difference and similar letters accompanying

means show no difference between them by Tukey’s multiple comparison range test at 5% significant level.

Table 2. Development time (in days) of C. pomona on different host plant species

plants was recorded to be the highest in January

(20.61%) and lowest in August (1.64%), respectively. In

case of C. tora host, the highest proportion was recorded

in the month of June (17.24%) and the lowest in the

month of January (5.33%) (Figure. 3). Occurrence of

larvae showed that 4800 leaves each of C. sophera,

C. occidentalis and C. tora that were surveyed during the

year, contained 984, 563 and 647 larvae, respectively

(mean + SEM: C. sophera: 0.21 + 0.01 larva per leaf,

C. occidentalis: 0.12 + 0. 02 larva per leaf and C. tora:

0.13 + 0.01 larva per leaf, ANOVA: F = 6.909,

df = 2,14397, P = 0.0001) (Table 1).

Developmental time and seasonal variation

Developmental time of different immature stages

(egg to pupae) of C. pomona was found to vary in

different seasons but did not show difference in any one

season between different host species (Figure 4).

Development time was recorded to be the longest at

lower temperature and lower relative humidity

corresponding to the month of December (controlled

condition: average temperature=18°C, average relative

humidity=51.33%) and shortest at higher temperature

and higher relative humidity in August (average

temperature=27.91°C, average relative humidity

=77.07%) (Table 2).

Survival rate and K factor analysis

Results showed that in field about 30% of the

eggs deposited by C. pomona developed in to pupae

during the months of July and August (average

temperature 31.09°C, average humidity 70%, mean

rainfall 7.45 cm). Developmental success was limited to

13.04% in the month of December (average temperature

19.330C, average humidity 51%, rainfall 0 cm).

Regression analysis of survival rate showed positive

correlations with average temperature (y =1.08 + 0.87x,



Figure 4. Development time (in days) of C. pomona on

different larval host plants in different months of a

year. Similar alphabets accompanying bars denote no

significant difference between the mean values in that

month.

Figure 3: Mean number of larvae of C. pomona

recorded on different host plants.

Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9P

rop

ort

ion

of h

ost p

lan

ts s

ele

cte

d fo

r o

vip

ositio

n

Months

C. occidentalis

C. sophera

C. tora

Figure 2. Proportion of host plants of three Cassia

species recorded with eggs of C. pomona in different

months of the year in the study area.

r2=0.74) (Figure 5a), average relative humidity

(y = 3.87 + 0.33x, r2 = 0.52) (Figure 5b), and with mean

rainfall (y =20.07 + 1.64x, r2 = 0.64) (Figure 4c).

Number of eggs that developed successfully in fields

(24.03+1.46; n=240) and in semi natural condition

(76.36+0.90; n=240) showed significant difference

(t =30.54, df =478, P =0.000).

K-value analysis showed maximum mortality in

development (0.46) in the month of December and

minimum (0.16) in the month of September. K - values

of the eggs recorded in different seasons were found to

be very high (0.21) and very low (0.09), respectively,

during these two months. Analyses showed that mortality

in the egg stage influenced the total K value the most

(Figures. 6a, b).

DISCUSSION

Natural populations of phytophagous insects

including butterflies frequently encounter wide choice of

host plants of differing suitability (Badeness et al., 2004;

Dennis et al., 2006). The dominant strategy among

herbivorous insects involves specialization on a set

of closely related plants that will maximize offspring

survival and fitness (Ward and Spalding, 1993; Gibbs et

al., 2006), and also to the phenological characteristics of

host plants. It is evident from the present study that

C. pomona butterflies utilize three species of Cassia for

oviposition and larval development in Trishna study

area. Among these host plants, maximum number of

C. pomona eggs were found in C. sophera with higher

proportion recorded during hot and wet months, and

lowest in dry and cooler months of the year. During dry

and cool months, females choose C. occidentalis in

higher proportion for oviposition followed by C. tora.

This might be due to the availability of more young

leaves in C. occidentalis and C. tora compared to

C. sophera in dry and cooler months of the year. Results

indicated that common emigrants preferred C. sophera

than the two other host plants but utilized three hosts

throughout the year depending on the host plant

phenology, and made the larval host range wider.

Patterns of host use have several effects on butterfly



Figure 5. Regression analysis between successful

development (%) of C. pomona eggs and climatic

factors: (a) average temperature (oC), (b) average

relative humidity (%), and (c) mean rainfall (cm).

population dynamics (Hanski and Singer, 2001). Food

plant-insect herbivore association is based on resource

size and optimal synchronization of their respective

life-cycles. If resource size in time and given space is

large, insects will show monophagism. In comparison, if

resource size is short and patchy, then insect herbivores

are generally polyphagous or oligophagous (Price, 1997;

Dixon, 1998; Nylin et al., 2009). In this study,

availability of taxonomically closely related Cassia

plants in time and given area under study on which

C. pomona successfully completed their life history

attributes might widen their host range. This finding is in

conformity with the optimisation theory of species in

relation to host plants in time and space (Begon et al.,

1996; Scheirs and Bryn, 2002).

Population of C. pomona showed strong

relationships with climatic factors. They took longer time

for development in the dry and cooler months when the

suitable habitat for oviposition and larval development

were minimum than in wet and hot months. But,

developmental time on the different host plants did not

differ during a particular season that suggested possible

qualitative similarity between host plants. However,

several studies showed that ovipositing females of

phytophagous butterflies typically show a preference for

host plants that are capable of supporting fast larval

growth (Thompson, 1988a, b, c; Janz et al., 1994).

Climatic factors are well known for their

significant influence on population dynamics of animal

communities (Leonard et al. 1998). Analysis of K-value

in this study has revealed that the average temperature,

the average relative humidity and the mean rainfall

showed strong positive relationships with survival rate of

C. pomona. In the present study no biotic factors such as

parasites, predators were noticed which can also

influence the population dynamics of C. pomona

butterfly.

CONCLUSION

Results revealed that C. pomona females

occurred and laid eggs throughout the year on three host

plant species of Cassia. It preferred C. sophera host over

C. occidentalis and C. tora for oviposition and larval

development. Pattern of egg distribution i.e. oviposition

was found to be linked with host plant phenology. Egg

mortality was the major influencing factor in

determination of survival rate. The k-value of egg

mortality (k1) and total mortality factor (K) showed

strong positive relationship.

ACKNOWLEDGEMENT

Authors are thankful to the Head, Department of



Figure 6. Key- factor analysis of development of

C. Pomona: (a) mortality in developmental stages

expressed as k values, (b) regression fit of mortality in

egg stage (k1) to the total K value.

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Jou

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Biology

Anti-inflammatory activity of lycopene isolated from Chlorella marina on

carrageenan-induced rat paw edema

Keywords: Microalgae, Chlorella marina, lycopene, anti-inflammation.

ABSTRACT:

Even though role of lycopene (all-trans) in controlling inflammation was reported, lycopene (cis and all-trans 40:60) isolated from green algae Chlorella marina was not reported so far. In this present study inflammation was induced in male Sprague dawley rats and edema was produced acutely by injecting 0.1 ml of carrageenan into the plantar region of the right hind paw of the rats subcutaneously. Intra peritoneal administration of algal lycopene (AL) at the dose of 10 mg/kg b.wt showed maximum (83%) inhibition on paw edema. The anti- inflammatory effect was significantly (P< 0.05) higher in rats fed with algal lycopene when compared to the standard drug voveran (71%) and all- trans tomato lycopene (TL) (63%). Carrageenan induced rats showed elevated levels of cyclooxygenase (COX) and lipoxygenase (LOX) activities in monocytes. Myeloperoxidase (MPO) in serum, C- reactive protein (CRP) and ceruloplasmin activity in plasma was also high in carrageenan induced rats when compared to normal. Lycopene from Chlorella marina showed significant effect in reducing the above parameters to that of the standard drug while tomato lycopene showed less effect when compared to algal lycopene. Therefore algal lycopene from Chlorella marina would be recommended for the treatment of anti-inflammatory disorders.

886-894 | JRB | 2013 | Vol 3 | No 3






Research Journal

Authors:

Renju GL and

Muraleedhara Kurup G.

Institution:

Department of Biochemistry,

University of Kerala,

Trivandrum, India.


Muraleedhara Kurup G.

Email:

[email protected].

Phone:

+919447251408.

Fax:

91-471 2308078.


Dates: Received: 02 Feb 2013 Accepted: 22 Feb 2013 Published: 23 Apr 2013

Article Citation: Renju GL and Muraleedhara Kurup G. Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema. Journal of Research in Biology (2013) 3(3): 886-894


Original Research

INTRODUCTION

Inflammation is a response which protects and

heals the host tissue after infection or injury. (Nathan,

2002). However, it is frequent that the inflammatory

response to several insults erroneously leads to the

damaging of normal tissues. Prostaglandin-E2 is

generated from arachidonic acid by the enzyme

cyclooxygenase (COX) at sites of inflammation in

substantial amounts and can mediate many of the

pathologic features of inflammation (Serhan and Levy,

2003). One of the early cellular events in inflammation is

the margination of leukocytes, primarily neutrophils and

this can be measured by myeloperoxidase activity

(Goulet et al., 1994).

Currently, non steroidal anti-inflammatory drugs

(NSAIDs) were used for inflammatory diseases. Even

though this drugs transiently suppresses inflammation,

but their long term use cause ulceration in the

gastrointestinal tract and renal morbidity (James and

Hawkey, 2003). However research focused on finding

newer drugs with pharmacological actions without side

effects.

Several antioxidants have been reported to

have anti-inflammatory and anti-arthritic activities

(Maxwell et al., 2006). In the present study a culturable

marine edible algae Chlorella marina was selected to

evaluate the anti-inflammatory activity of lycopene.

Generally tomatoes are the source of lycopene, but it has

many disadvantages (Shi and Le mague, 2000). The

content of lycopene in tomato is very less and the

configuration of lycopene is all-trans. Even though

lycopene from algae has been reported (Ishikawa and

Abe, 2004), no attempt has been made so far for the

commercialization of algal lycopene. It can be seen that

marine sources especially algae are the least exploited

for their bioactive molecules (Pinky and Goswai, 2012).

Work in our laboratory has shown that the lycopene

content in algae is comparatively high, when compared

to tomato lycopene. The most interesting observation

was that algal lycopene contain cis-configuration

(5-cis, 9-cis, 13- cis and 15-cis). Recently it has been

reported that the cis form of lycopene is more

biologically active than the trans form (Stahl and Sies,

1996).


Chemicals

Lycopene, carrageenan, linoleic acid,

Histopaque, arachidonic acid other fine chemicals were

purchased from Sigma, St. Louis, MO, USA. Diclofenac

sodium (Voveran) was obtained from Novartis, India.

Salt and vitamin mixtures were purchased from Merck,

Germany. All other chemicals and reagents were

purchased from Sisco Research Laboratory Pvt.Ltd

(SRL), India, and were of analytical grade.

Algal source

Marine algae Chlorella marina Butcher was

collected from the Vizhinjam coast of Kerala, located at

Latitude 08° 22’ North Longitude. 76° 59’ East on the

south west coast of India and was cultured under

laboratory conditions. The microalgae were identified by

the botanist (Dr. G. Valsaladevi, Department of botany)

and a voucher specimen (No. KUBH 5812) has been

deposited in the Department of Botany, University of

Kerala, India.

Culture medium

Walne’s medium (1970) was used as a basal

medium for the cultivation of Chlorella marina. 5 g /L

glucose was added to the basal medium. Flasks were

incubated at 25°C with continuous illumination. The pH

was adjusted to 7.5. Nicotine (10 µM/ L) was sterilized

by autoclaving and was added to 5 days old cultures for

the production of lycopene.

Biomass harvest

Chlorella marina cells were grown in suspension

cultures up to 30 to 40 days. The cells were harvested at

stationary phase by withdrawing the cultures in 50 ml

polypropylene tubes and centrifuged at 5000 rpm for

Renju and Kurup., 2013


10 minutes. Removed the medium and the pellets were

freeze dried, weighed and stored under nitrogen at -20°C.

Isolation of lycopene from Chlorella marina (AL) and

analysis

Harvested biomass (5g dry weight) was

suspended with 5 ml of 80% cold acetone and kept

overnight under 4°C for better and easy recovery of

carotenoids. The mixtures were vortexed for 2 minutes

and centrifuged at 5000 rpm for 20 minutes. After

repeated extractions (4 times), the supernatants were

pooled and the colorless cell pellets were discarded. The

extracts were dried over anhydrous sodium sulphate and

reduced to a minimum volume by evaporating the

solvents using N2 stream. The crude extracts were kept

for further separation of carotenoids in amber colored

containers under nitrogen at -20°C. All operations were

done at subdued light under nitrogen atmosphere. The

absorbance in the solvent phase was quantified by

spectrophotometric method at 470 nm as described by

Lichtenthaler (1987).

Isolation of all-trans lycopene from tomato (TL)

Tomatoes obtained from the local market,

Trivandrum, India were used. The all-trans lycopene

from tomato was extracted and evaluated according to

the procedure of Fish et al., (2002).

Determination of lycopene by HPLC

Lycopene extracted from algal cells and tomatoes

were determined by HPLC method at 450 nm as

described by Shaish et al., (1992). HPLC analysis of

lycopenes were performed using a silia chrom® column

(250 x 4mm + 5 x 4, NCLIOSIL 100-5-C18 5.0µm),

K 1001 type pump and the UV detector type of K 2600,

Germany. Elution was performed isocratically with

methanol: acetonitrile (9:1) v/v at a flow rate of

1 ml min-1. A UV detector with a wavelength of 450 nm

was employed. Lycopene (95%) obtained from Sigma

chemicals were used as standard. The retention time was

recorded and peak areas of standards and tests were

noted on each run and used for calculation of

concentrations of different fractions. All samples were

injected in duplicate.

Experimental animals

Male Sprague Dawley rats with the average body

weight of 150- 200 g of the same breed were selected for

the study. These animals were housed in the department

animal house and provided standard pellet diet and water

ad libitum and maintained with temperature at 25 ± 1°C,

humidity (55-60%) and photoperiod (12:12 h) light and

dark cycle. Experimental procedures conducted on rats

were approved by the Animal Experiment Committee

(218/CPCSEA) for animal care of Kerala University

according to Government of Indian law on animal use

and care.

Induction of acute inflammation-Carrageenan

induced rat paw edema

Carrageenan-induced rat paw edema assay was

conducted according to the procedure as described by

Winter et al., (1962). Five groups of six rats were

treated as AL and TL with doses 10 mg/kg and reference

drug Voveran, a Diclofenac sodium preparation

(20 mg/kg) were given orally and intraperitoneally (i.p),

1 h before the injection of carrageenan. Control rats were

given 0.1 ml 1% carrageenan. Inflammation was

induced by 0.1 ml, 1% carrageenan suspension in

0.9% NaCl solution was injected into the right hind paw

after 1 hour. The volume of the right paw was measured

by paw edema meter before and after injection in

the third and fifth hour. The paw edema and inhibition

was calculated by the equation: Activity= 100 - (100 ×

average drug treated/average for control).

Treatment Protocol and Experimental Design in

Acute Inflammation

Edema was induced on rat right hind paw by

aponeurosis injection of 0.1ml of 1% carrageenan in

0.9% saline. The experimental groups consisted of 30

rats were divided in to five groups.

Group I: control (received saline only),

Group II: carrageenan alone



Group III: carrageenan + algal lycopene (AL groups,

10 mg/kg i.p)

Group IV: carrageenan + tomato lycopene (TL groups,

10 mg/kg i.p)

Group V: carrageenan + Voveran (VOV groups,

20 mg/kg i.p.)

