Abstract - dspace.stir.ac.uk · concentntiona caused cell shrinkage in C. vuigaris and A#, aeruginosa after 24 h of preservation. The abilities of carp fry and fingerlings to ingest

Abstract

The filter feeding capebilitiei o f the Indian nuyot <=“ P* rohiia (Ham.) and Calla

catla (Ham.) and the ChincM caip Hypophthabnichthys moUtrix (Val.) were itudied

uboreloiy conditioni. An ultraatnictunl and histological study o f the pharyngeal

appaiatus associated with filter feeding o f laboratoiy reared fish between 30 and 90

mm standanl length (S L ) showed that a large number o f mucus cells, both small goblet

and large clavate cells, were present in the bucco-pharyngeal region o f all species

studied and on the wall o f the suprabranchial organ o f H. moUtrix. The density o f taste

buds was observed to be much greater on the roof o f the pharynx and comparatively

low on the roof o f the mouth in all species. Among the carpa studied, H. moUtrix had

the greatest concentration o f Uute buds on both the roo f and floor o f the bucco-

pharyngeal region.

An important part o f the itudy wai the development o f a sound methodology for

counting algal cells and colonies. An automated electronic particle counter, the Coulter

Multisizer, was used to increase the speed o f counting. A study was also carried out

to determine the effecte o f the preservatives Lugol’s iodine and buffered formalin at

different concentrations on both long-tenn and thort-term storage o f Fediasirum

boryamtm. Microcyttii aentginoia and Chlorella vulgaris. Both Lugol's iodine and

buffeted formalin o f different concentrations caused significant decreases bi the

numbers o f cells « id colonies during praservation. Lugo l's iodine o f differetb

concentntiona caused cell shrinkage in C. vuigaris and A#, aeruginosa after 24 h o f

preservation.

The abilities o f carp fry and fingerlings to ingest a range o f algal species were

investigated. H. moiitrix was found to be the most efficient among the carps studied

in ingesting P. boryanum and M. aeruginosa, although C. catia was found not to be

able to ingest M. aeruginosa. Ingestion rate was found to be positively correlated with

algal density. It was also observed that algal size had a positive effect on ingestion

rates. In a separate trial it was found that silver carp did not ingest a toxic strain o f M.

aeruginosa.

An attempt was also made to evaluate whether these filter feeding carp could detect

and ingest unattached bacteria by exposing them to the bacterium Chromobacterium

vioiaceum in suspension. Examination o f the gut contents o f the exposed fish showed

that the numbers o f H. moiitrix and L. rohita ingesting viable bacteria increased with

the concentration o f the bacterial suspension and with exposure time. However C . catla

ingested far fewer bacteria per unit time than the other two species.

UiAcknowledgement

1 would like to thank my luperviaor Dr Malcolm C M Beveridge for hia advice,

encoungement and worthy criticiam throughout the courie o f thii research and during

the writing up o f this thesu. I am also grateful for his patience and help during my

problems.

I would like to acknowledge my gratitude to Miss Anne Nimmo, Mr Allan Porter and

Ms Maiguerite Mason for their help and technical assistance. In addition I would like

to thank my fellow friends and Mr Brian Howie for their help.

I would like to acknowledge the help from the Department o f Biological Sciences,

University o f Dundee for analysis o f water samples for the toxin microcystin and for

supplying toxic algae for a number o f the trials.

I gratefully acknowledge the flnartcial support o f the Association o f Commonwealth

Scholarship Commission in the United Kingdom and the Registrar, Bangladesh

Agricultural Univenity, Mymensingh for granting me study leave from my Job,

Finally I would like to thank my w ife Happy, who helped me throughout the course

of my research «Id aU other actWltias in Scotland by offering her endless love. Thanks

« e also due to my daughter, son, father and m oth « for their help and ancouragamaM.

List of Tables

Table Filler feeding flahea arranged according to Order,1.1 Family and Speciea

2.1 Channelizing count loaaea (% ) al differenl counting ralea and total count m l' (xIO*) at different range o f counting rate

2.2 Numbera o f colotuea or cella m l'’ (± S.E.) obtained by different counting methoda

2.3 Equivalent apherical diameter (ESD), volume and aize o f algae meaaured by light mictoacope and Coulter Multiaizer

2.4 Reaulta o f aruUyaia o f variartce for the effecta o f different concentialioni o f preaervalivea after different duralkma o f preaervatioo on (a) F. boryanum, (b ) Af. aeruginosa (c ) C. vulgaris

2.5 Percentage decreaae in numbera o f coloniea and cell o fdifferent algae during different duration o f preaervation

3.1 Comparative dau on taate bud denaity (No.Aiim’ ) indifferent oro-pharyngeal regiona and taale bud aizea (pm) o f aame aize rohu, calla and ailver carp.

4.1 Student’a I valuea for initial veraua final algalconcentraliona o f experimenlal treatmenta o f (a ) L. rohita, (b ) C. casta and (c ) H. molitrix

4.2 Reauha o f Student'i l-leal carried out on F. boryanum colony diameter before and after grazing, (a) 1.4 g L. rohila, (b ) 7.7 g L. robila

4.3 Reaulu o f Snidem'a t- teat carried out on F. boryanum colony divneter before and after grazing, (a ) 1.7 g C. calla, (b ) 8.6 g C. casta

4.4 Reaulu o f Shidera'a l-leet carried out on F. boryanum colony d im eter before and after glazing, (a) 1.1 g H. molitrix, (b ) 7.1 g H. molitrix

27

28

29

32

3S

81

104

103

106

107

List o f Figures

2.1 The Coulter Muhiiizec (a ) Muhiiizer, (b ) tampUng 14tland; (c ) vacuum control unit (reproduced fiotnCoulter manual)

2.2 Multiiizer front view (reproduced from Coulter ISmanual)

2.3 Sampling itand front view (reproduced from Coulter 16iTuuiual)

2.4 Vacuum control unit (reproduced from Coulter manual) 17

2.5 Displayed data on the Multisizer screen (reproduced 18fixxn Coulter matuial)

2.6 Equivalem Spherical Diameter (BSD) (reproduced from 20Coulter manual).

2.7 Colony distribution pattern o f (a ) F. boryanum, (b ) M. 30aeruginosa and (c ) C. vulgaris following different duratiotu in ISOTON n.

2.8 Effects o f different concentrations o f both buffered 33formalin (colurtuu 1-4) and Lugol’a iodine (columru S-8) on (a ) F. boryanum (b ) A#. aeruginosa and (c ) C. vulgaris. Bars indicate ± S.E.

2.9 Effects o f different duratiotu o f preservation on (a ) F. 34boryanum (b ) M. aeruginosa and (c ) C. vuiguris. BartinrUcate ± S.E.

2.10 Colony size distribution pattenu o f ^.bo/yrvium before 36preeervation (BP ) and after 24 hours preservation in (a) buffered formalin o f concenttatioru O.S%, 1%, 2%, and4% (b ) LugoTs iodine o f concentrations 0.03%, 0.1%.0.2% and 0.4%.

2.11 Colony size distribution pattern o f M. aeruginosa 37before preservation (B P ) and after 24 hours preservation in (a) buffeted formalin o f concentrations 0.3%, 1%. 2% and 4% (b ) Lugol'tiodine o f concentratioru 0.03%, 0.1%, 0.2% and 0.4%.

Vil2.12 Cell lize diilribution petteni o f C. vulgaris befóte 38

pneervalion (B P ) and after 24 houii preaervation in (a) buffeted foinalin o f concanttaliona O.S%, 1%, 2%, and 4% (b ) Lu go l'i iodine o f concentratKxu 0.03%, 0.1%,0.2% and 0.4%.

3.1 Traiuvetie section o f skull showing the sub-division o f 32the oro-pharyngeal region.

3.2 The relationships between (a) g ill taker length, (b ) gill 63taker space and fish standard length o f rohu.

3.3 The telationships between (a) g ill taker length, (b ) gill 69taker space and fish standard length o f catla.

3.4 The relationship between gill taker length and fish 72standard length o f silver carp.

4.1 Diagram o f the system used in the Tilter feeding trials. 97

4.2 Ingestion rale G ) as a function o f food concentration 108(C ) for L. rohita (1.4 g ) feeding on M. aeruginosa.Fitted curve u I - 2.1E+08.e ’ ‘̂ ' ^ , (s' - 0.88).