At the end of third hour, the animals were

sacrificed by euthanasia. Blood was removed to ice cold

containers for various biochemical analyses.

Activity of Cyclooxygenase (COX) and

Lipooxygenase (LOX) in Peripheral Blood

Mononuclear Cells (PBMC)

Mononuclear cells were isolated the procedure

described by Radhika et al., (2007). Cox activity was

measured by the method of Shimizu et al., (1984).

15-LOX activity was determined by the method of

Axelrod et al., (1981).

Biochemical analysis

Serum myeloperoxidase (MPO) activity was

measured by Mullane et al., (1985). CRP in plasma was

determined by using Immunoturbidometric kit (Diasys

Diagnostics, Germany). Ceruloplasmin was estimated by

the method of Ravin (1961). Protein was determined by

the methods of Lowry et al., (1951).


The Statistical package for social sciences

(SPSS/PC+), version 11.5 (SPSS Inc; Chicago. IL, USA)

was used to analyze the results for statistical significance

using one-way ANOVA followed by Duncan’s test.

P value < 0.05 was considered as significant.


Sub plantar injection of carrageenan into the foot

of rats caused a time-dependent increase in paw volume.

The localized inflammatory response as evidenced

visually by the edema reached a maximum intensity at

third hour after carrageenan induction and this maximal

effect was seen until the fifth hour. Administration of AL

and TL has showed significant effects in decreasing

carrageenan-induced paw edema. Algal lycopene showed

maximum edema inhibition compared to all-trans tomato

lycopene and drug. AL exhibited 70% and 83% edema

inhibition at third/fifth hours, respectively. This effect

was comparable to the reference drug Voveran which

exerted 54% and 71% edema inhibition at third and fifth

hour, respectively. TL showed 51% and 63% edema

inhibition at third and fifth hour after carrageenan

induction (Figure. 1).

COX activity in PBMC was significantly

(p<0.05) increased in carrageenan treated rats when

compared to control rats (Figure. 2). Treatment with AL

showed significant (p<0.05) decrease in COX activity

when compared to carrageenan induced rats.

Prostaglandin is formed by the interaction of two distinct

but related enzymes, COX-1 and COX-2 and plays an

important role in promoting the signs and symptoms

of inflammation (Otterness and Bliven, 1985;

Ibegbulem et al., 2012). The activity of COX in PBMC

was decreased (p<0.05) in AL treated group when

compared to TL and voveran treated group. Reduction of

paw swelling and decreased activity of COX showed the

immunological protection rendered by the algal

lycopene. These results showed the anti-inflammatory

potential of the AL.

The activity of 5-LOX and 15-LOX in PBMC

was significantly (p<0.05) increased in carrageenan

induced rats when compared to normal rats

(Figure.3 and 4). Algal lycopene treatment significantly

reduced (p<0.05) in 5-LOX and 15-LOX activity, when

compared to CII rats. The effect was significantly higher

(p<0.05) than TL and drug treated groups.

Lipoxygenases are a family of key enzymes in the

biosynthesis of leukotrienes that are postulated to play an

important role in the pathophysiology of several

inflammatory diseases (Henderson, 1994; Yamamoto,

1992). In the normal situation, cellular leukotriene

production is suppressed by selenium dependent

peroxidases (Werz et al., 1997). On receiving



inflammatory stimuli, leukotriene production is elicited

through the arachidonic acid cascade, causing micro

vascular injury, vasoconstriction and production of

pro-inflammatory cytokines (Peskar, 1991). Studies have

shown that LOX and leukotrienes have a profound role

in carrageenan-induced inflammation (Henderson, 1994;

Gamache et al., 1986). In the carrageenan-induced

inflammation model, AL significantly reduced

carrageenan-induced 5-LOX and 15-LOX activities in

mononuclear cells, indicating decreased leukotriene

production and hence a protective effect.

MPO activity in serum was significantly

increased (p<0.05) in carrageenan induced rats when

compared to normal group (Table 1). Treatment with AL

showed significant decrease (p<0.05) in MPO activity

when compared to carrageenan induced rats. The MPO

activity was significantly decreased when compared to

TL and drug treated groups. The activity of MPO is a

marker of neutrophil infiltration (Bradley, 1982), and

was found to be significantly increased in the paw tissue

of carrageenan-induced rats. AL significantly decreased

(p<0.05) the elevated MPO activity, an indicator of

neutrophil in inflamed paws, suggesting that inhibition of

neutrophil infiltration might be another mechanism by

which AL achieves its anti-inflammatory effect.

Table 1 also shows the variations in serum CRP

and ceruloplasmin level in the test animals compared to

control. Serum CRP and ceruloplasmin levels were

significantly increased (p<0.05) in carrageenan induced

rats when compared to normal rats. Supplementation

with AL significantly decreased (p<0.05) the serum

CRP and ceruloplasmin levels when compared to

carrageenan induced rats. The levels of CRP and

ceruloplasmin were decreased significantly (p<0.05),

when compared to TL and Voveran treated groups.

C-reactive protein is an acute phase protein that has been

identified as an important biomarker for various

inflammatory, degenerative, and neoplastic diseases.

Elevated levels of CRP have been found in the blood

during virtually all diseases associated with active

inflammation or tissue destruction, particularly in

patients with rheumatoid arthritis (Pepys and Hirschfield,

2003; Kushner, 1991). In our study the increased levels



Figure 1: Effect of algal lycopene on carrageenan-

induced paw edema in normal and experimental rats.

Figure 2: Effect of algal lycopene on activity of COX in

PBMC of normal and experimental rats COX activity

is expressed as an optical density increase

(OD increase) per mg protein per minute. Val-

ues are expressed as mean ± SEM of six rats in each

group. a – Statistical difference of Control group with CII

group when p < 0.05. b – Statistical difference of CII group with group AL,

TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL

and group TL when p < 0.05. d –Statistical difference of TL group with

AL when p <0.05.

of CRP level was found to be significantly decreased in

algal lycopene treatment when compared to TL and

Voveran treatments.

The serum protein, ceruloplasmin is a powerful

free radical scavenger that oxidizes iron from the ferrous

to ferric state. Ceruloplasmin levels increase under

conditions leading to the generation of oxygen products

such as the superoxide radical and hydrogen peroxides

(Revnic, 1995). Serum ceruloplasmin level was

significantly increased in carrageenan induced rats when

compared to normal rats. Treatment with AL showed

significant decrease in the concentration of

ceruloplasmin. The increased levels of ceruloplasmin in

carrageenan induced rats could be decreased

significantly on treatment with algal lycopene when

compared to TL and standard drug Voveran might be

having a protective response against free radical

mediated lipidperoxidation.

Lycopene from edible marine microalgae

C. marina showed higher anti-inflammatory activity than

all-trans tomato lycopene and standard drug Voveran.

These effects might be due to the presence of two

isomeric form of lycopene (cis and all-trans) in the

microalgae. Reports available indicate that the

cis-lycopene has a high antioxidant potential when

compared to all-trans lycopene (Stahl and Sies 1992;

Clinton et al., 1996). Algal lycopene isolated from

C. marina could reduce cell influx, oedema formation



Figure 3: Effect of algal lycopene on activity of

5- LOX in PBMC of normal and experimental

rats

5-LOX activity is expressed as an optical density

increase (OD increase) per mg protein per min-

ute. Values are expressed as mean ± SEM of six

rats in each group. a – Statistical difference of Control group with CII

group when p < 0.05. b – Statistical difference of CII group with group

AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group

AL and group TL when p < 0.05. d –Statistical difference of TL group with AL

when p <0.05.

Figure4: Effect of algal lycopene on activity of

15- LOX in PBMC of normal and experimental

rats

15-LOX activity is expressed as an optical den-

sity increase (OD increase) per mg protein per

minute. Values are expressed as mean ± SEM of

six rats in each group. a – Statistical difference of Control group with

CII group when p < 0.05. b – Statistical difference of CII group with group

AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with

group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL

when p <0.05.

and release of mediators associated with inflammatory

condition, and therefore has the potential to be used as

an anti-inflammatory agent. Further studies are in

progress to evaluate the molecular mechanism of its

anti-inflammatory activity.

ACKNOWLEDGEMENT

We express gratitude to Dr. Anantha Lekshmi,

Veterinary Doctor, Department of Biochemistry,

University of Kerala, Kariavattom, India for helping us

with the animal experiments.

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Groups MPO

(µm/min/mg)

CRP

(mg/ml)

Ceruloplasmin

(mg/dl)

Control 5.85 ± 0.37 22.0 ± 1.24 0.10 ± 0.006

CII 20.52 ± 1.11a 97.71 ± 3.80a 0.34 ± 0.014a

AL 7.45 ± 0.37bc 45.94 ± 2.01bc 0.19 ± 0.007bc

TL 13.75 ±0.48bcd 80.64 ±3.18bcd 0.28 ± 0.016bcd

VOV 9.74± 0.39b 56.89 ± 2.42b 0.22 ± 0.010b

Table 1: Levels of CRP, Ceruloplasmin in plasma and

MPO in serum of experimental animals.

Values are expressed as mean ± SEM of six rats in

each group.

a – Statistical difference of Control group with CII

group when p < 0.05.

b – Statistical difference of CII group with group AL,

TL and VOV when p < 0.05.

c – Statistical difference of VOV group with group AL

and group TL when p < 0.05.

d –Statistical difference of TL group with AL when

p <0.05.

Table 2: Statistical table of Myeloperoxidase in one

way ANOVA followed by Duncan’s test

MPO

Sum of

Squares df

Mean

Square F

Between

Groups 827.642 4 206.911 91.002

Within

Groups 56.842 25 2.274

Total 884.485 29

Where df is degrees of freedom, F is F- ratio.

Table 3: Statistical table of CRP in one way ANOVA

followed by Duncan’s test


CRP

Sum of

Squares df

Mean

Square F

Between

Groups 20984.623 4 5246.15

6 121.093

Within

Groups 1083.082 25 43.323

Total 22067.705 29

Table 4: Statistical table of CRP in one way ANOVA

followed by Duncan’s test

Ceruloplas-

min

Sum of

Squares df

Mean

Square F

Between

Groups .196 4 .049 59.148

Within

Groups .021 25 .001

Total .217 29


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Identification of Animal Pasteurellosis by PCR Assay

Keywords: Culture lysate, genomic DNA, Pasteurella multocida, PCR .

ABSTRACT:

Diagnosis of pasteurellosis has become difficult, as there are five different capsular types and 16 somatic types. Molecular techniques like PCR are adapted nowadays for rapid and accurate diagnosis in early stage of the disease and also it provides useful information for epidemiological studies. The present study was conducted to study the efficiency of polymerase chain reaction (PCR) in the identification of P. multocida isolates and evaluation of different PCR methods viz., (i) PCR using genomic DNA (ii) PCR using culture lysate and (iii) PCR by colony touch method. In the present study P. multocida specific PCR was performed by using KMT1SP6 and KMT1T7 oligos. These oligos amplified the genomic DNA from P. multocida isolates only. All the three methods produced PCR amplified product at 460 bp and colony touch method was found to be the best method.

895-899 | JRB | 2013 | Vol 3 | No 3






Research Journal

Authors:

Venkatesan PS,

Deecaraman M and

Vijayalakshmi M.

Institution:

Department of IBT,

Dr. M.G.R. Educational &

Research Institute,

Department of IBT,

Maduravoyal,

Chennai - 600095.


Venkatesan PS.

Email:

[email protected]


Dates: Received: 04 Feb 2013 Accepted: 05 Mar 2013 Published: 30 Apr 2013

Article Citation: Venkatesan PS, Deecaraman M and Vijayalakshmi. Identification of animal Pasteurellosis by PCR assay. Journal of Research in Biology (2013) 3(3): 895-899


Original Research

INTRODUCTION

The various forms of pasteurellosis caused by

Pasteurella multocida are the major health problem for

livestock population worldwide. Diagnosis of

pasteurellosis has become difficult, as there are five

different capsular types and 16 somatic types. Molecular

techniques like PCR are adapted nowadays for rapid and

accurate diagnosis in early stage of the disease and also it

provides useful information for epidemiological studies.

Pasteurellosis has high impact on economic status of

Indian farmers. The overall incidence rate of

haemorrhagic septicaemia (HS) was reported as

6.4 per lakh population during 1974-86, resulting in

losses exceeding ten million rupees annually

(Dutta et al., 1990; singh et al., 1996).

Isolation and identification of P. multocida from

specimens like fresh tissues or heart blood followed by

the performance of various biochemical and serological

methods have been used to study P. multocida. These

include catalase, indole, oxidase and sugar fermentation

tests. Due to time consuming procedure and limitations

of these methods, molecular techniques like polymerase

chain reaction (PCR) were adapted nowadays. PCR has

advantages over the conventional techniques in rapidity,

sensitivity and specificity to identify the P. multocida.

The present study was conducted to assess the efficiency

of PCR in the identification of P. multocida from poultry

and ruminants and to evaluate the different methods in

PCR assay viz. PCR using genomic DNA, PCR using

culture lysate and PCR by colony touch method.


Isolation and Identification of P. multocida

Fifty two samples were collected from various

geographical areas of Tamil Nadu, India. Specimens

such as heart blood swab, liver, spleen and long bones

collected from various animals, were streaked directly

onto 5% sheep blood agar and Pasteurella multocida

selective agar as reported earlier (Moore et al., 1974) and

incubated at 37°C with 5-10 % CO2 for 24-48 h. Plates

were examined for colonies, the suspected colonies were

subjected to grams staining, and biochemical test as

per standard techniques. Standard vaccine strain of

P. multocida P52 (B:2) was taken as reference strain.

Pathogenicity test in mice were carried out for all the

fifteen isolates and PCR was performed for all the

isolates.

Isolation and Purification of Genomic DNA

A 900 µl cell suspension of each sample were

resuspended in 100 µl of 10x Tris-EDTA (TE) buffer

(pH 8.3) with 10 mg of lysozyme and were incubated at

37°C for 1.5 h. Bacterial cultures were treated with 10 µl

of proteinase K (10 mg/ml) and incubated at 50°C

for 1 h. The nucleic acid was extracted with

phenol-chloroform-isoamyl alcohol followed by ethanol

precipitation as per the method of Sambrook et al.,

(1989) and Sachithanandam et al., (2011).

PCR Using Culture Lysate

One Milliliter of 18 h broth culture or take few

freshly grown pure colonies from blood agar plate and

suspend in 500 µl sterile distilled water and centrifuge at

4000 g for 1 minute and collect the pellet. The pellet was

washed with sterile distilled water, resuspended in 100 µl

sterile distilled water and boiled for 10 min. The samples

were centrifuged to sediment cell debris and 10 µl of the

supernatant was used in the PCR reaction.

PCR Using Colony Touch Method

A single pure colony grown on agar plates was

used to perform PCR. A pipette tip was lightly touched

onto a colony and then suspend in PCR amplification

mixture.

PCR Technique

The species-specific primers KMT1SP6 and

KMT1T7 designed by Townsend et al., (1998) were used

in this study to amplify the gene sequences in

P. multocida.

Primers 1 KMT1SP6 5’-GCT GTA AAC GAA CTC

GCC AC- 3’

Venkatesan et al., 2013


Primers 2 KMT1T7 5’- ATC CGC TAT TTA CCC AGT

GG-3’

PCR mixture was prepared using PCR kit

obtained from FINNZYME, Finland. The 50 µl of

reaction mixture was prepared with 10 µl template DNA,

10ng of each primers, 200 µM concentration of each

dNTPs, 10x PCR buffer and 1 unit Taq DNA

polymerase. PCR amplification was carried out in an

automated thermal cycler (Perkin Elmer Gene AMP PCR

system 2400) with the following thermal programme.

Initial denaturising at 95°C for 4 min followed by

30 cycles of denaturising at 95°C for 1 min., annealing at

55°C for 1 min., extension at 72°C for 1 min. and final

extension at 72°C for 9 min, were carried out. After

amplification, PCR products were checked in

1.5% agarose gel electrophoresis along with the standard

molecular weight marker (Lambda DNA Hind III digest

and ϕ X 174 DNA Hae III digest; FINNZYME,

Finland).

The biochemical tests were carried out as per the

standard procedure followed in Arun kumar et al., (2012)

RESULTS

Out of total collection of 52 suspected samples,

procured from cattle sheep, goat and poultry, 15 samples

were confirmed as P. multocida based on biochemical

tests (Table 1) and PCR. All the P. multocida isolates

were pathogenic to mice and dies within 24 h. PCR was

performed for all the 15 isolates by 3 methods viz.,

colony touch method, culture lysate and with genomic

DNA. P52 strain of P. multocida, obtained from the

Institute of veterinary preventive medicine (IVPM)

Ranipet, Tamil Nadu, taken as a positive control

and the following bacteria Escherichia coli,

Clostridium chauvoei, Salmonella enteritidis,

Salmonella typhimurium, Bacillus anthracis,



Tests Name of the Isolates

D1P D2P FP GP HP KP LP NP OP AS CS TS YS BG MC

Hemolysis on Blood agar

- - - - - - - - - - - - - - -

Growth on MacConkey agar

- - - - - - - - - - - - - - -

Motility - - - - - - - - - - - - - - - Gelatin Liquefaction - - - - - - - - - - - - - - - Methyl Red Test - - - - - - - - - - - - - - - H2S (Hydrogen sulphide)

+ + - - + + + - + + + + + + -

Catalase + + + + + + + + + + + + + + + Oxidase + + + + + + + + + + + + + + +

Nitrate Reduction + + + + + + + + + + + + + + + Indole + + + + + + + + + + + + + + + Lysine Decarboxylase - - - - - - - - - - - - - - - Ornithine Decarboxylase

- + + + + + - + + + + + + + +

Urease - - - - - - - - - - - - - - - Pyrase - - - - - - - - - - - - - - - Esculin Hydrolysis - - - - - - - - - - - - - - -

VT (Voges Proskaeur - - - - - - - - - - - - - - - Phenylalanine - - - - - - - - - - - - - - - β-Galactosidase (ONPG)

- - - - - - - - - - - - - - -

β-Glucuronidase + + + + + + + + + + + + + + + α-Galactosidase + + + + + + + + + + + + + + + β-Xylosidase - - - - - - - - - - - - - - - N-acetyl β-D-glucosaminedase

- - + + + + - + - - + + + + +

Table 1. Biochemical Profiles for the Identification of Pasteurella multocida Isolates

+ : Positive, - : Negative,



Staphylococcus aureus and Klebsiella spp. used as

negative controls. The expected amplification size of

460 bp was obtained in all the 15 isolates. PCR

amplification was noticed at approximately 460 bp by all

the three methods and in all the 15 isolates as like that of

positive control (figure 1). No amplification product was

observed in negative controls (figure 1). Molecular

weight of PCR product was estimated based on the

standard molecular weight marker.

DISCUSSION

The 15 isolates of P.multocida collected from

different places and sources of origin produced

approximately 460 bp amplified product as that of

reference strain P52, but no amplified product was

noticed among the negative controls. It is concluded that

the primers were highly specific to P. multocida isolated

from various sources. The above result agrees with the

previous reports of earlier workers (Townsend et al.,

1998; Hunt et al., 2000; Miflin and Blackall, 2000; OIE

manual, 2000; Dutta et al., 2001). In this study the

amplified product of approximately 460 bp was observed

using three different methods viz. colony touch method,

culture lysate method and purified genomic DNA

method (figure 1). The intensity of the amplified PCR

product varies (figure 1), due to the variation in DNA

concentrations. Townsend et al., (1998) reported that

PCR using colony touch method produced amplification

Figure 1: Pasteurella multocida – specific PCR (PM-PCR) assay

CS TS YS BG MC PC NC M

460bp→

D1P D2P FP GP HP PC NC M

460bp→

KP LP NP OP AS PC NC M

These figures illustrate fragments specifically amplified by PCR in all the P. multocida isolates by means of the

primers KMT1SP6 and KMT1T7. Variation in the intensity of the amplified product was observed, due to variation in

DNA concentration of each sample.

D1P, D2P, FP, GP, HP, KP, LP, NP, OP, AS, CS, TS, YS, BG, and MC are the names of P. multocida isolates.

product approximately at 460 bp and the intensity of the

amplified product varied due to inconsistency of the

DNA concentration. Dabo et al., (2000) reported that the

boiled cell extract method has the advantages of

simplicity and rapidity in the identification of

P. multocida isolates. Since the PCR amplified product

of 460 bp was noticed in all samples of poultry and

ruminants, using oligos KMT1SP6 and KMT1T7, the

oligos are considered as specific to P. multocida

affecting all species of poultry and ruminants.

Considering the cost and time involved in the preparation

and purification of genomic DNA, the colony touch

method has advantages of simplicity and rapidity for

epidemiological surveys involving large number of

P. multocida isolates. PCR using colony touch method

would be an adaptable easy to perform method in

regional laboratories for rapid diagnosis of HS and FC

from field cases without the need to obtain pure culture

and extensive biochemical and serological tests.

ACKNOWLEDGEMENT

The authors thank the Head, department of

microbiology, Madras Veterinary College, Chennai, for

providing the facilities to carry out this work.

REFERENCE

Arun Kumar JM, Lakshmi A, Sangeetha Rani V and

Sailaja B. 2012. Isolation and characterization of feather

degrading bacteria from poultry waste. Journal of

Research in Biology. 2(7): 676-682.

Blackall PJ and Miflin JK. 2000. Identification and

typing of Pasteuruella multocida; A Review. Avian

Pathology 29(4):271-287.

Dabo SM, Confer A and Lu YS. 2000. Single primer

polymerase chain reaction fingerprinting for

Pasteurella multocida isolates from laboratory rabbits.

American Journal of Veterinary Research 61(3):305-309.

Dutta J, Rathore BS, Mullick SG, Singh R and

Sharma GC. 1990. Epidemiological studies on

occurrence of haemorrhagic septicaemia in India. Indian

Veterinary Journal 67(10): 893-899.

Dutta TK, singh VP and Kumar AA. 2001. Rapid and

Specific diagnosis of animal pasteurellosis by using PCR

assay. Indian Journal of Comparative Microbiology,

Immunology and Infectious Diseases 22(1):43-46

Hunt ML, Alder B and Townsend KM. 2000. The

molecular biology of Pasteurella multocida. Veterinary

Microbiology 72(1-2):3-5.

Manual of Standards for diagnostics tests and

vaccines. 2000. Office International Des Epizootics

Manual, France. 446-456.

Sachithanandam V, Mohan PM, Dhivya P,

Muruganandam N, Baskaran R, Chaaithanya IK and

Vijayachari P. 2011. DNA barcoding, phylogenetic

relationships and speciation of Genus: Plectropomus in

Andaman coast. Journal of research in Biology. 1( 3):

179-183.

Sambrook J, Fritisch EF and Mamiatis T. 1989.

Molecular cloning: a laboratory manual, Cold spring

harbor press, Plainview, N.Y. 2nd ed. 3.

Singh VP, Kumar AA, srivastava SK and Rathore

BS. 1996. Significance of Haemorrhagic Septicaemia in

Asia: India. International workshop on diagnosis and

control of Haemorrhagic Septicaemia. Bali, Indonasia.

May 28-30.

Townsend KM, Frost AJ, Lee CW, Papadimitrion

JM and Dawkins HJS. 1998. Development of PCR

assays for species and type specific identification of

Pasteurella mutlocida isolates. Journal of Clinical

Microbiolgy 36(4):1096- 1100.




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Source of light emission in a luminous mycelium of the fungus

Panellus stipticus

Keywords: Bioluminescence, Panellus stipticus, luminous mycelium, confocal microscopy.

ABSTRACT:

Mechanism of bioluminescence and light-emitting sources in higher fungi remain as an open question for a long time. We investigated the mycelium of cultivated luminous Panellus stipticus using confocal microscopy. No excitation light was imposed on the sample. Two types of sources of bioluminescence and their location were determined in the substrate mycelium. One were small 0.1-3 µm local formations disposed on the surface of hyphae, the other - relatively vast areas in bulk of the nutrient medium. No luminescence signal was recorded inside the hyphae. This may mean that the components of luminescent reaction are spatially separated within the cells, or the intracellular conditions block the reaction. The origin and formation of the light-emitting structures are discussed.

900-905 | JRB | 2013 | Vol 3 | No 3






Research Journal

Authors:

Puzyr Alexey,

Burov Andrey and

Bondar Vladimir.

Institution:

1. Institute of Biophysics SB

RAS, Krasnoyarsk.

2. Special Design-

Technology Bureau "Nauka"

KSC SB RAS, Krasnoyarsk.

3. Institute of Biophysics SB

RAS, Siberian Federal

University Krasnoyarsk.


Burov Andrey.

Email:

[email protected]

Web Address: http://jresearchbiology.com/

documents/RA0345.pdf.

Dates: Received: 02 Apr 2013 Accepted: 27 Apr 2013 Published: 06 May 2013

Article Citation: Puzyr Alexey, Burov Andrey and Bondar Vladimir. Source of light emission in a luminous mycelium of the fungus Panellus stipticus. Journal of Research in Biology (2013) 3(3): 900-905


Original Research

INTRODUCTION

Bioluminescence in fungal cells, which involves

the emission of light generated by a chemical reaction,

has long attracted attention of scientists (Harvey, 1952;

Shimomura, 2006; Desjardin et al., 2008). Researchers

studying bioluminescence of fungi focus their efforts on

three key areas: (i) methods of cultivation under

laboratory conditions and characteristics of the light

emission (Weitz et al., 2001; Prasher et al., 2012; Dao,

2009; Mori et al., 2011), (ii) the molecular organization

of luminescence system and light emission mechanism

(Shimomura, 2006; Airth and McElroy, 1959;

Kamzolkina et al., 1983; Oliveira and Stevani, 2009;

Bondar et al., 2011), (iii) - application of fungal

luminescence in analytical techniques (Weitz et al.,

2002; Mendes and Stevani, 2010).

There has been little research conducted to

determine sources of luminescent light in the fungal

structures. To the best of our knowledge, only the

mycelium of Panus stipticus and Armillaria fusipes,

growing on agar were investigated for light source

detection (Berliner and Hovnanian, 1963). The used

photographic process allowed to record light from a

single hypha.

However, a low resolution of the technique

limited by the emulsion grain size denied localizing the

source of light. The authors of this, obviously, pioneer

work, suggested that the light was emitted over the entire

cell. Given the size of the objects under study, such

research should employ methods of microscopic

investigations. Calleja and Reynolds, who studied

Panus stipticus and Armillaria mellea by optical

microscope with EMI 4-stage image intensifier tube,

came to the conclusion that light emission in an

individual hypha was limited to a segment removed from

the apical point (Calleja and Reynolds, 1970). Absence

of later works related to structural and morphological

studies of mycelium of luminous fungi with microscopy

is astonishing as all known microscopic methods are

widely used to investigate non-luminous fungi

(Riquelme and Bartnicki-Garcia, 2008; Roberson et al.,

2011; Steinberg and Schuster, 2011).

In this report the mycelium of luminous

Panellus stipticus was studied using confocal

microscopy to determine and localize the source of light

emission. In our opinion it is important to find in

luminous fungi structures (or formations), which are the

light-emitting sources, and their location. On the one

hand, this can provide additional knowledge about

morphology of luminous fungi, on the other - might give

insight into molecular-cellular organization of fungal

luminescent system and mechanism of light emission.


In this work we studied the culture of

Panellus stipticus luminous fungus (Bull:Fr.) Karst.,

IBSO 2301 (Figure 1). The mycelium was grown in

plastic Petri dishes at temperature 22°С on a commercial

nutrient medium Potato Dextrose Agar (HiMedia

Laboratories Pvt., India), or on richer medium containing

in 1 liter: 10 g of glucose, 5 g of peptone, 3 g of yeast

extract, 3 g of malt extract, 20 g of agar-agar. The

specimens exhibiting the highest light intensity were

selected for the experiments.

For confocal microscopy, a confocal laser

scanning microscope (LSM-780 NLO, Carl Zeiss,

Gottingen, Germany) equipped with a high sensitivity

GaAsP was used. Bioluminescence was recorded in the

accumulation mode with the 491–631 nm filter. The laser

was turned off (laser power = 0.0%) so that no excitation

light was imposed on the sample. This was done to avoid

fungal autofluorescence - emission of light by biological

substances such as flavins, lipofuscins and porphyrins

when excited by ultraviolet, violet, or blue light (Zizka

and Gabriel, 2008).

Images were processed using ZEN 2010 software

(version 6.0; Carl Zeiss). To prepare a specimen for

microscopy a fragment of agar with mycelium was cut

Puzyr et al., 2013


out and transferred to the cover glass.


Figure 2 shows a 3D projection of the mycelium

by producing a Z-stack with 82 sections, 0.208 μm thick

each. No bioluminescence was detected from the aerial

mycelium. The light emission was recorded from the

surface of specimen to a depth of ~ 16 μm with

maximum intensity localized at the depth of Z= 6-8 μm

where the main body of mycelium was located. Only

isolated signals were detected at Z=8-16 μm that

confirmed that the agar did not contribute to the observed

bioluminescence.

Two types of sources emitting luminescent

signals could be distinguished. One light source were

small 0.1-3 µm local formations, associated with the

substrate hyphae, the other – vast areas in bulk of agar

(Figure 3). Light intensity recorded in the agar was much

higher than that of the local sites in the area of hyphae.

The use of the larger magnification (Figure 4) and bright

field microscopy (Figure 5a) suggests that the local

luminous sites are cellular excretions located on the

hyphae surface while vast luminescent areas are formed

by their aggregation in agar.

While presence of luminous sites on the surface

of hyphae could be assumed, finding of luminescent

areas in the agar came as a surprise. It is uncontroversial

that the recorded bioluminescent signals result from the

interaction of mixing light components synthesized by

the fungal cells. Luminescent signals were recorded by

the confocal microscope only when these components

were outside the cells. No bioluminescence inside

hyphae may mean that inside the cells the components of

luminescent reaction are spatially separated and do not

interact with each other, or the intracellular conditions

(pH, oxygen concentration, presence of inhibitors, etc.)

block the reaction.

One could argue that the surface of glowing

structures should be either hydrophobic or they have a

membrane enclosing the internal volume. Only under

these conditions components necessary for the

luminescent reaction do not mix with the water phase

contained within the nutrient medium. This suggestion is

based on the sharp boundaries exhibiting by both small

local formations on the walls of hyphae and vast areas in

the nutrient agar (Figure 5b).