4.3 Ingestion rale ( I ) as a ftmction o f food concentration 109(C ) for L. rohita (1.4 g ) feeding on F. boryanum.Fitted curve is I - 2.1E+08.e ’ '^ * ^ , (H - 0.88).

4.4 ingestion tale ( I ) as a function o f food concentration 110(C ) for L. rohita (7.7 g ) feeding on M. aeruginosa.Fitted curve u I - l.lE +08.e ’ * '® ’*̂ , (H - 0.97).

4.3 Ingestion rate ( I ) as a function o f food concentration 111(C ) for L. rohita (7.7 g ) feeding on F. boryanum.Fitted curve U I - 1.9E+08.e (t* - 0.90).

4.6 Ingestion tale ( I ) >s a ftinction o f food concentration 112(C ) for C. calla (1.7 g ) feeding on F. boryanum. Fitted curve is I - 1.8B+08.e ‘ (s’ - 0.98).

4.7 Ingestion rale ( I ) as a ftinctlon o f food concentration 113(C ) for C. calla (8.6 g ) feeding on F. boryanum. Fitted curve is I - 2.0E+08.e (r* - 0.88).

4.8 Ingestion m e ( I ) as a ftmction o f food concenimion 114(C ) for H. moliirix (1.1 g) feeding on M. aeruginosa.Fitted curve Is I - 2.1&»<W.e ‘ (t* - 0.88).

4.9 ingestion rale ( I ) as a ftmction food concenimion (C ) 113for H. moliirix (1.1 g ) feedhu on F. boryanum. Fitted curve is I - 2.7E+08.e ’ '■‘" 'S (t* - 0.89).

List of Plates

Plate 7

Plate 8

Plate 9

SEM veiw o f lipa and retpiiatofy valve (a ) rohu, (b ) 54catla, (c ) silver carp and (d ) closer veiw o f the base o f the respiratory valve o f catla as tnaifced by d in figure b.

SEM survey o f buccal floor (a) rohu, (b ) calla and (c ) 56silver catp.

SEM study o f pharyngeal roof (a) rohu. (b ) catla and 58 (c ) silver carp.

SEM o f the pharyngeal floor (a ) tohu, (b ) catla and 60 (c ) silver carp

SEM survey o f pharyngeal surface o f gill rakers (a) 62rohu, (b ) calla and (c ) silver carp

Histological section o f supra-branchial organ o f silver 74 carp (a) transverse section (b ) cross section.

Transverse section o f palatal organ 76

SEM survey o f microridges in the bucco-pharyngeal 77 region (a) rohu, (b ) catla and (c ) silver carp.

SEM study o f taste buds (a) type I, (b ) type D and (c ) 89type IV.

3.3 Résulte3.3.1 Introduction: tcnninology and

subdivisions o f the oro-phatynx3.3.2 Labeo rohlta3.3.3 Catta catia3.3.4 Hypophthalmichtkys moUtrix3.3.5 ligh t and scanning electron

microscopy o f the oro-phaiyngeal wall

3.3.6 Specializations o f the oro- phaiyngeal epithelium

3.4 Discussion

Chapter IV Filter-feeding o f carps on planktonic algae

4.1 Introduction

4.2 Materials and methods4.2.1 Introduction4.2.2 Algae4.2.3 Fish4.2.4 Experimental system4.2.3 Experimental protocol4.2.6 Sample analysis4.2.7 Determituttion o f Ingestion rates

4.3 Results4.3.1 Algal ingestion4.3.2 Toxic algal ingestion triids

4.4 Discussion

Chapter V Filter-feeding o f carps on bacteria

3.1 Introduction

3.2 Materials and Methods3.2.1 Fish3.2.2 Bacteria3.2.3 PtepiuBtion o f agar media and

inoculation3.2.4 Experimental system3.2.3 Prsliminary taivaatigation o f carp

92

92

CHAPTER I

Introduction

1.1 Evolution and extent o f filter feeding among flehex

The oldeit teleoetean fUhet evolved u generalized predators feeding on compualively

Urge prey (macrophagy) (OoiUne, 1971). The main divergence ftom due baik pattern

has been a trend towards feeding on smaller prey (microphagy), such as plankton. The

evolution from rrracrophagy to microphagy is mruked by the development o f

specUlized feeding stntctirres and the regression o f others (Lazzaro, 1987). This has

itKiuded, for example, modiflcation o f the jaw ftom fixed to ptottusible, replacement

o f teeth by eUborate gill rakers on the btanchUI arches, modification o f some gill

rakers into tm epibratKhUl organ on the roof o f the mouth, turd lengthenttrg o f the

digestive tract in older to process larger amounts during the digestive pmrse between

meals (MarahaU, 1963; NeUon, 1967; Durbin and Durbin, 1973). The evolution,

feeding behaviour and selectivityes o f planktivorous fishes has been reviewed by

Lazzruo (1987).

The most specialized o f the microphagous feeders are the filter-feeding species. The

menhaden, a filter-feeding clupeid microphage, reperUs most o f the evolutionary steps

mentioned above during lu life cycle, as do a number o f other species. The selective

predation o f larval menhaden is directed towartU Individual zooplankton, mainly

copepods (June & Carlion, 1971), and during metamoiphoau, paniculate feeding ia

gradually replaced by filler-feeding on zooplankton, phytoplankton, and fine detritui

(Durbin A Duibin, 1975).

Only a unall proponion o f the energy produced by phytoplankton can be tranifened

through the food web to the higher trophic levels, usually predatory macrophages.

Hence a considerable proportion is available to plankton feeding fishes. Planktivores

are involved in the main commercial fisheries o f the world (anchovies, sardines,

herrings, menhadens and many others). Today, teleosts include more than 95% o f the

living species o f fish (Marshall, 1%5) and microphagy is apparent in most

(fieshwaler, at least) orders even in the Acipensifoimes (Actinoplerygia) (see Table

1 . 1 ) .

Most fishes feed on plankton during at least some period o f their lives. Some fishes

feed on plankton during their entire lives, whereas others feed on plankton only during

the early stages. Some fishes are obligate planktivores, feeding exclusively on plankton:

others are facultative planktivores feeding on plankton as well as on other food items.

1.2 Cyprinids and flilcr-fccding

Cyprinids are generally known under the common names o f carps, barbs, minnows,

roach, ludd, dace, bitterling, rasbora, danios and gudgeon. Their dominant

chancteristica are the lack o f jaw teeth and the poaseaaion o f a (usually) protnisile

upper jaw and pharyngeal teeth. In all, there are some 1700 valid species belonging to

at least 220 genera, making the fMnily the most specioae o f all freshwater teleost

fishes. The cyprinids have a wide geographical distribution and occur in much o f

mainland Eurasia, Japan, moat o f the East Indian islands, A frica and North America

(Howes, 1991). Cyprinids contributed some 32% o f g lobal aquaculture production

(tonnes) in 1988 o f which silver carp alone contributed 1.1% (FAO 1990). Am ong the

cyprinids, there are a number o f groups iiKluding the Indian major carp species such

as Labeo rohita (Ham.), Catla calla (Ham.) and Chinese carp species (eg .

Hypophthalmichthys molitrix (Val.), Aristichthys nobilis (Richardson) which are of

great economic importaiKe in the fisheries and aquaculture industries o f Bangladesh

luid other Asian countries.

Composite fish culture, or polyculture, o f Indirui major carps together with Chinese

ciups and the conunon carp has been found to result in high fish production in

freshwater ponds (Sukumiuan el al. 1968; Alikunhi et ai., 1971; Chaudhuri ef a/.

1975). At higher densities, however, there is growing evidence that the Chinese carps

adversely affect the growth o f the Indian major carps such as calla and rohu

(Lakshmanan el a/.,1971; Dey el H/.I979). Although no specific studies have been

undertaken, the dominance o f Chinese carp over the Indian major carp is widely

believed to be due to their greirier competitive advantage in exploiting food resources.