So far it is not clear whether the luminous

structures containing components necessary for the

emission are formed within the fungal hypha or on/in

their surface. In the first case it requires a transport

system providing for the mechanism excreting the

Puzyr et al., 2013


Figure 1 View of culture of Panellus stipticus (IBSO 2301) growing on agar in natural light (A) and in the dark (B).

forming structures outside the cell. This is plausible

because the Golgi apparatus, that synthesizes secretory

vesicles containing products of vital functions and

excretes them from the cell, is well known. In the second

case on/in the wall cell there should exist structural

elements performing specialized secretory function.

On the basis of the results above we hypothesize

the following. Cells of P. stipticus synthesize and

localize the components required for bioluminescence in

structures which can originate within the cell and then

are moved on the outside surface of the hyphae by

a mechanism analogous to the mechanism of transport

via the Golgi complex. They can be also assumed to

form directly on/in outside surface of the hyphae by

structural elements of the cell possessing secretory

function. Such enclosed structures make possible to

concentrate the necessary components within a small

volume. Separation of luminous structures from the

surface of hyphae and their subsequent diffusion into the

bulk of the nutrient medium produce the vast

Puzyr et al., 2013


Figure 2 Fragment of 3D pattern of bioluminescence produced by P. stipticus.

Figure 3 Confocal luminescence image of the

P. stipticus mycelium. Figure 4 Confocal luminescence image of an

individual hyphae.

20µm

5µm

areas of luminescence in the agar.

CONCLUSION

Confocal microscopy due to its high resolution

and ability to record low light signals offers new

opportunities in investigation of fungal bioluminescence

system. Using this technique the sources of light

emission were identified for the first time in the

mycelium of P. stipticus (IBSO 2301) cultivated on agar

medium. One source were local formations disposed on

the surface of the substrate hyphae, the other – vast areas

in bulk of agar formed by aggregation of these luminous

structures. Further study is required for a detail

understanding whether the discovered structures are

specific for this fungus or they are common among other

luminous fungi.

ACKNOWLEDGEMENTS

The authors thank Mr. Barinov A.A. (OPTEC,

Novosibirsk) and Dr. Baiborodin S.I. (TsKP for

microscopic analysis of biological objects, SB RAS,

Novosibirsk) for technical assistance with confocal

microscopy. We are grateful to Dr. Medvedeva S.E.

(IBP SB RAS, Krasnoyarsk) for the cultivation of

luminescent fungi.

This work was supported: by the Federal Agency

for Science and Innovation within the Federal Special

Purpose Program (contract No 02.740.11.0766); by the

Program of the Government of Russian Federation

«Measures to Attract Leading Scientists to Russian

Educational Institutions» (grant No 11. G34.31.058); by

the Program of SB RAS (project No 71).

REFERENCES

Airth RL and McElroy WD. 1959. Light emission from

extracts of luminous fungi. J Bacteriol.;77(2):249-250.

Berliner MD and Hovnanian HP. 1963.

Autophotography of luminescent fungi. J Bacteriol. 86

(2):339-341.

Bondar VS, Puzyr AP, Purtov KV, Medvedeva SYe,

Rodicheva EK, Gitelson JI. 2011. The luminescent

system of the luminous fungus Neonothopanus nambi.

Doklady Biochem Biophys.;438(1):138-140.

Calleja GB, Reynolds GT. 1970. The oscillatory nature

of fungal bioluminescence. Trans Br Mycol Soc. 55:149-

154.

Dao TV. 2009. Pilot culturing of a luminous mushroom

Omphalotus af. illudent (Neonothropanus namibi).

Biotechnology in Russia. 6:29-37.

Desjardin DE, Oliveira AG, Stevani CV. 2008. Fungi

bioluminescence revisited. Photochem Photobiol Sci.;7

(2):170-182.

Harvey EN. Bioluminescence. New York: Academic

Press. 1952.

Puzyr et al., 2013


Figure 5 Confocal luminescence (A), bright field (B) and overlay (C) images of the substrate. Scale bar = 20 μm.

Kamzolkina OV, Danilov VS, Egorov NS. 1983.

Nature of luciferase from the bioluminescent fungus

Armillariella mellea. Dokl Akad Nauk SSSR.;271:750-

752.

Mendes LF and Stevani CV. 2010. Evaluation of metal

toxicity by a modified method based on the fungus

Gerronema viridilucens bioluminescence in agar

medium. Environ Toxicol Chem. ;29:320-326.

Mori K, Kojima S, Maki S, Hirano T, Niwa H. 2011.

Bioluminescence characteristics of the fruiting body of

Mycena chlorophos. Luminescence. 26(6): 604-10.

Oliveira AG and Stevani CV. 2009. The enzymatic

nature of fungal bioluminescence. Photochem Photobiol

Sci. 8(10):1416-21.

Prasher IB, Chandel VC, Ahluwalia AS. 2012.

Influence of culture conditions on mycelial growth and

luminescence of Panellus stipticus (bull.) P. Karst. J Res

Biol. 2(3):152-9.

Riquelme M and Bartnicki-Garcia S. 2008. Advances

in understanding hyphal morphogenesis: ontogeny,

phylogeny and cellular localization of chitin synthases.

Fungal Biol. Rev.;22(2):56-70.

Roberson RW, Saucedo E, Maclean D, Propster J,

Unger B, Oneil TA, Parvanehgohar K, Cavanaugh C,

Steinberg G, Schuster M. 2011. The dynamic fungal

cell. Fungal Biol. Rev.;25(1):14–37.

Shimomura O. Bioluminescence: chemical principles

and methods. Singapore: World Scientific, 2006.

Weitz HJ, Ballard AL, Campbell CD, Killham K.

2001. The effect of culture conditions on the mycelial

growth and luminescence of naturally bioluminescent

fungi. FEMS Microbiol Lett. 202(2):165-170.

Weitz HJ, Colin D, Campbell CD, Killham K. 2002.

Development of a novel, bioluminescence-based, fungal

bioassay for toxicity testing. Environ Microbiol. 4(7):

422-429.

Puzyr et al., 2013



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Biology

Local people’s attitude towards conservation and development around

Pichavaram mangrove ecosystem, Tamil Nadu, India.

Keywords: Mangrove ecosystem, Livelihood, Attitudes, Conservation, Development.

ABSTRACT Studies in mangrove ecosystem are often focused on biological or ecological criteria and interdependency between mangroves and people is normally neglected. The situation is similar in Tamil Nadu; India which has a coastline of about 950 km. One of the major mangrove forests in Tamil Nadu is situated in Pichavaram, Cuddalore district. The present study was carried out in the seventeen hamlets, which are directly or indirectly dependent on the Pichavaram mangrove wetlands for their livelihood and survival. These seventeen hamlets consist of over 2600 households many of whom derive their principal income from fishing and related activities. Individual surveys were carried out for 10% of the households in each of the selected hamlets. Semi-structured questionnaires were used for surveys to study the attitude and perception of the community on the conservation and importance of mangrove wetlands and resources. The study was conducted to assess the awareness, attitudes and views of people dependent on the mangrove ecosystem towards conservation issues and development options. It was observed that a large percentage of the sampled population showed a positive inclination towards conservation of the ecosystem and were well aware of their responsibility towards it.

906-910 | JRB | 2013 | Vol 3 | No 3



www.jresearchbiology.com Journal of Research in Biology


Research Journal

Authors:

Lakshmi Kodoth and

Ramamoorthy D.

Institution:

Department of Ecology &

Environmental Sciences,

Pondicherry University,

Puducherry.


Lakshmi Kodoth.

Email:

[email protected]


Dates: Received: 08 Aug 2012 Accepted: 26 Aug 2012 Published: 06 May 2013

Article Citation: Lakshmi Kodoth and Ramamoorthy D. Local people’s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India. Journal of Research in Biology (2013) 3(3): 906-910


Original Research

An International Scientific Research Journal

INTRODUCTION

The Mangrove ecosystem has been studied

extensively by scientists more in the ecological and

biological sense. During the 1980s and early 1990s, more

attention was given to research involving the human

interactions with the forested wetlands (FAO, 1985;

Hamilton et al., 1989; FAO, 1994; Cormier-Salem,

1999). Mangrove wetlands are a dominant feature of the

intertidal areas of the tropical and subtropical regions in

between 25°N and 25°S latitudes. The mangrove

ecosystem provides a number of ecological services:

provision of plant and animal products (Macnae, 1974;

Rasolofo, 1997; Spaninks and Beukering, 1997),

sediment trapping and nutrient uptake and transformation

(Furukawa et al., 1997; Hussain and Badola, 2008), they

provide detritus food for the aquatic fauna, harbour

migratory and aquatic birds, serve as spawning ground

for fishes, mussels and prawns. They also act as a natural

shield against storms and tidal waves (Kathiresan and

Rajendran, 2005).

The coastal communities are largely dependent on

the mangrove forests for firewood, timber, honey, fodder

and for its fishery resources. Most coastal communities

in the tropics are significantly dependent on the harvest

of marine and coastal resources for sustaining their

livelihoods (Kunstadter et al., 1986). The majority of

people living near the mangrove areas derive their

income predominantly from fishing and related activities.

Hence, the present study was carried out as it is essential

to understand people’s attitude and perception towards

the mangrove ecosystem as they derive their livelihood

from it; it helps us in formulating better policies and

enhances the developmental plan for the ecosystem.

Study Area

India has a coastline of 7,516 km of which Tamil

Nadu has about 950 km. Extensive mangrove wetlands

are located in two places namely, i) in Pichavaram,

Cuddalore district and ii) Muthupet in Thivarur and

Tanjore districts.

The Pichavaram mangrove wetland is located in

the northern extreme of Cauvery delta between the

Vellar and Coleroon estuaries (figure 1). Geographically,

it is located between 79°47’E longitude and 11°27’N

latitude. The Pichavaram mangrove forests have an area

of about 1,350 ha, which are colonised by 13 true

mangrove species. Rhizophora Sp and Avicennia Sp are

the predominant mangrove species present in the

Pichavaram mangrove forests. Pichavaram mangrove

wetland is also rich in its fishery resources (figure 2).

Annually about 245 tons of fishery resources are

harvested from this mangrove wetland, of which prawns

alone contribute 85% of the catch (Selvam et al., 2003).

Methods

People belonging to 17 hamlets surrounding the

Kodoth and Ramamoorthy, 2013


Figure 1 Glimpse of the Pichavaram mangrove forest

Figure 2 Fishing in the mangrove backwaters

Pichavaram mangroves wetland were selected for survey.

For each selected hamlet 10% of the households were

picked up randomly for the household survey. Using

semi-structured questionnaires, information on the

demography, land use, income and occupational pattern

as well as local dependence on the mangrove resources

were gathered (Badola and Hussain, 2003; Glaser,2003) .

Few open ended questions were also included to

determine the attitude and perception of villagers

towards development and conservation issues. A total of

324 households were surveyed. The responses we got

were mostly in terms of yes, no and we don’t know.


Assess the awareness and views towards conservation

The results (Table 1) showed that majority of the

respondents i.e. 91% (n=324) were aware that

Pichavaram mangrove was as declared protected area

and this awareness was gained largely because of,

NGO’s working in that area and the forest department.

An overwhelming percentage (84%) of the local

population felt responsible towards the protection of the

mangrove ecosystem and another 76.7% are in favour of

eco-development projects in the area. Out of the 324

respondents, 67% of the people are willing to cooperate

with the forest department for the same. Only a small

percentage of people feel their rights being violated

because of the protected area status if the ecosystem.

When questioned regarding their views on

eco-development initiatives and its implementation, a

majority of the respondents (44.7%) were in favour of

the community led initiatives. 32% felt that NGO’s

should take lead in eco-development and the rest

23% felt that the government should take up

eco-developmental projects by itself ( Table 2).

The importance of the mangrove forests to the

local population was emphasized when a majority of

people were against cutting down of the forests. A

majority of the respondents (71%) felt that more

mangrove plantations need to be carried out, while

28.4% felt that the present conditions of the mangrove

forests were good (Table 3).



Table 1: Attitude of people towards Pichavaram Mangrove Ecosystem and conservation (n= 324)

Questions Yes (%) No (%) Don’t Know (%)

Are you aware that Pichavaram Mangrove Ecosystem is declared

as Protected area? 91 9 -

Do you feel any sense of responsibility for the protection of the

ecosystem? 84 13.8 2.2

Do you feel your rights have been violated after the declaration of

PA? 11.9 80.5 7.6

Do you face any problem because of PA? 15.8 78.6 5.6

Are you in favour of the implementation of an ecodevelopment

project? 76.7 15.3 8

Would you like to co-operate with the forest department with regard

to the ecodevelopment project? 67 23 10

Table 2: View of respondents towards

Eco-Development itiatives (n = 324)

Views Frequency Percentage

Want through govt.

initiative 75 23.1

Want through

Community initiative 145 44.7

Want through NGO

initiative 104 32

Table 3: View of local people towards various

management alternatives (n = 324)

Management Alternatives Responses (%)

Forests should be cut and land used

for other purposes 0.6

Current situation of protecting the

forests is good 28.4

Increase in mangrove plantations

needed 71

The findings in this study are similar to that of

the study in Bitarkanika mangrove ecosystem in Orissa

(Badola and Hussain, 2003) which shows that the

villagers are well aware of their responsibility to the

ecosystem and willing to participate in the conservation

efforts of both the government and NGO’s.

Developmental Options

Recently, eco tourism has been promoted to a

large extent in Pichavaram mangrove forests. Majority of

the respondents (76%) were in favour of developing eco

tourism as it will improve job opportunities for the local

population. Shrimp farms are not favoured in the area as

83% of the responses were against setting up of such

farms. This is primarily due to the fact that shrimp farms

in the area are the reason for increase in salinity of the

canal water (Table 4).

Ecological functions and values identified by local

community

The respondents were given a list of ecological

functions to find out how much they were aware of the

functions and its direct or indirect importance in their

livelihoods.

Table 5 shows ranking of use values, 76% gave

highest ranking to contribution of mangroves towards

fishing. 63% gave agriculture as their second preference.

Incase of ranking ecological functions performed by

the Pichavaram mangrove ecosystem, 77.8% of the

responses favoured Tsunami/cyclone mitigation. 67.2%

gave second preference to nutrient cycling (Table 6).

The results show that the respondents were aware

of both the direct and indirect benefits of the mangrove

ecosystem. It is evident from the results that people value

the uses or function which more beneficial to them in

their day today lives.

CONCLUSION

The results showed that in general people have a

positive attitude towards conservation and are aware of

their responsibility in sustaining these mangrove forests.

The socio economic and market conditions influence the

people’s attitude towards the resources. Eco

developmental plans were in favour with the local

population since it will be helpful in formulating

sustainable policies for ecosystem. The promotion of eco

tourism in the area had a largely positive response hence

it should be capitalised on to improve local economy.

Inclusion of the local people in decision making process

can lead to successful management of the Pichavaram

mangrove ecosystem.

REFERENCE

Badola R and Hussain SA. 2003. Valuation of the

Bhitarkanika mangrove ecosystem for ecological security

and sustainable resource use. Study report. Wildlife

Institute of India, Dehra Dun.



Table 5: Ranking of the use values in Percentage

(n=324)

Use values Rank 1 (%) Rank 2 (%) Rank 3 (%)

Fishing 76 18 6

Agriculture 26 63 11

Tourism 35 56 9

Table 6: Percentage ranking of various functions

(n=324)

Ecological

functions

Rank 1

(%)

Rank 2

(%)

Rank 3

(%)

Fish 59.4 34.3 6.3

Aesthetic 38 59 3

Tsunami/cyclone

mitigation 77.8 22.2 0

Nutrient 32.2 67.2 0.6

Table 4: View of local people towards various developmental options (n = 324)

Queries Yes (%) No (%) Don’t know(%)

Are you in favour of developing eco tourism in the area 76 16 8

Are you in favour of shrimp farms 8 83 9

Has Shrimp farms been useful to you? (n=14) 47 46 7

Cormier-Salem MC. 1999. The Mangrove: an area to

be cleared…for social scientists. Hydrobiologia. 413:

135-142.

FAO. 1985. Mangrove management in Thailand,

Malaysia and Indonesia. FAO Environment Paper 4,

Food and Agriculture Organization of the United

Nations, Rome.

FAO. 1994. Mangrove forest management guidelines.

FAO Forestry Paper 117, Food and Agriculture

Organization of the United Nations, Rome.

Glaser M. 2003. Interrelations between mangrove

ecosystem, local economy and social sustainability in

Caete Estuary, North Brazil. Wetland Ecology and

Management. 11:265-272.

Furukawa K, Wolanski E and Mueller H. 1997.

Currents and sediment transport in mangrove forests.

Estuarine, Coastal and Shelf Science. 44:301-310.

Hamilton LS, Dixon JA and Miller GO. 1989.

Mangrove forests: an undervalued resource of the land

and of the sea. In: Borgese EM, Ginsburg N, Morgan JR.

(Eds.), Ocean Yearbook 8. University of Chicago Press,

Chicago. 254-288.

Hussain SA and Badola R. 2008. Valuing mangrove

ecosystem services: linking nutrient retention function of

mangrove forests to enhanced agroecosystem production.

Wetlands Ecology and Management. 16:441-450.

Kathiresan K and Rajendran N. 2005. Coastal

mangrove forests mitigated tsunami. Estuarine, Coastal

and Shelf Science. 65:601-606.

Kunstadter P, Bird ECF and Sabhasri S. (Eds.). 1986.

Man in the Mangroves. United Nations University,

Tokyo.

Macnae W. 1974. Mangrove forest and fisheries. FAO/

UNDP Indian Ocean Fishery Programme. Indian Ocean

Fishery Commission. Publication IOFCDev. 74:34-35.

Rasolofo MV. 1997. Use of mangroves by traditional

fishermen in Madagascar. Mangroves Salt Marshes.

1:243-253.

Selvam V, Ravichandran KK, Gnanappazham L and

Navamuniyammal M. 2003. Assessment of

community-based restoration of Pichavaram mangrove

wetland using remote sensing data. Current Science.

85:6,794-798.

Spaninks F and Beukering PV. 1997. Economic

Valuation of Mangrove Ecosystems: Potential and

Limitations. CREED Working 14.




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Biology

Biodegradation of phenol at low and high doses by bacterial strains indigenous to

Okrika River in the Niger Delta of Nigeria

Keywords: Biodegradation, phenol, bacteria, Okrika River.

ABSTRACT: Assessments on biodegradation at low and high doses of phenol by bacterial strains indigenous to Okrika River in Niger Delta of Nigeria were carried out. Growth at low dose of 0.01 µg/l phenol showed that highest and lowest cell density values of OD540nm of 0.15 and 0.09 in Pseudomonas sp. SD1 and Citrobacter sp. RW1 while at 1.0 µg/l phenol concentration the highest cell density values of OD540nm of 0.28 was observed in Staphylococcus sp. RW2. The highest specific growth rate of 0.019 h-1 at 500 mg/l of phenol was obtained for Pseudomonas sp. SD1 while Citrobacter sp. RW1 had the lowest specific growth rate of 0.014 h-1 at 500 mg/l of phenol. The specific phenol degradation rate ranges from 55.35 to 130.98 mg/(L.h.OD). The order of specific phenol consumption rate at 1000 mg/l by the organisms is: Bacillus sp. SD3>Pseudomonas sp. SD1>Citrobacter sp. RW1>Staphylococcus sp. RW2. Citrobacter sp. RW1 exhibited highest growth yield in 250 mg/l of phenol with the growth yield of 6.24 (x 10-4 A540 unit.l/mg). The results showed that the test organisms might be the most suitable bacterial strains for removal of phenols at low and high doses in phenolic polluted media.

911-921| JRB | 2013 | Vol 3 | No 3



www.jresearchbiology.com Journal of Research in biology


Research Journal

Authors:

Nwanyanwu CE 1*

Abu GO2.

Institution:

1.Department of

Microbiology, Federal

university of Technology,

P.M.B.1526, Owerri,

Nigeria.

2.Department of

Microbiology, University of

Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria.


Nwanyanwu CE.



Dates: Received: 26 Dec 2012 Accepted: 17 Jan 2013 Published: 06 May 2013

Article Citation: Nwanyanwu CE and Abu GO.

Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria. Journal of Research in Biology (2013) 3(3): 911-921


Original Research

INTRODUCTION

Contamination of aquatic environment brought

about by the discharge of wastewater resulting from

anthropogenic activities clearly continues to be a major

environmental issue. Effluents are very important

sources of chemicals entering aquatic ecosystems. They

may contain hundreds, or even thousands of chemicals,

but only a few of them are responsible for effluent

toxicity (Tisler et al., 1999). High strength wastewaters

have been reported to be associated with chemical

processing industries. Wastewaters generated from these

processing industries such as petrochemical, oil

refineries, coke-processing plants, etc contain a large

number of organic and inorganic pollutants at high

concentrations that exhibit adverse effect on the

environments when released (Papadimitriou et al., 2009).

The presence of high level of these contaminants formed

the major pollutant in the water body as a result of

continuous discharge of effluents by industries into the

ecosystem. In water these pollutants of the discharged

effluent sorbs onto particulate materials and if not

degraded eventually end up in sediments. As an ultimate

respiratory of most xenobiotic contaminants that enter

water bodies, sediments act as both carrier and sources of

contaminants in aquatic environment (Akan et al., 2010).

Thus, the contaminated sediments may represent a

continual threat of recontamination of the aquatic

environment as the adsorbed pollutants if not degraded,

in turn lead to the exposure of aquatic life to organic

pollutants such as phenol (Mort and Dean-Ross, 1994).

On the other hand, the release of contaminants from

sediments could increase the amount of toxic compounds

in the waters making them more available to organisms

and affecting their life cycles, reproduction, metabolism

and physiology. Microorganisms being ubiquitous in

nature exploit many carbon and energy sources in its

niche for growth. Several species of micro-organisms

inhabiting hostile ecological niche have been reported by

Colwell and Walker (1977), Atlas (1981), Heinaru et al.

(2000) and Polymenakou and Stephanou (2005).

Microorganisms indigenous to aquatic environment are

crucial for the biodegradation of organic matter and the

cycling of nutrients, while these microorganisms are

susceptible to toxic pollutants from industrial effluent

discharges, especially petroleum refinery. Therefore,

perturbations of aquatic microbial communities could

have consequences for the higher trophic levels and for

the overall aquatic environment.

The composition of effluents from petroleum

refineries varies according to their origin, storage and

treatments as these wastewaters are enriched with

different pollutants. Phenol and its derivatives along with

other organic and inorganic compounds is one of the

most common contaminants present in refinery effluents

(Jena et al., 2005) which renders refinery effluents its

toxic nature. Phenols as constituents of industrial

effluents may remain in water body for much longer

period if it is continually or consistently released into the

aquatic environments from sources thereby increasing its

elevation in the environment. The toxic nature of phenol

and its derivatives to microbial cells is well documented

(Kahru et al., 2002; Keweloh et al., 1990). Owing to

toxic nature of phenol, its contact with microorganisms

always results in the decrease of microbial enzyme

activity (Nwanyanwu and Abu, 2011) as well as leading

to death of organisms at higher concentration.

A large number of microbial genera possess the

capability to degrade organic pollutants. Among the

bacterial genera implicated in the degradation of phenol

include Pseudomonas, Bacillus, Corynebacterium

species etc. The ability of organisms to degradation

phenol and other toxicants is related to adaptation of the

microorganisms to the compound of concern and

adaptation is associated with synthesis of new enzymes

capable of transformation of the toxicant to harmless

substances (Jaromir and Wirgiliusz, 2007). The resultant

effect of biodegradation of phenol and other organic

compounds is growth as the organic pollutants are used

Nwanyanwu and Abu, 2013


as the source of carbon and energy.

This research assessed the growth and utilization

of phenol at low and high doses by bacterial strains

indigenous to Okrika River in the Niger Delta of Nigeria.


Chemical reagents

All chemical reagents used in the study were of

analytical grade and were obtained from sigma chemical

company, St Louis Missouri, USA, BDH chemicals,

Poole, England and HACH chemical company.

Sample collection and analysis

The Okrika River is a small tidal river that

empties into Bonny estuary in Niger Delta of Nigeria.

The River is highly polluted as a result of effluent

discharges from Port Harcourt petroleum refinery

industry sited along its bank (IAIA09 Conference

Proceeding, 2009). Sediment and water samples were

collected from the river as described by Nweke et al.,

(2007) and the samples analyzed within few hours of

collection. The results of the physicochemical analysis of

the samples are as shown in Table 1.

Isolation and identification of bacterial strains

The bacterial strains used in this work were

isolated from the samples by spreading one tenth of

decimally diluted sediment suspension and water

samples on mineral salt agar-phenol (2.5 mM) medium

and the isolated organisms identified as described

elsewhere (Nwanyanwu et al., 2012). The isolates were

designated according to their sources (RW for River

water, SD for sediment) and were then maintained on

nutrient agar slants.

Preparation of inoculum

The bacterial strains used for the assay were

grown in 100 ml of sterile nutrient broth media for 48 h.

The turbid culture medium were harvested, washed

and suspended in deionized distilled water then

followed by standardization of the suspensions

spectrophotometrically to an optical density of

0.4 at 540 nm and used as inocula.

Assay for isolates growth in very low concentrations

of phenol

The ability of the isolates to grow and utilize

phenol at low concentrations (0-1.0 µg/l) was assessed in

sterile Bushnell Haas (BH) mineral salt broth medium.

The assay was carried out as described by

Nwanyanwu et al., (2012) with little modification. The

medium without agar was used instead for the assay.

After inoculation of the flasks, growth profile of the

organisms was monitored by the optical density

(OD540nm) on daily basis.

Growth and biodegradation of phenol at high

concentration

Degradation of phenol at high concentration by

the organisms was carried out in sterile BH medium

contained Erlenmeyer flasks. The flasks were

supplemented with aliquot of sterile phenol (2000 mg/l)

to bring the final phenol concentrations in the flasks to

250, 500, 750 and 1000 mg/l. The flasks after inoculation

with the test organisms were incubated at 30oC in an

incubator. At predetermined time, samples were

withdrawn to determine cell growth and phenol

concentration. Controls, one without phenol and another

without cells in BH medium were set up. At

predetermined time, samples were removed and used to

measure for cell growth (optical density, OD540nm) and



Table 1: Physicochemical characteristics

of Okrika River

Parameter/unit

Sample source

Water Sediment

pH 8.90 6.90

Elect. conduc. (µscm-1) 364 615

Oil and grease (mg/l) 16.0 103.0

BOD (mg/l) 8.16 -

COD (mg/l) 84.0 -

PO4 (mg/l) 0.15 0.90

SO4 (mg/l) 118 117

Phenol (mg/l) 6.1 15.5

Zn (mg/l) 0.03 3.48

Cu (mg/l) <0.01 0.06

Pb (mg/l) <0.01 <0.01

phenol residue (4-amino antipyrine) in cell free samples.

Analytical methods

C e l l g r o w t h w a s d e t e r m i n e d

spectrophotometrically while phenol was analyzed by

photometric method using 4-aminoantipyrine as the

colouring agent and measuring the absorbance at 500 nm

(Folsom et al., 1990).

Data Analysis

Specific growth rate

The specific growth rate (µ) for each

concentration of phenol was calculated from the slope of

linear logarithmic plots of optical density against time as

expressed in equation 1 (Gokulakrishnan and Gummadi,

2006):

Specific degradation rate

The specific degradation rate (Qs) was

determined through the relationship of equation 2 (Loh

and Wang, 1998):

Where: [Ph] denotes phenol concentration

(mg/l), t denotes incubation time (h) and X denotes cell

concentration (optical density, OD540 nm).

Yield factor

Yield factor (Y) of the biomass was calculated

using equation 3 (Bajaj et al., 2009):

Where dX is the change in cell biomass related to

the change in substrate concentration dS. X was replaced

with the OD at 540 nm.


The phenol content of Okrika River water and

sediment were 6.1 and 15.5 mg/l while oil and grease of

the River water and sediment were 16.0 and 103.0 mg/l

respectively (Table 1). This level of oil and grease as

well as phenol in the River water and sediment were

much higher than the previously reported levels of

10.56 and 15.23 mg/l (oil and grease) and 5.13 and

16.0 mg/l (phenol) (Otokunefor and Obiukwu, 2005).

This indicated that these compounds have accumulated

in Okrika River over time and pose the major pollutants

of the river.

Figure 1 shows the growth of the test organisms

in low concentration of phenol amended mineral salt

medium. All the organisms showed progressive growth

in low phenol concentration medium. Highest growth of

the organisms was observed in phenol concentration of

1.0 µg/l followed by 0.1 µg/l. The least growth was

observed in 0.01 µg/l. Among the test organisms,

Staphylococcus sp. RW2 showed the highest growth in

0.1 and 1.0 µg/l of phenol with optical density (OD)

values of 0.23 and 0.28 respectively while Citrobacter

sp. RW1 showed the least growth in all the low

concentrations (0.01, 0.1 and 1.0 µg/l) of phenol

amended medium with OD values of 0.09, 0.11 and 0.13

respectively. Growth of microorganisms especially

bacterial species at phenol concentration as low as

microgram per litre have been reported by many authors.

Chesney et al., (1985) have reported growth of water

microorganism in water sample supplemented with 0.001

to 1.0 µg/ml of phenol. Also Goldstein et al., (1985)

have reported the growth of Pseudomonas sp. in a

112

12

tt

XXIn

2/

X

dtPhdQs

3dS

dXY



Table 2: Yield factor (Y) of biomass after growth at

different initial phenol concentrations

Bacteria

Yield factor, Y(x 10-4A540

unitsa. l/mg)

Phenol concentration (mg/l)

250 500 750 1000

Citrobacter sp. RW1 6.24 4.46 2.69 3.11

Staphylococcus sp.RW2 4.96 3.80 3.28 3.00

Pseudomonas sp. SD1 4.96 3.80 3.28 3.00

Bacillus sp. SD3 3.28 4.46 2.69 3.11 a A540 units = optical density at 540 nm

medium amended with 1.0 and 10.0 µg/l concentration of

2, 4-dichlorophenol. Pahm and Alexander (1993) found

that Pseudomonas sp. K, Flavobacterium sp. M4,

Flavobacterium sp. M1 and Pseudomonas sp. SP3 grown

in p-nitrophenol (PNP) of concentration of 0.1 µg/l

reached a total viable count of 105 and 106 cells/ml.