Competition i l reputedly greateil between the planktivorous specie! o f cups such as

between catla (C. calla) and bighead carp CA. nobUis) and between rohu (L. rohita) and

silver carp (/f. molilrix) (e.g. Dewan el al., 1991).

1.3 Filter-feeding behaviour

Plankton feeding Ashes shows two distinct behaviours with regard to feeding on

plankton: paniculate feeding and Alter feeding. Paniculate plankton feeding Ashes

attack single individual prey items which they visually select prior to engulfment from

the water column (Werner. 1977; Janssen, 1980, 1981). By contrast, both pump Alter

feedere and tow-net Alter feedeii do not visually select individual prey items, but

engulf a volume o f water containing the food organisms, then remove the planktonic

prey and panicles by passing the water through Altering structurea (e.g. gill takers,

tnicrobranchiospines on the gill arches and branchial tooth platea). Tow-net Alter

feeders surround the prey items with their mouths which are held fu lly agape while

swimmirtg rapidly (Dutbin and Duibin, 1975; Colin, 1976; Rosen and Hales, 1981).

Pump Alter feeders use rtiythmlc suction actions to capture pcey items, while

swimming slowly or remaining relatively stationary (Motiatty el al., 1973; Dtcnner,

1977; Janssen, 1976).

Janssen (1976) de scribed an imetmediate feeding mods, gulping, fo r the alswifa, Alota

pseuttoharengus. «nd the cieco, Coregonus artedii, where fuh ute thort sequence* o f

several pump actions which alternate with pauses o f about O.S seconds.

1.4 Filler-feeding mechanisms

The use o f intricale gill rakers in the filler feeding o f planktivorous fuhes has been

weU documented (Iwata. 1976; Shen, 1969; Hossler et al., 1979; Cremer and

Smilheiman, 1980; O ’ConneU. 1981; Lammens, 1985; Lammens et al., 1987;

Hoogenboezem et al., 1990. 1991; Northcolt et al., 1991). However, noi all plankton

feeding fishes possess well-developed gill-raken. Suyehiro (1942) noted thst a number

o f families contain planktivorous species which possess very coarse gill-rakers. The

families Syngnathidae and Fistulariidae have totally degenerate gill-rakers, yet their

chief food* are plankton and benthic diatoms. Many o f the lilapias (Cichlidae) feed on

plankton without the benefit o f well developed gill-rakers (Fryer and He*. 1972; Pauly,

1976; Northcolt 1988; Northcolt and Beveridge 1988.)

Some worker have beUeved that gill raker* functioned as passive sieves in filler

feeding (Durbin and Durbin, 1975; Rosen and Hales, 1981; Drenner et al., 1984;

Mummert and Ditnner. 1986; Hoogenboezem. 1991). Gill raker morphology o f rohu,

calla Mid silver carp suggesU that the structures represent efficient passive sieves tai

view o f their elaborate structure. Smith (1989) has also suggested that the gill rakers

o f silvar carp act as passiva, mechanical sieves during filler feeding.

Sanderton « al., (1991) have propoaed that the gill arch itnictufe o f another cypiinid.

the blackfuh, Onhodon microlepidoms, acU ai a barrier during feeding which direcu

particle-laden water towards the mucus covered roof o f the oral cavity, where particles

are retained. Oili-arch structures Iwd previously been assumed to be the site o f particle

retention in suspension feeding fishes. Flow velocity o f water and its direction inside

the oral cavity o f vertebrate suspension feeders, particularly at the level o f filtering

elements, U important. Labarbera (1984) suggested that virtually aU suspension feeders

capture particles from the water al low Reynolds number with cylindrical filtering

elemenu. Oerristen and Porter (1982) observed that the surface chemistry o f filtering

appendages as well as food particles are important for the crasucean zooplankler

Daphnia magna feeding rrt low Reynolds numbers.

Other than sieving, the flow o f water and iu direction within the oral cavity, and

cylindrical filtering elemenu. Greenwood (1953) has proposed mucus entrapment o f

phytopUnkton in the filler feeding mechanism o f Ortochromis esculentus. Mucus

entrapment mechanisms in suspension feeding have subsequently been suggested for

other fishes (Robolham, 1982; Sibbing and Uribe, 1985; Beveridge t l al.. 1988;

Noithcott and Beveridge. 1988; Sanderson era/.. 1991). The presence o f large numben

o f mucus cells in the bocco-pharyngeal region o f rohu and calls has been reported

(Krqioor, 1957; Sinha, 1975; Sinha and Moitra, 1975).

Filter feeding fuh do not visually delect individual prey items, but engulf a volume o f

water containing the food organisms along with non-food items. Taste buds on the oro

pharyngeal surface help the fish to select food from non-food particles during filter

feeding. The role o f taste buds in selection and detection o f food particles in fishes has

been proposed by several workers (Sibbing and Uribe. 198S; Sibbing, 1987;

HM iowskyj el al., 1989).

The resulu o f quanliutive filler feeding studies have been used to create functional

response models. A number o f curves have been fitted to data from filter feeding

studies (Iv lev, 1961; MuUin « al., 1975; Seale and Wassersug, 1979; Seale « al.,

1982; Kiprboe el al., 1985; Drenner el al., 1987). Kiprboe el al., (1985) described a

sigmoid curve which best fitted data on ingestion rates o f a planktoruc copepod.

However. Mullin el al., (1975) demonstrated stalUlically that all three curves

(rectilinear, Ivlev and Michaelis-Menten) could be used justifiably to describe

zooplankton data. Most o f the work on the functional responses o f filter feeding has

been done on zooplankton, whereas little information exisU on filter feeding fish

species.

Particles o f different sizes and types, including free-living unattached bacteria, attached

bacteria, phytoplmklon (sometimes including toxic strains), zooplankton and suspended

organic matter, occur in aquatic ecosystems (Bowen. 1976; Schroeder, 1978;

Opuzynaki, 1981a, Reynolds,I984). Adamek and Spinier (1984) repotted that silver

carps are capable o f ingeating particlea o f < I - 3.37 pm. Kuznetaov (1977) auggeated

that ailver carp may be able to ingeat bacteria fiom the water in aaaociation with algae.

Detection and ingeation o f unattached bacteria in auapenaion by a number o f other

flahea haa alao been reported (Beveridge et al., 1989, 1991).

1.5 The preaent study

G iven the above information and the lack o f atudiea on filter-feeding caipa it waa

decided to inveatigate the filter feeding mechaniama o f the economically important

apeciea rohu, catla and ailver carp in a aeriea o f expérimenta:

i) an hiatological and ultraatructural inveatigation o f the bucco-pharyngeal region

and the orgaru aaaociated with filter feeding o f rohu, catla and ailver carp

ahould be carried out in order to help deacribe and understand the filter feeding

mechaniama involved.

U) a quantitative atudy o f filter feeding on phytoplankton o f two aizea and five

denaitiea (9 - 193 pm’ ) xIO* bio-volume per ml ahould be undertaken in order

to generate functional response curves. The chosen density were the more or

leas similar natural planktonic density at different season o f the year o f different

freshwater habitat o f Bmgladeah. In addition, both toxic and non-toxic atialna

at MicrocyMtU aeruginosa were used to asacas the filter feeding strategies o f one

tpeciet (tUver caip) in the presence o f toxic algee.

Ui) • study on the ingestion o f bncteria would be carried out to deteimme

whether fishes were able to detect or ingest the unattached bacteria in

suspension.

With regards to experiment (ii) in which large volumes o f daU are likely to be

generated it was first necessary to develop and evaluate automated counting procedures

for planktonic algae.

12

C H APTER II

Investigation o f automated particie counting and sizing methods for

aigae

2.1 Introduction

For various reasons, aqtiatic ecologists and others must often resort to working with

preserved phytoplankton nuUerial artd use autonuued counting ruid sizing methods in

order to rapidly process large nitmbers o f samples. In grazing experiments it is very

important to obtain tKcurate counts, sizes turd bio-volumes o f rdgal cells in order to be

able to interpret results.