Figures 2 and 3 showed typical profiles of cell

growth and biodegradation of phenol at high

concentrations by bacterial strains of Okrika River

ranging from 250 to 1000 mg/l. The lag phase of the

organisms in phenol fortified medium was short. The

short in lag phase period depends on the pre-exposure of

the organism. Phenol was completely utilized by the

isolates within 180 h of incubation. Phenol

concentrations of 500, 750 and 1000 mg/l was degraded

completely within 96, 132 and 156 h by Pseudomonas

sp. SD1 while same concentrations of phenol was

degraded completely within 108, 144 and 180 h by other

test organisms. Time-dependent degradation of organic

compounds has been reported to be linked with

concentration of the organic compound as observed by

many authors (Colwell and Walker, 1977; Kotresha and

Vidyasagar, 2008). This may be due to changes in the

transport mechanism of the substrate across the cell

membrane in response to high phenol concentration

hence diminished capacity to catabolize phenol. This is

in line with the reports of Gilbert and Brown (1978),

Keweloh et al., (1990), Collins and Daugulis (1997) and



Figure 1: Growth profile of the bacteria in mineral salt medium fortified

with phenol concentrations

Time (h)

Ab

sorb

an

ce (

A54

0 n

m)

Nwanyanwu and Abu (2011) who observed the toxic

effect of phenol at the membrane level, thereby

disrupting the activity of enzymes in phenol-utilizing

bacteria. Also, Joseph and Joseph (1999) and Ye and

Shen (2004) reported that phenol toxicity depends on the

sensitivity as well as source of organism.

The growth profiles of the pure cultures expressed

as optical density and phenol residues at different initial

concentrations are shown in figures 2 and 3. The cells

gradually increase in number as the phenol residues of

the medium progressively decreased. This may be due to

high phenol concentration made available more carbon to

the organism for growth. Pseudomonas sp. SD1

degraded 1000 mg/l of phenol in 160 h with a cell

biomass (OD540nm) of 0.363.

The dependence of specific growth rate on

phenol concentration is shown in Figure 4. From this

plot, the specific growth rate increased with increase in

the initial phenol concentration upto 250 mg/l and then a

progress decrease started with increase in phenol



Ab

sorb

an

ce (

A54

0 n

m)

Figure 2: Biodegradation and cell growth profile of planktonic bacteria of Okrika River in high

phenol concentrations

Time (h)

Ph

en

ol

(mg

/l)

concentration. In the present study, at 500 mg/l of phenol

concentration, the specific growth rate of Pseudomonas

sp. SD1 is increased (highest µ =0.017 h-1). For

concentration higher than 500 mg/l, the specific growth

rate of Pseudomonas sp. SD1 decreases and became

almost constant at 750 mg/l (µ = 0.011 h-1) and

1000 mg/l (µ = 0.011 h-1) of phenol. This is quite similar

to the result obtained by Dey and Mukherjee (2010) who

observed increase in specific growth rate (0.093 h-1) of

mixed microbial culture up to 300 mg/l of initial phenol

concentration and then started decreasing to a constant

(0.057 h-1) at 600 and 700 mg/l of phenol. This trend

suggested that the phenol is an inhibitory substrate. Thus

the parameter has been found to be a strong function of

initial phenol concentration. At 250 and 500 mg/l, the

highest specific growth rate values of 0.026 and 0.017 h-1

were observed in Citrobacter sp. RW1 and Pseudomonas

sp. SD1 respectively while the lowest specific growth

rate of 0.016 and 0.014 h-1 at the same concentration of

phenol was observed in Pseudomonas sp. SD1 and



Figure 3: Biodegradation and cell growth profile of sediment bacteria of Okrika River in high

phenol concentrations

Time (h)

Ph

en

ol

(mg

/l)

Ab

sorb

an

ce (

A54

0 n

m)

Citrobacter sp. RW1 respectively. However, the

growth rates of the test organisms are similar

to that of Pseudomonas aeruginosa and

Pseudomonas pseudomallei degrading phenol in saline

solutions (Afzal et al., 2007).

The specific rate of phenol degradation of the

organisms is depicted in figure 5. The specific

degradation rate (Qs), was estimated by correlating

phenol concentration versus culture time using

regression technique in Microsoft Excel to obtain the

equation of best fit of the degradation curve. The

correlation were differentiated with respect to time and

then divided by the cell mass (Loh and Wang, 1998).

The specific degradation (consumption) rate of a

compound was suggested to be a measure of microbe

performance. The highest specific consumption rate of

phenol was observed in Bacillus sp. SD3 with specific

degradation rate value of 130.98 mg/(L.h.OD) at

1000 mg/l while Staphylococcus sp. RW2 showed the

least specific consumption rate of phenol with a specific

degradation rate value of 99.83 mg/(L.h.OD) at the same

concentration. The organisms in this work showed a

robust decrease in specific degradation rate as the phenol

concentration decreases. This is in line with the work of

Cho et al., (2000) who observed an increase in specific

degradation rate as phenol concentration increases in

their assessment of influence of phenol on

biodegradation of p-nitrophenol by freely suspended and

immobilized Nocardioides sp. NSP41. Agarry and

Solomon (2008) also made similar reports in their work

on kinetics of batch microbial degradation of phenols by

indigenous Pseudomonas fluorescence.

Table 2 shows the growth yield of the test

organisms expressed as absorbance, A at 540nm unit litre

of cells produced per mg of phenol substrate utilized.

The growth yield varied among the test organisms

ranging from 2. 69 to 6.24 (x 10-4A540 units. l/mg). High

growth yield were obtained at low concentration of

toxicant (phenol) while low values of growth yield were

obtained at high phenol concentration. At 250 mg/l

highest and lowest growth yield were observed in

Citrobacter sp. RW1 and Bacilllus sp. SD3 with cell

yield coefficients of 6.24 and 3.28 (x 10-4A540 units.l/mg)

respectively. The higher value of Y observed in

Citrobacter sp. RW1 indicate that phenol was degraded

very efficiently by the organism. All the growth yields



Phenol, So (mg/l)

Figure 4: Specific growth rate of the organisms

at different initial phenol concentrations

Sp

ecif

ic g

row

th r

ate

(h

-1)

Figure 5: Specific degradation rate at different

initial phenol concentrations by the bacterial strains

Phenol, So (mg/l)

Sp

ecif

ic d

egra

dati

on

rate

(m

g/(

L.h

.OD

))

reported here were lower than those reported by other

authors. Yield coefficients of 0.14 and 0.16 have been

reported (Bajaj et al., 2009). The yield coefficients

reported by Yoong et al., (1997) are 0.16 and 0.27.

As Citrobacter sp. RW1, Staphylococcus sp. RW2,

Pseudomonas sp. SD1and Bacillus sp. SD3 shown high

specific phenol consumption rate, they have

demonstrated strong potential to utilize and grow in

phenol of low and high phenol concentrations of upto

1000 mg/l. This indicated that these strains have great

potential for application in the treatment of phenolic

wastewater and in the bioremediation of phenol impacted

media.

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Advantages


[email protected]




Jou

rn

al of R

esearch

in

Biology

Phenol and Heavy Metal Tolerance Among

Petroleum Refinery Effluent Bacteria

Keywords: Phenol, heavy metals, refinery effluent bacteria.

ABSTRACT: Bacterial isolates from petroleum refinery effluent were evaluated for growth in increasing doses of phenol and heavy metal ions. All the test organisms were able to grow in mineral salt medium with phenol concentration of 15.0 mM (≈ 1412.0 mg/l) except Pseudomonas sp. RBD3. Aeromonas sp. RBD4, Staphylococcus sp. RBD5 and Pseudomonas sp. RBD10 showed the highest tolerance to 15.0 mM of phenol followed by Corynebacterium sp. RBD7 while Escherichia coli RBD2 and Citrobacter sp. RBD8 showed the least tolerance to 15.0 mM of phenol. The minimum inhibitory concentrations (MICs) ranged from 1.0 mM for mercury and 4.5 mM for chromium, nickel, lead and copper. The bacterial strains were most susceptible to mercury toxicity. Viable counts of the organism on mineral salt-phenol agar showed a typical growth pattern for inhibitory substrate. The threshold concentration is 0.5 mM for Bacillus sp. RBD1, Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter sp. RBD8, Streptococcus sp. RBD9, Pseudomonas sp. RBD11 and Escherichia coli RBD12 and 1.0 mM for Pseudomonas sp. RBD3, Aeromonas sp. RBD4, Staphylococcus sp. RBD5, Corynebacterium sp. RBD7 and Corynebacterium sp. RBD10. The results suggest that microorganisms isolated from petroleum refinery effluent are potentially useful for detoxification of phenol impacted systems in the presence of heavy metals.

922-931 | JRB | 2013 | Vol 3 | No 3



www.jresearchbiology.com Journal of Research in Biology


Research Journal

Authors:

Nwanyanwu CE,

Nweke CO, Orji JC,

Opurum CC.

Institution:

Department of

Microbiology, Federal

University of Technology,

P.M.B. 1526, Owerri,

Nigeria.


Nwanyanwu CE.


Web Address:

http://jresearchbiology.com/

documents/RA0317.pdf.

Dates: Received: 24 Dec 2012 Accepted: 09 Jan 2013 Published: 10 May 2013

Article Citation: Nwanyanwu CE, Nweke CO, Orji JC, Opurum CC. Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria. Journal of Research in Biology (2013) 3(3): 922-931


Original Research

INTRODUCTION

Petroleum refinery effluents are wastes liquids

that resulted from the refining of crude oil in petroleum

refinery. The effluents are composed of oil and grease

along with many other toxic organic and inorganic

compounds (Diya’uddeen et al., 2011). Among the toxic

components of these effluents are heavy metals. Heavy

metals include cobalt, chromium, nickel, iron,

manganese, zinc, etc. They usually form complexes with

different non metal donor atoms which account for their

participation in various microbial metabolisms in the

environment (Kamnev, 2003). Some of these heavy

metals such as cobalt, chromium, nickel, iron

manganese, zinc, etc. are required in trace amount by

microorganisms at low concentration as nutrients, since

they provide vital co-factors for metalloproteins and

enzymes and are known as essential metals while others

such as cadmium, mercury, lead, etc have no

physiological functions and are known as nonessential

metals (Sevgi et al., 2010). At high concentration both

essential and nonessential heavy metals exert an

inhibitory action on microorganisms by impairing the

essential functional groups as well as modifying the

active conformation of biological molecules. This results

in reduction of microbial activity leading to increased lag

phase as well as slow growth rate (Aleem et al., 2003).

It is expected that petroleum refinery effluents

will contain some of these metals in reasonable quantity

as well as aromatic compounds such as phenols. Organic

and inorganic mixed pollutants are known to be

commonly present in industrial effluents and also other

contaminated sites. In this case, apart from affecting the

viability of the microbiota, the metal activity may have

synergistic effect on biodegradation processes of the

aromatic compounds. Thus studies related to the

association of the bacterial tolerance properties to metals

and degradation of phenolic compounds may be relevant

to applications in bioremediation processes (Silva et al.,

2007).

Discharge of these metals into natural waters at

increased concentration in refining operations can have

severe toxicological effects on aquatic environment and

humans. Heavy metals as well as phenol are known to

be harmful pollutants emanating from industrial

wastewaters that have negative effects on

microorganisms. These metals are in the form of

inorganic and metallo-organic compounds while phenol

appears to be a soluble component of the industrial

effluents (Nwanyanwu and Abu, 2010; Hernandez et al.,

1998). These environmental pollutants which are

environmentally mobile tend to accumulate in organisms,

and become persistent because of their chemical stability

or poor biodegradability (Emoyan et al., 2005).

Contamination of wastewater with high concentration of

heavy metals caused a significant decrease in

the numbers of bacteria in biological system

(Otokunefor and Obiukwu, 2005). It is obvious that

heavy metals are one of the toxic contaminants in

wastewaters and causes disorder in biological wastewater

treatment (Sa’idi, 2010). Microorganisms being

ubiquitous in nature have been reported to be found in

inhospitable habitats such as petroleum refinery

effluents, coke effluents, etc (El-Sayed et al., 2003;

Hidalgo et al., 2002) as the effluents are characterized by

the presence of phenols, metal derivatives, surface active

substances and other chemicals (Suleimanov,1995).

Bruins et al., (2000) in their work reported that

organisms in such inhospitable environment must have

developed metal resistance systems in an attempt to

protect sensitive cellular components. On the other hand,

utilization of phenol and other pollutant is enhanced by

adaptation and production of appropriate enzymes by

organisms for the removal of the toxicants

(Nwanyanwu et al., 2012).

This study investigated the tolerance to heavy

metals and phenol by bacterial population in petroleum

refinery effluent.

Nwanyanwu et al., 2013



Sample collection

Petroleum oil refinery effluent was collected

from Biological treatment plant unit (Rotary biodisk,

RBD) of Port Harcourt oil refinery complex and

transported to the laboratory for physicochemical

analysis which includes pH, total dissolved solids,

biological oxygen demand (BOD), chemical oxygen

demand (COD), phosphate (PO4), nitrate (NO3), oil and

grease, phenol, electrical conductivity and heavy metals

content. The methods used for the analysis were as

shown elsewhere (Nwanyanwu et al., 2012).

Microbiological analysis

Microbiological counts were estimated by plating

0.1 ml of the 102 - 106 decimally diluted effluent samples

in physiological saline on appropriate agar plates. Total

heterotrophic bacterial count was done on nutrient agar

plates while phenol-utilizing bacterial count was done on

phenol-agar medium of Hill and Robinson (1975). The

inoculated plates were incubated for 24 h at 30oC for the

heterotrophic bacterial count and 72 h for phenol-

utilizing bacteria count.

Isolation and identification of bacterial strains

The discrete bacterial colonies that developed on

phenol-agar medium were purified, characterized

biochemically and identified as described by

Nwanyanwu et al., (2012).

Preparation of inoculum

The organisms were grown in nutrient broth

medium contained in Erlenmeyer flasks (100 ml) at

28±2oC for 48 h. Thereafter, the cells were harvested and

washed in sterile deionized distilled water. The cell

suspensions were standardized by adjusting the turbidity

to an optical density of 0.1 at A540.

Screening of isolates for phenol tolerance

Into 5.0 ml mineral salt broth medium contained

in 15.0 ml screw capped glass culture tubes were added

aliquots of phenol stock solution (200 mM). The tubes

were sterilized by autoclaving at 121oC for 15min and

allowed to cool at room temperature (28±2oC).

Thereafter, 0.1 ml aliquot of cell suspensions were

seeded into the tubes and incubated at 30oC for 96 h. The

final concentrations of phenol in the tubes ranged from

0.1-100 mM. Controls included cells in mineral salt

medium without phenol and mineral salt medium

supplemented with phenol but without cells.

Development of turbid culture depicted tolerance to

phenol stress. Isolates that exhibited phenol tolerance

from 5.0 mM and above were used for further phenol and

heavy metal toxicity assay.



Table1: Physicochemical and microbiological

analyses of biological treatment unit of petroleum

refinery wastewater

Parameter/ unit Value

pH 8.18

Elect. conduct (µs/cm) 485

Oil and grease (mg/l) 15.0

TDS (mg/l) 250

BOD (mg/l) 8.0

COD (mg/l) 76.0

Phenol (mg/l) 13.6

PO42- (mg/l) 0.14

NO3- (mg/l) 1.20

Metal concentration

Zn2+ (mg/l) 0.02

Cu2+ (mg/l) <0.02

Cr2+ (mg/l) 0.05

Pb3+ (mg/l) <0.01

Ni2+ (mg/l) 0.02

Cd2+ (mg/l) <0.01

Microbial load

THBC (CFU/ml) 2.52 x 108

TPUBC (CFU/ml) 1.14 x 108

% TPUBC (%) 45.24

THBC = Total Heterotrophic bacterial count; TPUBC = Total phenol-utilizing bacterial count

Growth on phenol-mineral salt agar

The isolates were tested for their ability to grow

on mineral salt agar medium (MSM) amended with

increasing phenol concentrations. An aliquot (100 µl) of

decimally diluted standardized inoculum of each isolate

in physiological saline was spread plated onto surface

of MSM plates with 2.0-20 mM of phenol

concentrations. Control included cells in MSM plates

without phenol. The culture was incubated at 30oC for

72 h (Kahru et al, 2002). The number of the colony that

developed was enumerated as colony forming unit per ml

(CFU/ml).

Minimum inhibitory concentration (MIC)

determination

Stock solutions of Cd, Zn, Hg, Cu, Pb, Ni, Co

and Cr as salts of CdCl2, ZnSO4, HgCl2, CuSO4, PbCl2,

Ni(NO3)2, CoCl2.6H2O and K2Cr2O7 were prepared in

deionized distilled water. All the chemicals used were

analytical reagent grade.

The minimum inhibitory concentrations (MIC) of

eight heavy metal ions at which no growth was observed

were determined at pH 7.2 against each bacterial isolate

using tube dilution method (Hassen et al., 1998) with

little modifications. Graded concentrations of each heavy

metal ranging from 0.05 mM to 10.0 mM were prepared

in tryptone soy broth (TSB) contained in screw capped

culture tubes. The supplemented TSB-heavy metal

medium was sterilized by autoclaving at 121oC for

15 min. On cooling to room temperature (28±2oC), the

tubes were seeded with 100 µl of the bacterial

suspension and incubated at 30oC for 72 h. Inoculated

medium free of heavy metal ions and uninoculated

medium with metal ions served as positive and negative

controls respectively. The MIC of the metal to the test

isolates is the lowest concentration that totally inhibited

growth of the organisms.


The physicochemical and microbiological

properties of the petroleum refinery effluent are shown in

Table 1. Phenol-utilizing bacteria represented 45.24% of

the microbial load of biodisk effluent. The high

population of phenol-utilizing bacteria obtained could be

related to natural selection and adaptation to phenol at

the unit. The concentration of heavy metals in the

effluent present in the effluent may be as a result of

physicochemical treatment (oxidation and reduction,

chemical precipitation, etc) given to the raw wastewater

before been channeled into the biological treatment unit.

The result of screen test for phenol tolerance is

shown in Table 2. With the exception of Pseudomonas

sp. RBD3 that tolerated phenol up to 10 mM, all the

organisms are able to tolerate phenol stress up to

15.0 mM. The growth of the isolates in the medium with

phenol concentrations above 10.0 mM may be attributed

to previous exposure to phenolic raw wastewater influent

into the biological treatment unit (RBD). This is in line

with the report of Santos et al., (2001) in which they

related the growth of Trichosporom sp. in phenolic

amended medium of 10.0 mM concentration to previous

phenolic wastewater shock load from stainless steel

industry. Moreso, the tolerance of the organisms to high

concentration of phenol (15.0 mM) may be the ease with

which the isolates open the phenol ring for its subsequent

uptake as carbon and energy source (Ajaz et al., 2004).

Gurujeyalakshmi and Oriel (1989) in their work have

reported that Bacillus stearothermophilus strain BR219

could grow on phenol at levels up to 15 mM. In contrast,

growth inhibition of Bacillus, Pseudomonas and

Citrobacter species at phenol concentration above

1.0 mM has been reported by many authors

(Obiukwu and Abu, 2011). Janke et al., (1981) reported

inhibition of phenol hydroxylase activity in

Pseudomonas species at 0.25 mM phenol concentration.

Yang and Humphrey (1975) found that the growth

of Pseudomonas putida was strongly inhibited

above phenol concentration of 0.5 mM. Buswell and

Twomey (1975) reported that growth of



Bacillus stearothemophilus was inhibited at phenol

concentration above 5.0 mM.

The effect of increasing doses of phenol

(0.05 - 15.0 mM) on the population of the test organisms

are shown in Figure 1. Generally, the viable counts

increased with the concentration of phenol until a certain

concentration when the growth of the organisms was

inhibited. The growth of the organisms on phenol

followed a substrate inhibition pattern. Increasing phenol

concentration resulted in decrease in microbial growth

and eventually very minimal growth was detected at the

highest phenol concentration (15.0 mM) in all the test

organisms. The growth of Bacillus sp. RBD1,

Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter

sp. RBD8, Streptococcus sp. RBD9, Pseudomonas sp.

RBD11 and Escherichia coli RBD12 with a total viable

count of 7.1 x 106, 8.0 x 106, 7.2 x 106, 7.8 x106,

7.5 x 106, 8.8 x 106, 7.4 x 106 and 7.4 x 106 CFU/ml

respectively were stimulated at phenol concentrations up

to 0.5 mM (≈ 47.06 mg/l). Similarly, at phenol

concentration up to 1.0 mM (≈ 94.11 mg/l), the growth

of Pseudomonas sp. RBD3, Aeromonas sp. RBD4,

Staphylococcus sp. RBD5, Corynebacterium sp. RBD7

and Corynebacterium sp. RBD10 with a total viable

count of 8.4 x 106, 7.5 x 106, 7.2 x 106 and

8.4 x 106 CFU/ml respectively, were stimulated.

Thereafter, the total viable counts progressively

decreased as the phenol concentration increases. This

growth pattern is typical of in an inhibitory substrate like

phenol. The inhibition of bacterial growth by phenol is

well-documented. However, some bacteria are more

tolerant to phenol than others. For instance, the growth

inhibition constant (Ki) for bacteria degrading phenol

have been reported as 54.1mg/l (0.57 mM)

(Monteiro et al., 2000), 129.79 mg/l (1.379 mM) (Kumar

et al., 2005), 2434.7 mg/l (25.87 mM) (Arutchelvan et

al., 2006) and 7.818 mM (Wei et al., 2008). In this study,

all the test organisms tolerated phenol up to 10.0 mM

(≈ 941 mg/l) and with the exception of Pseudomonas sp.

RBD 3, all the bacterial strains tolerated 15 mM

(≈ 1412 mg/l). This is in line with the report of Worden

et al., (1991) that Bacillus stearothermophilus BR219



Table 2: Phenol tolerance of the test isolates in different concentrations of phenol

Bacteria

Growth in mineral salt broth with added phenol

Phenol concentration (mM)

0.1 0.2 0.5 1 2 5 10 15 20 50 100

Bacillus sp. RBD1 + + + + + + + + - - -

Escherichia coli RBD 2 + + + + + + + + - - -

Pseudomonas sp. RBD 3 + + + + + + + - - - -

Aeromonas sp. RBD 4 + + + + + + + + - - -

Staphylococcus sp. RBD 5 + + + + + + + + - - -

Bacillus sp. RBD 6 + + + + + + + + - - -

Corynebacterium sp. RBD7 + + + + + + + + - - -

Citrobacter sp. RBD8 + + + + + + + + - - -

Streptococcus sp. RBD9 + + + + + + + + - - -

Pseudomonas sp. RBD10 + + + + + + + + - - -

Corynebacterium sp. RBD11 + + + + + + + + - - -

Escherichia coli RBD12 + + + + + + + + - - -

+ = growth ; - = no growth

tolerated phenol concentration of 15.0 mM. Similarly,

Corynebacterium species was reported to resist 15 mM

phenol while Staphylococcus, Corynebacterium, Bacillus

and Proteus were found to resist 10 mM of phenol

(Ajaz et al., 2004). However, many authors have

reported inhibition of microorganisms at such high

phenol concentration (Hossein and Hill, 2006; Kotturi et

al, 1991). Li and Humphrey (1989) as well as

Gurujeyalakshmi and Oriel (1989) have reported

microbial growth inhibition at relatively low

concentrations of 2.0 mM and 0.25 mM respectively.



Phenol (mM)

Tota

l V

iab

le C

ou

nt

(x 1

06 C

FU

/ml)

Figure 1: Growth of bacteria on mineral salt agar medium supplemented with increasing doses of phenol.

0

2

4

6

8

10

Pseudomonas sp. RBD3

0

2

4

6

8

10

0 4 8 12 16

Citrobacter sp. RBD8

The tolerance levels of refinery wastewater

phenol-utilizing bacteria to heavy metals expressed as

minimal inhibitory concentrations (MIC) are shown in

Table 3. The test isolates in this study showed similar

trend of susceptibilities to heavy metal ions based on

minimal inhibitory assay. The high MIC values obtained

in the study may be as a result of long term exposure of

the organisms to metal ions in the refinery effluent.

Highest MIC values were exhibited in Chromium,

Copper and Nickel while the least MIC was shown in

mercury among the isolates with a maximum value of

>3.0 mM and minimum value of <2.0 mM.

Pseudomonas sp. RBD3 showed maximum MICs value

range of 1.5 - 4.5 mM whilst Escherichia coli RBD12

showed minimum MICs value range of 1.0 - 3.5 mM in

all the metals tested. The MICs are higher than that

reported by El-Deeb (2009) for some phenol-degrading

bacteria. However, the MIC values are similar to the

values reported elsewhere (Nieto et al., 1989,

Nweke et al., 2006a, Akinbowale et al., 2007). The MIC

of metal ranging from 0.5 - 2.5 mM, 1.25 - 2.5 mM,

5.0 - 12.0 mM, 1.0 - 1.25 mM, 0.25 - 1.0 mM and

1.25 - 5.0 mM against hydrocarbon-utilizing bacteria was

reported for cadmium, chromium, lead, cobalt, mercury

and copper respectively (Nweke et al., 2006a). These

reported MICs in most cases corroborates the values

observed in this study. The MIC in growth inhibition

assay is analogous to the concentration of metal ion

that exhibited 100 % inhibition in dehydrogenase

activity assay. Thus, the MIC of zinc against river

water planktonic bacteria have been reported as

1.558 ± 0.037 mM, 1.283 ± 0.068 mM,

2.469 ± 0.045 mM and 1.328 ± 0.094 mM for

Escherichia, Proteus, Micrococcus and Pseudomonas

species respectively (Nweke et al., 2006b). Likewise, the

concentration of zinc that gave 100% inhibition of

dehydrogenase activity in sediment Bacillus and

Arthrobacter species are 1.442 ± 0.062 mM and

1.199 ± 0.042 mM respectively (Nweke et al., 2007).

Also, Hassen et al., (1998) have reported MIC values of

0.1, 0.8, 1.5, 1.6 and 1.8 mM for Mercury, Cobalt, Zinc

and Cadmium, Copper and Chromium respectively on

Pseudomonas aeruginosa, Citrobacter freundii,

Staphylococcus aureus, Streptococcus sp. and

Bacillus thurieniensis. Hassen et al., (1998) in their work

reported 3.0 mM chromium as the MIC for

Nwanyanwuet al., 2013


Organism

MIC of metal (mM)

Cd Zn Hg Cu Pb Ni Co Cr

Bacillus sp. RBD1 3.5 2.0 1.5 4.0 4.5 3.5 2.0 4.0

Escherichia coli RBD2 3.5 2.5 1.0 4.0 3.0 4.0 2.5 3.5

Pseudomonas sp. RBD3 4.0 3.0 1.5 4.5 3.0 4.5 3.0 4.5

Aeromonas sp. RBD4 3.5 3.0 1.0 3.0 4.0 4.0 2.0 4.0

Staphylococcus sp. RBD5 4.0 2.0 1.0 3.5 3.0 4.0 3.0 4.0

Bacillus sp. RBD6 3.0 2.5 1.5 3.5 4.0 4.0 3.0 4.0

Corynebacterium sp. RBD7 3.0 2.0 1.5 3.0 3.5 3.5 2.5 3.5

Citrobacter sp. RBD8 3.5 1.5 1.0 2.5 3.0 3.0 2.0 2.5

Streptococcus sp. RBD9 4.0 2.0 1.0 3.5 2.5 4.0 3.0 2.5

Pseudomonas sp. RBD10 3.5 3.0 1.5 4.5 4.0 4.5 3.5 4.0

Corynebacterium sp. RBD11 2.5 2.0 1.0 4.0 4.0 3.0 4.0 4.5

Escherichia coli RBD12 3.0 1.5 1.0 3.0 3.5 3.5 2.5 3.0

Table 3: Minimal inhibitory concentrations of heavy metals

Pseudomonas aeruginosa S8 and Citrobacter freundii

S24. The variation in the tolerance of heavy metal could

be attributed to the bacterial strain involved, assay

technique or culture conditions. However, the study has

proved that heavy metals such as mercury, zinc and lead

do indeed have toxic effect on bacteria. Although it may

vary from one species to another, there is no doubt that

heavy metals do inhibit bacterial growth.

Metals as toxic contaminants of various

environmental sites have been reported to have adversely

affected potential biodegradation processes occurring in

the environment (Said and Lewis, 1991). Amor et al.,

(2001) reported that the level of metal inhibition of

microbial growth depends on concentration as well as

nature of the metal and the type of microbial species.

Sandrin and Maier (2003) reported that metals such as

copper, zinc, cadmium, chromium, nickel, mercury and

lead are known to inhibit biodegradation of organic

pollutants by microorganisms. Phenol biodegradation

have also been reported to be inhibited by metals

(Nakamura and Sawada, 2000; Alves de Lima

et al., 2007, El-Deeb, 2009). Due to accumulative

behaviour of heavy metals, the effluents from petroleum

refinery industries could constitute enriched media to

propagate and spread microbial populations which are

resistant to metallic ions. Thus, microorganisms isolated

from petroleum refinery effluent having combined

abilities to grow in high concentration of phenol medium

and resistance to metals is potentially useful for

detoxification of phenolic wastewater co-contaminated

with heavy metals.

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Jou

rn

al of R

esearch

in

Biology

Effect of Chromolaena odorata leaf extract on haematological profiles in

Salmonellae typhi infested Wistar rats

Keywords: Salmonellae typhi, Chromolaena odorata, Blood cells, Anti-haematotoxic, Rats.

ABSTRACT: Haematological indices provide an insight about the internal environment of a given organism. In this present study, the possible anti-haemototxic effect of Chromolaena odorata on Salmonellae typhi – induced haematotoxicity in rats were investigated. The experimental animals were divided into three groups. Group A received only food and water (control). Group B and C received in addition to food and water, single dose of stock Salmonellae typhi at a dose of 106cfu/ml. The animals in group B and C were allowed to be infected with Salmonellae typhi for 7 days and confirmed by widal test, after which group C was treated with 750mg/kg body weight/day ethanolic extract of Chromolaena odorata for 10 days. The result showed a significant (p < 0.05) decrease in Red Blood Cells (RBC) count, packed cell volume (PCV), haemoglobin (Hb), mean corpuscular haemoglobin (MCH), Mean Corpuscular haemoglobin Concentration (MCHC), neutrophil and increase in platelet, total White Blood Cell (WBC) and lymphocytes in animals infected with Salmonellae typhi when compared to the control non-infected group. Treatment of animals in group C with ethanolic extract of Chromolaena odorata showed a significant (P < 0.05) increase in mean values of RBC count, PCV, Hb, MCH, MCV, MCHC and decrease in platelets, WBC and lymphocytes when compared to the group infested with Salmonellae typhi only. The results above suggest the anti-haematotoxic potential of ethanolic extract of Chromolaena odorata in Salmonellae typhi infested rats.

932-939 | JRB | 2013 | Vol 3 | No 3





An International

Scientific Research Journal

Authors:

Nwankpa P1, Ezekwe AS1,

Ibegbulem CO3 and

Egwurugwu JN2.

Institution:

1. Department of Medical

Biochemistry Imo State

University, Owerri, Nigeria

2. Department of

Physiology, Imo State

University, Owerri, Nigeria

3. Department of

Biochemistry, Federal

University of Technology

Owerri, Nigeria.