There rue several types o f apparatus used for counting o f algae, including the Lund

chamber (Lund, 1959). haetrucytomeler and Sedgewick-rafter cell (Nalewrtjko, 1966:

Bailey-Waiu. 1978; Northcolt et at., 1991). In recent yean electronic particle courUen

have been used in biological research for precise particle counting (Parsons, 1973;

Vturderploeg. 1981; Breteler, 1985; Kersting, 1985; Robinson el at., 1990).

13

There we leveral types o f electronic pwticle counter in use, includiitg the Coulter

Multisizer (Coulter Electronics. Luton, Beds. U .K .). The Coulter Multisizer consists o f

three elements (Fig. 2.1): (• ) the Multisizer unit, (b ) sampling stand and (c ) vacuum

control unit. A cuvette containing the sample to be analyzed it placed in the platform

o f the sampling stand (F ig 2.3). A stirrer ensures thw the sample is well-inized. When

directed, a quantity o f the sample is drawn by vacuum pressure creWed by the pump

and regulated by the vacuum control unit (Fig. 2.4) through a small, precisely drilled

orifice into a glass mbe. The tube is selected on the basis o f particle size, each lube

being able to deal with particles 2-60% o f stated orifice size, although according to the

manufacturer, particles 20-40% o f orifice size give best resulU. As each particle pastes

through the lube apetture it crewes an electric pulse by changing the resistance o f the

tenting zone, the size o f the pulse being related to the size o f the panicle. The signal

is transmitted to the Multisizer unit (Flg.2.2) where it is processed and displayed on

the screen. The display (Fig. 2.S) gives infomuuion on N - panicle count, - panicle

count with coincidence correction (l.e. a statistical-based conection for two panicles

which may have passed through the tube orifice at the same lime), £ ■ channelizing

count with panicle size distribution. By manipuUtion o f the displayed data it is

possible to count only panicles within a defined size range. The Mullisizer can be

operated in a variety o f ‘ modes' in which panicles in a known volume o f sample are

counted (siphon mode) or panicle counting takes place over a defined period o f lime

(time mode).

F ig . 2 .2 M u l t i » i * * r f r o n t v i «w (Produc*d from C o u l t * r manual)

r i g 2 .4 Vacuum c o n tro l un it (reproducad from C o u lta r manual)

F ig . 2.S D isp layed data on tha M u lt ia iz a r acraan (rap rodu cad fro r C o u lta r inanual)

19

Detailed descriptions o f electronic panicle counters and the principles o f operation are

given in manufacturer’ s manuals (e.g. Coulter) and in more general texts (e.g. Parsons,

1973).

Whilst it is generally assumed that automated methods involve considerable savings

in time, reports on the advantages and disadvantages o f using electronic panicle

counters for algae are scant. Kersting (1983) reponed that one problem o f electronic

panicle counters (Coulter Electronics Ltd Model T A n. Model ZB and Chatuielizer C-

1000 coupled to Model ZB ) is count loss caused by dead time o f the instruments (time

elapsed between two electrical pulses at the maximum capacity o f the machirre to

generate the electrical pulses per second). In order to avoid this, a maximum counting

frequency o f 200 counts/second was recommended.

Light and electron microscopy are widely used for determination o f algal cell sixe

(Bellinger, 1974; Sicko-Ooad el at., 1977; Clarke et at. 1987). Electronic panicle

counters are also used for this purpose (Sheldon and Parsons, 1967; Vanderploeg,

1981; Bakker errs/., 1983). The Multisizer expresses panicle size in terms o f equivalem

spherical diameter (ESO), i.«. irrespective o f panicle size and shape the Muhisizer

expresses psuticle size in terms o f an idealized sphere (Fig.2.6) and uses this to

crUculale bio-volume. Vrurderploeg (1981) concluded that the bio-volume o f long thin

cells were significantly under-estimtUed by electrorric panicle counters unless cell

20

F ig 2 .6 E q u iva len t Spherica l D iameter (ESD) (rep rodu ced from C o u lte r m an u a l).

21

length« were «honer than tube orífice size. Bakker et at. (1985) «tated that uiing

monocultures or single species-dominated natural phytoplankton, cell counU and bio

volume estimations obtained by visual and electronic methods show reasonable

agreement. They also repotted under-estimation o f bio-volume by electronic counters

when algae are cylindrical, elongate o r needle-like.

Acid Lugol's iodine and buffered formalin are widely recommended as preservatives

o f fteshwater algae (Vollenweider 1974; Wetzel and Likens 1979, A PH A 1989),

although linle woik has been done on their effecu on algal cells and colonies. In a

study o f autotrophic and heterotrophic marine flagellates Breteler (1985) found drastic

losses o f cells and changes in cell size occurred when Lugol's iodine and other

preservatives (e.g. acetic acid, trichloríc acid, sublimate and benzoic acid-5hydroxy-

sulfo) were used. Both SUver and Davoll (1978) and Holtby and Knoechel (1981) have

reported the loss o f radioiaotope label as a result o f algal cell disintegration in Lugol s

icxliiK.

In this study it was decided to investigate the usefulness o f automated counting and

sizing methods for algal cells and to examine how numbers and sizes o f cells and

colonies o f fteshwater green algae (Chlorophycaea) and Cyanobacteria (blue-green

algae), as determined by the Coulter Multisizer, are affected by both short-term and

long-term storage in LugoLs iodine and buffered formalin. The investigation o f

ruitomtded ccxirMing and size and bio-vohime estimation was cairied outonly with P,

22

boryanum and M. aeruginosa, the two algae <

Chapter IV ).

1 in the ingestion expeiimenu (a

2.2 Materials and Methods

2.2.1 Algae

Cultures o f the freshwater blue-green alga (Cyanobacteria) Microcystis aeruginosa

Kutzing emend. Elenkin 1924 were obtained ftom the Department o f Biology.

Univeisity o f Dundee. The green algae Fediastrum boryanum (Turpin) Meneghini 1840

and CMorelta vuigaris Beijeriitck were obtained from the NERC Culture Collection o f

Algae and Protozoa, FBA, Windemrere, U .K . C. vulgaris, P. boryanum and M.

aeruginosa were cultured in Bold's Basal rrredium (Nichols 1973), Jaworski’s medium

and nitrate-supplemented BO-U medium (Staruer et at 1971) respectively (see

Appendix 1, 2, 3). Batch-culture methods were employed. Cultures were grown In 3

I flasks in an Ulumiruited cabinet maintained at 16-19 *C.

2.2.1 Counting and estimation o f cell size and blo-voiume

In order to compare the performances o f different counting techniques five replicate

counts were made by haemacytometer BS 748 tuid Sedgewick-Ralter cell S30 (Fisons)

for each o f samples o f M. aeruginosa and F. boryanum respectively. Standard Methods

23

(A PH A 1989) were followed for counting the algae by haemacytometer and

Sedgewick-iafter cell. The same samples were counted using the Coulter Multisizer,

the methods employed being based on those described by Parsons (1973). Ten different

count rates (count. s"‘ ) with five replicates were employed using siphon mode. Each

replicate coruisted o f 200 pi sub-samples transferred to 20 ml recommended

commercially-prepared electrolyte, a non-sterile but bacteriostatic, phosphate-buffered

saline solution; concentration ■ 0.85% NaCl (IS O TO N 11; Coulter Electronics. Ltd..

Luton. Beds.. U.K.).

At least one hundred cells or colonies o f each algal species were measured using an

Olympus BH2 phase contnat light microscope. The bio-volumes o f M. aeruginosa

cells were calculated using the generalized formula fo r a sphere as Northcott el at.,

(1991):-

V -4/3 itabc

where a - 0.5 x length; b • 0.5 x height; e - 0.5 x width. The bio-volume o f P.

boryanum was calculated by using the fotmula:-

V - xr’ L

where r • radius o f the colony; L • depth o f the colony. The Coulter Multisizer was

f i « « used for estimating the size and bio-volume o f the rUgae. A similar calculation for

24

P. boryanum wai lued by Bellinger (1974).

The lize distribution o f the algal cells was also deteimined by introducing the same

volume (200 fU) o f algae into 20 ml ISOTON n. CounU were deteimined immediately,

and after S min, 10 min, 20 min and 40 min following transfer to ISCXTON n.

2,2J Effects o f preservatives on algal cells

Acid Lugol's iodine was prepared according to the methods given in W etzel and

Likens (1979) aitd Standard Methods (A PH A 1989), in which 10 g iodine and 20 g

potassium iodide are added to 200 ml distilled water containing 20 ml glacial acetic

acid. Buffered formalin (20 g sodium borate 4 1 I 37% formaldehyde) was prepared

according to the method given in Standard Methods (A P H A 1989). The solutions were

stored in a refrigerator at 4*C and used within 3 weeks.

Four concentrations o f each preservative and seven preservation periods o f both short

and long duration were investigated. Samples taken ftom log-phase cultures o f algae

were preserved with Lugol's iodine at concentrations o f 0.0S%, 0.1%, 0.2% and 0.4%

and in buffered formalin at concentratioiu o f 0.5%, 1%, 2% and 4 %. Three replicate

samples were made for each concentration. Samples were stored at room temperature

in ambieiM light conditions, away ftorn direct sunlight.

23

Cell or colony numben and their size distribution for each sample were estimated by

Couher Multisizer fitted with tubes o f orifice size 70, and 200 pm. An initial count was

made using fiesh material. Distinct peaks o f particle number versus particle size were

generally observed, other parts o f the spectrum being assumed to represent bacteria and

cellular debris. Using the channel selection facility on the Multisizer it is possible to

exclude unwanted counts and thus only the peak corresponding to algal cells or

colonies was counted (see Figure 2.5). As background counts (i.r. counts o f particles

present in the electrolyte solution) were extremely low (70-90) in all cases, no

adjustmenu to counts were made.

Four sub-samples from each sample were examined immediately prior to preservation

and at intervals throughout the preservation period. For the purpose o f analysis, 2(K)

pi sub-samples from C. viilgarir, M. aeruginosa and F. boryanum were added to 20

ml o f the recommended ISOTON II. A ll counM were made between 1 minute and 5

minutes after introduction to the electrolyte.

2.2.4 Statistical methods

Multifactor A N O V A was used to verify significant treatment effecte and Duncan's

multiple range test used to compare individual treatments within the A N O V A design.

Student's t-test was used to verify differences between samples (Sokal and Rohlf.

1973; Bailey. 1981).

26

2.3 Results

Channelizing count loss (ditfeience between total count and channelizing count) was

estimated as 1% when the count was 200 - 300 panicle, s'’ and 22% at 2600 - 2800

panicle, s'* count (Table 2.1). A ll subsequent sample counu were maintained at 200-

300 panicles, s ' in order to minimise count losses.

Counts obtained from the Coulter Multisizer were slightly higher than those from the

haemacytomeler and Sedgewick-Rafter cell. A comparison o f counu using different

devices is presented in Table 2.2.

The diameter o f P. boryanum measured by light microscopical method was higher

than cell BSD measured by Coulter Multisizer. Bio-volume o f P. boryanum measured

by light microscopy was also higher than that estimated by Coulter Multisizer.

Similarly, the size and bio-volume o f M. aeruginosa determined by light microscopy

were higher than that given by the Coulter Multisizer (see Table 2.3).

No changes in the size dUtribution panem o f P. boryanum, M. aeruginosa and C.

vulgaris with time were observed following immersion in ISOTON n. (F ig. 2.7).

However, in all instances, sample colony number and cell number at different durations

o f preservation were slgnlficaitly different (P < 0.01) from firesh samples (Table 2.4).

T a b l* 2.1 Channelis ing count loaaas <%) «nd t o ta l count ml (x lO *) a t d i f f e r e n t range o f counting ra te .

Channelis ing count lo s s (%)

Count a’ * To ta l count ml* ‘ t S.E.

1 — 200 - 300 9T758 ± 0.612 350 - 450 90.37 ± 0.473 500 - 600 89.43 t 0.384 700 - 800 89.79 ± 0.836 900 ' 1100 68.62 ± 0.647 1200 - 1400 88.51 ± 0.7210 1500 - n o o 87.97 ± 0.8612 1800 - 2000 87.81 ± 0.8716 2200 - 2500 86.02 ± 0.9122 2600 - 2800 67.94 ± 0.68

T a b i« 2.2 Numb«r o f c o lo n i« « o r c o l la mi** (± S.B) obtainod by d i f fo r o n t countlng mothoda <n " 50 f o r a l l moans).

Countlngmathod

M icrocyMtit count mi**

P«di«afcrum count mi**

Timo roquirod fo r 10 aamplo

count w ith f i v o r o p lic a to

C ou ltorM u lt ls iz o r

70.34 X 10=* (± 0.21)

102.62 X 10’ <± 0 .8 4 )

50 m inuto«

Haomacytomotor 77.53 X 10* ( t 0.56)

- 140 minuto«

Sodgowick r a f t o r c o l l

97.26 xlO* (± 1 .32 )

175 minuto«

T a b i» 2.3 Bquivalant sp h arlca l diam atar (ESD), voluma and a lza o f a lgaa maasurad by l i g h t m icroacopa and C ou ltar M u lt ia ia a r .

L igh t a lcroa cop y Cou ltar Countar

PadiaatrumborymnuM

“ 135--------------Haan Voluma Diamatar

16015 ^m* 63.66±8.43

5Ì70TÌ7.08 15243 Mm>

M»

M ic ro cy s t isssruginoss

ESDHaan Volumalangthwidth

51.31 Min' S.0t2 M<n 4.0 M"

4.1311.03 39.06 Mm’

Mm

30

r i g . 2 .7 Colony d i f t r ib u t io n pa tto rn o f ( « ) P. borysnum, (b) M. * 0ru g in o »s and (c ) C. vu ig a r ia fo llo w in g d i f fa r o n t du ration * in ISOTON I I .

31

Reductioiu in average numbere o f P. boryanum cokxiiet were olwerved at all

concentrations o f preservative and in all durations o f preservation smdied (Fig.2.8a and

Fig.2.9a). A fter 24 h, the average number o f P. boryanum colonies was found to have

by 4.9 %. and after 70 days by 22.9% (Table 2.5). When mean colony sizes

were compared, no significant shrinkage o f P. boryanum colonies was observed during

24 h o f preservation (Fig.2.10).

A fter 24 h, the mean number o f M. aeruginosa colonies was found to have decreased

by 2.7%, and after 70 days it had decreased by 29.4% (Table 2.5). The rate o f

disintegration o f M. aeruginosa colonies appeared to decrease with tune between 2

days and 70 days preservation (Table 2.5 and Fig. 2.8b). Some shrinkage o f M.

aeruginosa ceUs. as evidenced by a shift in peak distribution, was observed after 24

h preservation at all concentrmions o f Lugol's iodine (Fig. 2.11). Decreases in average

colony numbers were observed to be significantly higher (P < 0.05) in 0.05% Lugol s

iodine (Fig.2.9b).