Promise Nwankpa


Dates: Received: 15 Feb 2013 Accepted: 05 Mar 2013 Published: 11 May 2013

Article Citation: Nwankpa P, Ezekwe AS, Ibegbulem CO and Egwurugwu JN. Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats. Journal of Research in Biology (2013) 3(3): 932-939


Original Research

INTRODUCTION

Enteric fever, also called typhoid fever caused by

the bacterium Salmonellae typhi, is an acute life

threatening febrile ailment (Kotton, 2007). Typhoid fever

is distributed worldwide and prevalent throughout the

tropics where it is the commonest cause of fever

(Wilcocks and Manson-Bahr, 1972). Literature reports

have shown that two million cases of typhoid and 200

thousand related deaths occur worldwide each year

(Steinberg et al., 2004). One challenge of development in

developing countries, is the provision of portable water

for the populace as poor sanitary condition and hygiene

has been reported to increase the prevalence of

Salmonellae typhi infection with reduced incidence in

developed countries (Kotton, 2007). Available reports

indicate that typhoid infection is the leading cause of

morbidity and mortality in a developing country like

Nigeria where water carriage method of sewage disposal

is inefficient (Crump et al., 2004). Salmonellae typhi

infection causes gastroenteritis which symptoms include

nausea, vomiting and diarrhea (Parry et al., 2002). The

affected organs include spleen, liver and other tissues

which habor the bacterium before entering the blood

(Jones and Falkow, 1996). During metabolism, bacterial

cells, release chemical toxins which interactions damage

the tissue of the host organism. This tends to disrupt the

blood components or blood forming tissues.

Blood is one of the specialized body fluid

responsible for the transportation of nutrients, oxygen,

hormones and other metabolites to the body’s cell and

metabolic waste products away from those cells to sites

of elimination. It is known to be the most important body

fluid that regulates various vital functions of the body

such as excretion, respiration, circulation, osmotic and

temperature balance etc. Mammalian circulation of blood

transports specific nutrients, gases, metabolic products

and hormones between different tissues and organs

(Baynes and Dominiczak, 2005). Literature reports

indicated that haematological profiles of different species

of animals may be influenced adversely by diabetic

condition (Edet et al., 2011), phenylhydrazine (Sanni

et al., 2005), some anti-retroviral drugs (Kayode et al.,

2011) and aqueous leaf extract of Ocimum gratissimum

(Obianime et al., 2011).

Chromolaena odorata (known as siam weed,

independent weed, killer weed) is a perennial shrub

which grow in rainforest, grassland and arid bushvelds

(Timbilla and Braimah, 2002). The leaves of the plant

has been reported to be widely used as herbal remedy for

the treatment of various ailments. Available reports have

shown a decotion of the leaf extract effective in the

treatment of malaria and cough (Suksamran et al., 2004).

Akah (1990) has reported the haemostatic and

anti-inflammatory property of the leaf extract while

Thang et al., (2005) has shown the stimulation of

granular tissue and re-epithelization of the epithelial

tissue during wound healing. Recently Nwankpa et al.,

(2012) reported the antioxidative effect of ethanolic leaf

extract of Chromolaena odorata in rats. Other medicinal

uses including anti-hypertensive, anti-diarrhoeal and

diuretic has been reported (Iwu, 1993).

In rural communities in Nigeria, the use of

Chromolaena odorata for treating Salmonellae typhi

infection is common but the effect of the plant on

haematological indices in typhoid fever is not known.

This study was therefore designed to assess the effect of

Chromolaena odorata on haematological profiles in

Salmonellae typhi infested rats.


Plant Material: The Chromolaena odorata leaves were

collected from a natural habitat in Owerri and

authenticated by professor S.C. Okeke, a taxonomist at

the department of Plant Science and Biotechnology, Imo

State University Owerri, Nigeria. The voucher specimen

was kept in the university herbarium for references.

Preparation of Extract: Large quantities of fresh leaves

of Chromolaena odorata, washed free of sand and

Nwankpa et al., 2013


debris, were dried under shade at room temperature at

27°C for 3 weeks. Electric blender was used to

homogenize the dried leaves to a powder form. A 700g

of the powder macerated in 1.1 litres of 80% (v/v)

ethanol were allowed to stand for 24 hours. A chess clot

was used to filter the mixture and the filtrate

concentrated in vacuo at 37-40°C to 10% its original

volume using a rotary evaporator. The concentrate was

evaporated in a water bath at 40°C to a solid residue, the

extract. The extract was dissolved in 100ml of 10%

ethanol to an approximate concentration used for the

experiment.

Salmonellae typhi: The stock Salmonellae typhi was

procured from Federal College of Veterinary and

Medical Laboratory Technology of the National

Veterinary Research Institute Vom, Jos, Plateau State,

Nigeria. Nutrient agar plate, cesteine lactose electrolyte

deficient plate (DCA) was used to sub-culture the micro-

organism which was incubated at 37°C for 24 hours and

examined for growth. The stock sample used for the

experiment was prepared as culture slants using

McCartney bottle and nutrient agar. Salmonellae typhi

from the sub-cultured medium was aseptically incubated

for 18 hours at 37°C.

Animals: Albino Wistar rats of both sexes weighing

between 150-200g were obtained from the animal house

of Faculty of Medicine, Imo State University Owerri,

Nigeria. They were maintained at room temperature and

acclimatized for 12 days to daily handling. They were

fed ad-libitum with commercial rat chow (Product of

Pfizer Nigeria Ltd) and had free access to water.

Induction of typhoid: Each rat was orally administered

with 1ml of Salmonellae typhi at a dose of 106cfu/ml to

induce typhoid (Kirby, 1960).

Experimental design: Twenty - four albino Wistar rats

were used for the study. They were randomly assigned

into 3 groups. Each group has 8 rats.

Group A: The rats in this group were fed with rat chow

and had free access to water. They were not administered

with Salmonellae typhi and serve to monitor successful

induction of typhoid.

Group B: The rats in this group served as control. They

were fed with rat chow and had free access to water.

Single dose of Salmonellae typhi at106cfu/ml was orally

administered to rats in this group but were not treated

with the plant extract.

Group C: The rats in this group were fed with

rat chow and had access to water. Single dose

of Salmonellae tysphi at 106cfu/ml were orally

administered to the rats in this group. After 7 days

of infection, 750 mg/kg ethanolic leaf extract of

Chromolaena odorata were orally administered to the

animals daily for 10 days.

Collection and preparation of blood samples for

analysis

At the end of the treatment, the animals were

fasted for 24 hours, re-weighed and sacrificed under

chloroform anesthesia. By cardiac puncture, blood

sample was collected from each animal with a sterile

syringe and needle, in EDTA anti coagulated bottle. The

anti-coagulated blood samples were used for

haematological analyses which were carried out within

24 hours of sample collection.

Haematological analysis

Full blood counts such as packed cell volume

(PCV), Haemoglobin (Hb), Red Blood Cell (RBC), Total

White Blood Cells (TWBC), Platelet count, differential

white blood cell (like lymphocytes, monocytes,

eosinophils, neutrophils) and red cell indices including

Mean Corpuscular Haemoglobin (MCH), Mean

Corpuscular Volume (MCV), Mean Cell Haemoglobin

Concentration (MCHC) were estimated using the

Sysmex® Automated Haematology Analyzer KX-2IN,

Sysmex Corporation, Kobe, Japan.


Data generated were statistically analysed by

one-way analysis of variance (ANOVA) of the SPSS

statistical programme of Microsoft Excel. Values were



declared significantly different at p<0.05.


Table 1 and 2 shows the effect of

Salmonellae typhi infection and subsequent treatment

with ethanolic leaf extract of Chromolaena odorata on

haematological parameters in rats. The results showed a

significant (P < 0.05) decrease in Red Blood Cells (RBC)

count, haemoglobin (Hb), Packed Cell Volume (PCV),

Mean Corpuscular Haemoglobin (MCH), Mean

Corpuscular Volume (MCV), Mean Corpuscular

Haemoglobin Concentration (MCHC) and percentage

nuetrophil levels in Salmonellae typhi infested rats

compared to the non-infested group (Table 1 and 2).

On the contrary, the total White Blood Cell (WBC),

platelets and lymphocyte levels in rats infested with

Salmonellae typhi showed a significant (P < 0.05)

increase compared to the non-infested group (Table 2).

Treatment of the rats in group C with ethanolic leaf

extract of Chromolaena odorata showed a significant

(P < 0.05) increase in RBC count, Hb, PCV, MCH,

MCV, MCHC and percentage neutrophil levels

compared to the Salmonellae typhi infested non-treated

group (Table 1 and 2) while treatment of rats in group C

with ethanolic leaf extract of Chromolaena odorata

showed a significant (P < 0.05) decrease in platelets,

WBC and lymphocyte levels compared to the non-treated

Salmonellae typhi infested group (Table 2). However the

results of this study showed no significant (P > 0.05)

difference in RBC, Hb, PCV, MCV, MCH, MCHC,

platelets, WBC, and lymphocytes in Salmonellae typhi

infested rats treated with Chromolaena odorata

compared to the non-infested rats (Table 1 and 2).

Haematological indices provide relevant

information regarding the internal milieu of an organism.

Nutritional, environmental and microbial infection are

among several other factors which have been reported to

have adverse effects on the haematological profiles of

most organisms. Vitamin B12 and folic acid deficiency

(Jee et al., 2005, Murray et al., 2007) and exposure to

environmental pollutants such as carbondisulphide,

insecticide, hexane, gasoline vapour, nitrocellulose

thinner has been reported (Dhembara and Pandhe, 2000;

Uboh et al., 2007; 2009; 2012 and Savithri et al., 2010).

Bacterial infection in living cells release toxins which

metabolism results to increase in release of free radical

species with attendant damage to the cells (Stipanuk,

2000). In this study, Salmonellae typhi infection

significantly decreases the level of RBC, PCV, Hb,

MCH, MCV, MCHC, neutrophils and increases the level

of WBC and lymphocytes. The observation made in this

study agrees with the report of Wilcocks and Manson-

Bahr (1972) in Salmonellae typhi infection and Kumar

and Kuttan (2005) on cyclophosphamide induced



Group Treatment RBC

X1012/L Hb

(g/dL) PCV (%)

MCV (fL)

MCH (pg)

MCHC (g/dL)

A Negative control/water

3.69 ± 0.21 14.43 ± 0.65 44.33 ± 2.13 63.12 ± 1.60 17.19 ± 1.12 31.27 ± 1.20

B Salmonellae typhi (Positive control)

1.62 ± 0.03a 10.09 ± 0.71a 33.26 ± 2.14a 54.85 ± 1.55a 12.52 ± 1.30a 24.12 ± 1.23a

C

Salmonellae typhi + Chromolaena odorata

3.49 ± 0.05bc 14.15 ± 0.79bc 43.40 ± 2.34bc 61.95 ± 1.32bc 16.55 ± 1.02bc 30.12 ± 1.33bc

Table 1: Effect of Chromolaena odorata on mean values of red blood cells, packed cell volume, hemoglobin and

red cell indices in both experimental and control groups.

Mean ± SD (n = 8) a Significantly different compared with negative control (P < 0.05). b Significantly different compared with Salmonellae typhi (positive control) (P < 0.05). c No significant difference compared with negative control (P > 0.05).

toxicity. The haematotoxic effect of Salmonellae typhi

infection may be explained by the interaction of the

bacteria or its toxins with the blood forming tissues/

organs which may inhibit the rate at which some specific

or generalized haemopoeitic committed stem cells are

synthesized by the tissues. Some reports have shown that

hexane, cyclophosphamide and benzene induced

haematotoxic effect is associated with the interaction of

their metabolites with the haematopoeitic tissues and

cause depression in their haematopoeitic activities

(Synder and Hedli, 1996; Kumar and Kuttan, 2005).

Increase in total white blood cells and lymphocytes as

well as decrease in neutrophils seen in this study is

consistent with the reports on effect of insecticides and

pesticides such as fenvalerate, lindane, aldrin among

others, on total white blood cells and the differential

counts in experimental animals (Synder and Hedli, 1996;

Kumar et al., 1996; Savithri et al., 2010). This may be

explained by increased lymphopoeisis and/or enhanced

release of lymphocytes from lymph myeloid tissue (Das

and Mukherjee, 2003). This response may be a direct

stimulatory effect of toxic substance on lymphoid tissue/

pollutant induces tissue damage and disturbance of the

non-specific immune system leading to increase in

production of leukocytes. Neutrophils are known to be

involved in the phagocytosis of foreign substances in the

body during which some of them are ruptured. This may

explain the decrease in neutrophil count on infection

with Salmonellae typhi.

Ethanolic extract of Chromolaena odorata

significantly increased the level of RBC, Hb, PCV,

MCV, MCH and MCHC thereby reducing and

ameliorating the anaemic condition induced by

Salmonellae typhi infection. The observed increase in

RBC, Hb, and PCV may be explained by the role of

Chromolaena odorata extract in reversing bone marrow

depression with attendant improvement in erythrocyte

membrane stability through the antioxidant potential of

the plant extract, thus reducing haemolysis (Krause and

Mahan, 1984; Naaz et al., 2007, Nwankpa et al., 2012).

The improvement on the haematopoetic activities of the

tissues and/or maintenance of red blood cell membrane

integrity relieves the anaemic condition observed in

Salmonellae typhi infection.

Consequently, increase in RBC count on

administration of Chromolaena odorata leaf extract

translates to an increase in MCV while increase in Hb

translates, to an increase in MCH and MCHC.

Furthermore, inhibition of microbial growth by the plant

extract has been reported. Okigbo and Ajalie (2005) and

Alisi et al., (2011) showed that Chromolaena odorata

leaf extract possess antibacterial activity which inhibit

the growth of Salmonellae typhi in cells. Decrease in

total white blood cell, lymphocytes and attendant

increase in neutrophils on administration of the plant

extract may be explained by the inhibition of growth of



Group Treatment Platelets X103μL-1

TWBC

X103μL-1 Lymphocytes

(%) Neutrophils

(%)

Eosinophils

(%) Monocytes

(%)

A Negative control/water

855.18 ± 2.11 16.24 ± 0.78 70.11 ± 2.01 20.19 ± 1.15 1.98 ± 0.6 2.51 ± 0.11

B Salmonellae typhi (Positive control)

880.13 ± 1.5a 25.85 ± 1.16a 82.14 ± 2.11a 11.56 ± 0.87a 3.20 ± 1.10 2.90 ± 0.55

C Salmonellae typhi + Chromolaena odorata

858.82 ± 1.46bc 17.14 ± 1.21bc 72.18 ± 1.88bc 19.26 ± 1.11bc 2.10 ± 0.80 2.6 ± 0.52

Table 2: Effect of CO on mean values of platelets, total white blood cells and differential cell counts in both

experimental and control groups

Mean ± SD (n = 8) a Significantly different compared with negative control (P < 0.05). b Significantly different compared with Salmonellae typhi (positive control) (P < 0.05). c No significant difference compared with negative control (P > 0.05).

Salmonellae typhi in the cell. The inhibition of growth of

the microorganism lead to the destruction of excess

WBC and lymphyocytes released by the cell in response

to bacterial infection (Nancy et al., 2005). Conversely,

increase in neutrophil count on administration of the

plant extract may be explained by reduced phagocytosis

of the microbial cell consequent upon drastic reduction

in the growth of microbial cell.

CONCLUSION

This study has established the anti-haematotoxic

pot en t ia l of ethanol i c lea f extract of

Chromolaena odorata against Salmonellae typhi induced

haematotoxicity in rats.

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