By comparison, C. vulgaris cells were found to be less affected after 70 days. After

24 h and 70 days, the average number o f C. vulgaris cells was found to have decreased

by 4.7% and 14.8% respectively (Table 2.5). Shrinkage o f C . vulgaris cells was also

observed after 24 h preservation in all concentrations o f Lu go l’ s iodine (Fig. 2.12). No

slgnlfkum differences in decreases in average cell numbers were observed among the

diffeient concentrations o f different preservatives (Fig. 2.9c)

32

Tab i« 2.4 R « «u lt « o f « n « l y « l « o f va rian e« fo r th « « f f « c t a o f d i f f « r « n t conc«n tratlona o f p r « « « r v a t iv «a at d if fa ra n t du ration « o f praaarvation on (a) F. borymnurn, (b) M. smrugino»*,(c ) C. vu lgsr is

Sourcas o f v a r ia t io n

— a r r r Sua o fSquares

MeansSquares

r -ra t io S iq . la va l

Concant rat ionDurationXntaractionErrorTo ta l

----7-----749126191

1902442€20175222152923119

r m i --------27177712657174

3 io .n1561.9472.74

P < .01 P < .01 P < .01

(b)

Sourcas o f v a r iâ t ion

a r r r Sua o f Squares

MeansSquares

r - r a t io S ig . la va l

Concant rat ionDurationXntaract ionErrorTo ta l

7749128191

r m v r n n r1.041Bf091.126B40922699063.597E4>09

2036996321487127682301600017734

1U 86.39~6365.741297.96

P < .01~ P < .01 P < .01

(c)

Sourcas o f V a ria tion

a r r r Sua o f Squares

MasnsSquares

P^ratio B ig . le v e l

Concantrat ionDurationXntaractionErrorTota l

-- -̂--749126191

3199260247160761665306562187435514316496

4920375----6737252017409917069

“ laTTT!3942.4S 10.19

P < .01 P < .01 P < .01

33

# , '%■ # , -e 1 t « • 1« 9t re

Duration In daya

b

» « e '# 91 re

Duration In daya

r i g . 2.6 E ffa c ta o f d i f fa r a n t du rationa o f p raaa rva tlon on tha maan c a l l nuinbar fo r a l l concantra tlon fo r both p ra aa rva tlvaa o f (a ) P. toory^num (b) M. •mruqino»» and <c) C. vulgmtim. Bara in d lca ta ± S.E.

'1_____________ ™

1 I « 0 0 * 0 « 01 0«Perfnaim tufoT* ioOm«

Conctntratlont in %

Conctntrallont in %

Concantratlont In %

r i g . 2.9 E f fe c t s o f d l f fa r o n t concon tra tiona o f both b u ffo rod form alin (column 1-4) and Lu go l'a iod in a (column 5-8 ) on tha maan c a l l numbar o f a l l tima aariaa o f (a ) P, b o r y n u m (b ) H. M Tug inoa » and (c ) C. vuJgaria . Bara in d ica ta ± S .E .

35

•>«9Or*-

« « « 0« B en •

N ^ ^ N *H

•

1r«4m

93 • r> m r» r»

o ^ NM M H

«>«9«tH

r» tH *9 03 93 <N

«n r4 Or-< fM *H

•

Ì»

03 O N fn <n

N O•H »

«>«9«

N <N «n tn ® íd r* o

m1

>%9•H

N «n lO 93 «n o

m m «o

o r* ^93 39 «9

o o

1U

i B ; «

Ï * :2 -5U 2 l iib 2 I f

li i i ti

36

r i g . 2 .10 Colony « I z o d is tr ib u t io n p s tto rn i o f P.borymnum bo foro p ro s e rva t io n (BP) and a f t a r 24 hours p rsssrva tion in (a ) b u ffs rsd fo rm a lin o f conesn tra tions 0.5%» 1%» 2%» and 4% (b ) L u go l's io d in s o f concsntrationa 0.05%» 0.1%» 0.2% and 0.4%.

37

r i g . 2.11 Colony • iz a d is tr ib u t io n pattarn o f M. aaruginoaa ba fo ra p raaa rva tion (BP) and a f t a r 24 hours p ra aa rva tio n in (a ) b u ffa ra d fo rm a lin o f concantrationa 0.5%, 1%, 2% and 4% (b) L u go l'a iod in a o f concantra tiona 0.05%, 0.1%, 0.2% and 0.4%.

3«

T iq . 2.12 C U . 1 « d is t r ib u t io n p . t t . r n

Of c o n c «n tr «t io n * 0.05%, 0.1%, 0.2% «nd 0.4 .

39

2.4 Discussion

Chsnnelizing count loss incressed with increasing counting rate (Table 2.1). Count loss

by electronic particle counter was also r^wited by Kersting (1983) and as stated in

Section 2.1»he leconunended arate o f 200 counts/second. He suggested the count loss

was due to dead time o f the machine i.e the machine fails to count every particle. The

channelizing count loss in this experiment may have been the result o f this

phenomenon. An alternative explanation is that there are errors inherent in the

equipment in assigning particles to specific channels.

The Coulter Multisizer gave slightly higher, althou^ not statistically significani, counts

than the haemacytometer and significantly higher (P < 0.03) counts than the

Sedgewick-rafler cell (Table 2.2). This may have been due to the instnunent counting

debris and other particles in addition to algal cells and colonies whereas the

haemacytometer and Sedgewick-rafter cell counts were for algae alone. It was

estimated in the present study that it required 40 minutes to count ten algal samples

by Couher Mullisizer, whilst the same counts by haemacytometer and Sedgewick-

Rafter ce ll required 120 minutes and 140 minutes respectively. Discussions with other

workers using automated and manual mathods (Y . Waddell, personal communication)

conArm that automated counting required signiOcantly Ims time than manual counting.

M oreover it is possible using the Coulter Multisizer to send data directly to a computer

for storage and hutfier statistical analysis, thus saving even greater amounts o f time.

40

The BSD and volumes o f M. aeruginosa and F. boryanum measured by microscope

were significantly different from those obtained from the Coulter Multisizer. Tftese

differences are [wobably due to differences in algal cell size and shape. Vandeiploeg

(1981) reported that the Coulter counter significantly underestimated the size o f long

and thin algae. Bakker et ai. (1985) also concluded that the Coulter counter may

underestimate the volume and BSD o f cylindrical, elongated or needle-like

phytoplankton. The present findings further confumed that electronic counting devices

underestimate the bio-volume o f long and thin algal cell and that their use in bio

volume estimation should be largely restricted to cells w colomes spherical in shape.

No changes in size distribution partem o f fresh F. boryanunt, M. aeruginosa and C.

vuigaris were observed following different durations o f immersion in ISOTON n (Fig.

2.7). This is probably because the concentration o f ISOTON II approximates that of

the culture media and thus docs not change the osmotic pressure inside the algal cell.

Visual observation o f samples showed that after 8 days preservation in 0.05% Lugol s

iodine. F. boryanum and A#, aeruginosa samples were cloudy, possibly as a result o f

algal disiniegratkxi or possibly because o f inadequate preservation and growth of

microorganisms. Examination o f samples by light microscopy confirmed that mwiy

cells had disintegrated.

41

P. boryanum wxl M. aeruginosa u m p la were mote advenely afTecled by both Lu go l'i

iodine and buffered fomialin at aU concentrationf than C. vulgaris aamplea, poaaibly

because o f diffeiencea in ce ll wall stnicture. Breteler (1985) obaerved that no loaaea

o f marine diatoms occuned when pieserved with Lugol's iodine, although flagellates

were drastically affected after only 24 h preaervatkm. Bieteler (ibid) concluded that this

difference was due to the diiuom silica cell wall.

There is a clear indication in this study that colonial and cellular disintegration

increases with preservation time. Published work, although scant (Weber 1968; Silver

and DavoU 1978; Holtby and KtK>chel 1981; Paerl 1984; Breteler 1985). indicates that

all commonly used algal preaervatives or Tixation methods, such as freeze-rixation, can

damage algal cells. Indeed, the warning is often given that snull, delicate cells may be

destroyed by preservatives. In some iiutances, modified versions o f preservatives may

be suggested: for example, the replacement o f acetic acid with sodium acetate is

recommended for Coccolithophores, whilst for long-term storage o f algae, it is

recommended that the concentration o f Lugol's iodine be increaaed and that formalin

also be added (AH PA , 1989). In the light o f the findings from the present study, it is

recommended that more w ork on the development o f algal preservatives be urgemly

carried out.

42

The advantages and disadvantages o f the automated counting method, as described in

the present study, can be summarised as follows. The Coulter Multisizer facilitates

ngiid counting. Moreover it gives additional infotmation on size distribution patterns,

equivalent spherical diameter (BSD), surface area and volume o f panicle. The Coulter

Multisizer can send daU directly to a computer, facilitating storage, analysis and

presentation. Minor changes in the size distribution pattern can readUy be detected by

Coulter Multisizer which would be difficult by other methods. However, the Coulter

Multisizer cannot readily distinguish between algae and other particles o f the same size

and it significantly underestimates the size and volume o f long, thin algal cells. It is

therefore best used with these disadvantages in mind and with routine compansons

with material examined by conventional light microscope.

43

CHAPTER I I I

Histology and Ultrastructure

3.1. Introduction

A t plankton feeden rohu, catia and ailver caip h «v e comparatively wellKleveloped gill

rakert. Some anatomical and •tnictural studiet o f g i l l raken o f eilver and bighead caip

have been made (W ilamoviki, 1972; Iwata, 1976; Cremer and Smithennan, 1980;

Jiraaek. el al., 1981;), moat having been carried out on adult fiahet. Spataiu, el

<i/.,(1983) reported that the average apace between the gill rakera o f ailver carp and

highead carp o f 15 - 30 cm suuidard length aize w a i 36 pm and 84 pm reapectively.

Smith (1989) deacribed the gill raker apparalua o f ailver caip (mean weight 32.Sg) aa

a mechanical, paaaive aieve, which cannot filter pautklea o f leaa then 10 pm, whereaa

Adamek and Spinier (1984) repoited that ailver caup (three yean old, average weight

790g) can ingeal paiticlea o f < 1 - 3.37 pm. B y compariaon, linie woik haa been

carried out on the gill raken o f ciula and lohu.

The role o f mucua cella in filter feeding haa been auggealed by many workera and

might be an explanation o f how paiticlea amidler than the iiuer-giU raker apacea can

be trapped. Greenwood (1953) described mucus entrapment o f phytoplankton by

Oreochromis esctdentus during filter feeding. Sinhe end M oitra (1975) found many

mucui cell* uiocuUed with the pharyngeal and anterior oesophageal region o f Labeo

rohita, and suggested that they played an important role in trapping and transpoiting

food particles during feeding. Sinha (1975) also observed the presence o f large

numbers o f mucus cells in the buccopharyngeal region o f Cor/n catia and Ctrrhina

mrigata. The presence o f different types o f mucus cells in the buccopharyngeal region

o f Cyprinus carpio has been reported by Sibbing and Uribe (1985). Mucus entrapment

o f food particles during suspension feeding in other fishes has also been suggested

(Robotham, 1982; Beveridge et al.. 1988; Notthcott and Beveridge,1988; Sanderson.

et a/..1991).

In many fishes, iiKluding cyprinids, food particles are ingested along with the water.

There are a number o f hypotheses concerning the selection o f food particles from the

water by fish. Sibbing and Uribe (1985) described the role o f taste buds in the feeding

o f Cyprinus carpio. Sibbing (1987) repotted that the taste buds in the pharyngeal roof

o f Cyprinus carpio lie on top o f a muscular cushion, the palatal organ, a thick,

ptotnided floppy structure suspended from the roof o f the buccopharyngeal cavity,

which is active in selection and food transport. Hlohowskyj et at. (1989) suggested that

all seven species o f the herbivorous cyprinid genua Hybognuahus bear taste buds or

papillae in the pharyngeal region and that they ate arranged in a panem that helps in

filtering or trapping diatoms and other small food items. Smith (1989) found that non

food panicles would not Induce feeding tat silver carp, whereas organic matter from the

gnvel filter, after being filtered through a S filter, induced vigorout feeding.

Catla and silver carp also possess a palatal organ. In the case o f rohu, the palatal organ

is slightly different and resembles a muscular cushion provided with several

honeycomb-like projections or py illa e (Sinha and Moitra, 197S). Boulenger (1901)

first described the base o f this palatal organ in Hypophthatmichthys spp. as the

suprabranchial organ. Wilamovski (1972) r^xMted that the involvement o f the

suprabraiK;hial organ in filter feeding o f silver carp was as a pun^. He also observed

that each g ill arch and its canal enters into a tube which extends into the palatal tissue

block to form the hollow, spiral suprabranchial organ surrounded by the muscle tissue

o f the palatal organ. Sibbing and Uribe (1983) observed that the surface o f the palatal

organ o f Cyprinus carpio is irregulaily but heavily papillaied and densely packed with

taste buds on the tops o f columnar-shaped papillae, even more dense in the lateral

areas than in the medial region, and plays an important role in distinguishing food

from non-food material during feeding. Sanderson et o/.(1991) reported that the mucus-

covered roo f o f the oral cavity plays an important role in the retmtion o f particles in

the suspension-feeding cyprinid blackfish, Orthodon microiepidotus.

A review o f published literature fails to provide a clear picture o f the Altering

i^yparatus and its associated orgaiu in rohu, catla and silver carp, particularly the fry

and fingeiiing stages. Most published studies to date have been limited to gill raker

morphometry mkI histology o f the alimentary canal o f aduh fish. In view o i this it was

decided to study the bucco-pharyngeal region and the Altering apparatus associated

46

with the filter feeding o f Labeo rohita. Cotta catla and Hypophthatmichthys motitrix.

The itudy included detailed ultiaatiucniral and hiatological invettigationa to elucidate

poaaible feeding mechanianu.


3.2.1 Fish

Two^day old fry o f Labeo rohila, Catla catla and Hypophthatmichthys moUtrix were

imported fiom the Fisheries Reseiuch Iiutitule. Mymeiuingh, Bangladesh in sealed

plastic bags. They were held initially in a static water quarantine system with aeration

for 4 weeks until preliminary diserue monitoring could be completed. They were then

traruferred to a recirculrttory system for rearing. They were fed on a mixed diet

composed o f rUgae. Anem ia and ground trout pellets. Fish o f standard length (S L ) 30 -

92 mm were used in the experiments.

3.2.2 Feeding apparatus morphometries

The buccopharyngeal feeding structures, (t^. gill arches) were carefully removed from

the fish with the aid o f a binocubr dissecting microscope (Olympus V M ). A ll

specimens used in this study had been previously preserved In 10% neutral-buffered

formalin. Preliminary examiruMion o f the gill apparatus showed a consirietable degree

47

o f variation among the gill arches. In order to standardize observational data gill arch

n was chosen fcM* mcMphometric study. Once it had been excised and |Mepared fot

morphometric data the following measurements were made using an Olympus BH2

light microscope :

(a) gill arch length (from the base o f the anterior most gill raker to the base o f

the posterior most gill raker);

(b ) the number o f gill rakers;

(c ) gill raker length;

(d) the distance between the gill rakers.

A t least 10 measurements for (c ) and (d ) were made from the mid-section o f gill arch

n. The lengths and distances between g ill rakers horn the mid-section o f gill arch II

were used to detennine the relationsh^ between the standard length and the chosen gill

raker data from the fish.

In order to obtain detailed descriptions o f the feeding apparatus and for a comparative

study among rohu, catla and silver carp the following data were taken from fishes o f

approximaiely 90 mm standard length:

48

(> ) length o f each gill arch;

(b ) number o f gill taken on each gill arch;

(c ) length o f all individual gill taken on each gill arch;

(d ) taste bud number and spacing on the gill raker in the mid-section o f gill arch

H;

(e ) number o f taste buds per mm’ on the roof o f the buccopharyngeal cavity.

Counting o f taste buds on the gill taker and roof o f the buccopharyngeal cavity was

carried out by both electron and light microscopy.

The area o f the filtering part o f individual gill arches was detetmined using the

following formula developed from Jirasek et al. (1982) and Oibson (1988);

Fa - (L a I) a 2

where Fa • filtering part o f the gill arch; L - length o f the gUl arch; I - average length

o f the pharyngeal surface o f gill taker.

49

Fifteen Rshea for each species were used in this puipose ranging fnrni 30 to 100 nun

Standard Length (S.L.).

3.2J Scanning electron microacopy (SEM )

For scanning electron microacopy (SEM ) silver carp, rohu and catla o f similar size

were selected and deheaded after being killed by an overdose o f 10% benzocaitte (Ross

and Oeddes, 1979). A standard method was used for the processing o f specimens

(Reutter t t at., 1974; Sibbing and Uribe 1983). The specimens (heads) were washed

in buffered physiological saline to remove mucus and fixed for seven days at 4*C in

3% glutaraldehyde, buffered at pH 7.2 in 0.1 M sodiumciKodylate containing 2.3%

glucose. The heads were then sectioned horizontally into a dorsal and ventral half to

investigate the floor and roo f o f the buccopharyngeal region and intact gill arches

carefully removed for detailed study. Specimens were then washed repeatedly in a

buffered saline solution prior to being transferred to 1 % osmium tetroxide, buffeted in

0 .1 M sodiumcacody late, for 6 h for final fixation. After repeated washing in the buffer

the specimens were then dehydrated through a graded acetone series, stored In 70%

acetone If necessary, and critical point dried In a Polaron E-1000 dryer before being

mounted on alumiitium stubs with silver conducting pair« and coated with a layer o f

gold In an Edwards SI30B sputter coaler. A PhiUips 300 Scanning Electron

Microscope was used for exrunination o f the specimens.

so

3.2.4 HUtoloKy

Speciment from L. rohita, C. catia and H. moUtrix were fixed in 10% neutral buffered

fonnalin for at least a week and then processed by automatic tissue processor and

embedded in wax (Appendix - 8). A series o f sections was made after trimming and

décalcification (4h in 13% H C l)o f the specimen. A ll sections were 3 pm in thickness.

Serial sections were stained with Haematoxylin-Eosin (H ftE ) for structural analysis.

Neutral as well as acid mucus is stained with Periodic Acid Schiffs reagents (P .A .S )

whereas Alcian Blue (A B ) at pH 2.3 was used for acid mucopolysaccharides and

glycoproteins (Drury and Wallington, 1980 ). A ll sections were examined using a light

microscope fitted with an eyepiece graticule.

An accurate assessment o f the density o f the mucus cells was not attempted. However,

the densities o f different types o f mucus cells in different areas were recorded for

comparative purpose.

3 J Results

3.3.1 Introduction; terminology and subdivisions o f the oro-pharynx

Using conventioniU terminology the oral cavity is assumed to extend from the lips to

SI

the level o f the lower jew . The buccal cavity extendi from here to the bate o f the Tint

branchial arches (F ig 3.1.). The pharyngeal cavity in cyprinids is sub-divided into the

anterior pharyttx (syn. btatKhial cavity), containing the gill arches and is involved in

respiration and food selection, and the posterior pharynx (syn. chewing cavity). The

respiratory rUamenti o f the gill arches project irtto the opercular cavity.

3.3J Labeo rohita

Oto-buccal cavity

Upr. The toothless ventral mouth is bordered by thick soft upper and folded lower lips,

the upper lip overlapping the lower lip. The lips are fringed and bear large numbers

o f taste buds (240Anm’ ) (Plate la ) on the tops o f closely-packed epithelial folds. Taste

buds are absent at the mouth angles.

Oro-buccal roof. A smooth, thin. crescetM-shaped respiratory valve projects from the

oral roof at the inner edge o f the upper jaw (Plate la ). No taste buds were found on

53

the dietal half o f the valve whereat tome pl^>illae with tatte buds were found on the

basal part (40Anm^).

Oro-buccai floor: Small fuyillae were found to extend inwards from the hcNtiy area o f

the lower jaw. LongitudiiuU folds were observed which run caudo-laterally towards the

first branchial slit (Plate 2a). These folds allow considerable extension o f the buccal

walls. A few taste buds (25/nwn^) were fouiKl. and only at the top o f these fokb. There

is no true tongue althou^ the central convex area o f the buccal floor which is o fto i

referred to at a tongue is supported by the mobile gloesohyal arch which it connected

to the hyoid arch. Many papillae with taste buds (65Anm’ > were present in this area

(PUte 2a).

Pharyngeal cavity

Anterior pharynx: The roof o f the anterior pharynx is formed by a muscular cushion,

the organ, covering the base o f the skull. The surface o f the palatal organ

closely fits the pharyngeal surface and curvature o f the gill arches. The surface o f the

palatal CM'gan is a honeycomb-like projection aiKi is densely packed with taste buds

(Plate 3a). The deiuity o f taste buds in the medial region is less (average up to 170

A im *) than the lateral area (average up to 320 Anm‘ ). In the posterior region o f the

palatal organ the number o f taste buds is markedly reduced (average up to 130 AnnH).

64

The floor o f Che anterior pharynx (Plate 4a) i i largely cocnpoaed o f Che gill iux:hes rurd

aasociated taken. The gill archea and the double rowa o f taken form a perforated

plane along the floor o f the phruyngeal cavity. The pharyngeal surfaces o f the gill

raken bear numerous small projectioru or conical papUlcK with taste buds (Plate Sa).

Tw o rows o f g ill raken are atUKhed by a thin muscular strip o f tissue tUong the gill

arches and the pharyngetU surface o f this tissue bean very few taste buds. The gill-

raken from subsequent arches are turanged in such a way thru they can be useds for

forxl selection.

O ill taker length (O R L ) and gill taker space (ORS) were observed to itwrease with

increasing fish standard length (S L ) and the relalioiuhip between fish standard length

and gill raker length; luid fish standard length and gill raker sptKX rue shown in Fig.

3.2 imd nuy be described by the erpiacioiu:

O RL - -1634.S + 1261.3 k>g.SL; r - 0.98

ORS > -8.10 -f 33.36 k>g,SL; r - 0.97

Fish stan0ar0 iangih (mm)

Fith tu n d a rd langih (mm)

F ig . 3.2 Tha ra la t ion a h ip a betwean (a) g i l l rakar langth« (b g i l l rakar apaca and f i a h atandard langth o f rohu.

66

3.3 J CaHa catia

Oro-buccAl cavity

Lip: The upturned (»ocnitile, toothlett mouth is bordered by thick folded with taste

buds (40Anm^) along the inner surface (Plate lb ), whereas the folded part is alntost

devoid o f taste buds. No taste buds are found at the mouth angles.

Oro‘buccai roof. A crescent-shaped respiratc»y valve projects from the oral roof at the

inner edge o f the upper jaw (Kate lb). No taste buds were found on the lespiratcxy

valve other than few at the base (40 taste buds/ mm^). At the base o f the respiratory

valve several oval-shaped papillae with taste buds are present (Plate Id). The roof o f

the oral cavity bears small papillae with taste buds (1 KVmm^).

Oro-buccalfloor: No mandibular valve is present. Large numbers o f longitudinal folds

bear arwl taste buds at the top o f the folds which run caudo-laterally towarcb

the first branchial slit (Plate 2b). There is no true tongue, although the central convex

area o f the buccal floor, which looks like a tongue, is supported by the mobile

glossohyal, connected to the vmtral elements o f the hyoid arch. Medium-sized papillae

with taste buds are universally conspicuous in this area (1 lO^nm*).

67

Phaiyngeal cavity

Anterior pharynx: The roof o f the anterior pharynx u formed by a bi-lobed palatal

organ. The lu iface o f the palatal organ clocely fiu with the pharyngeal turface o f the

gill raken.

The iiirface o f the bi-lobed palatal organ hai three ridgei at each ride which ate

heavily papiUated (Plate 3b) and detuely packed with tatte buds (270/ttim’ ). In between

the ridgei are found few papillae with low deniitiei o f taile budi (90Mun’ ). In the

poiterior part o f the palatal organ, the triangular area encloied between the gill atchea

arrd the chewing pad, the papillae are densely packed although there are markedly

reduced numbers o f taste buds (90Anm’ ).

The floor o f the anterior pharynx (PUte 4b) is irutinly composed o f the gill arches and

associated rakers. The gill arches and double rows o f rakers form a perforated bi-lobed

plane with four canals at each side that lie immediately adjacent to the palatal organ

when the floor o f the mouth is raised.

The pharyngeal surface o f the gill rakers bears two tows o f small papillae , each with

a single terminal taste bud (Plate Sb). The gill rakers are not fixed, and may be

adjusted during food selection.

68

O ill raker length (O R L ) and gill raker space (O RS ) were also observed to increase with

standard length (S L ). The relationship between fish standard length and gill raker

length and fish standard length and gill raker space are shown in Pig. 3.3 and can be

described by the following equations:

Q R L - -2221.0 -f 1969.1 k>g,SL; r - 0.96

ORS - 27.32 -f 32.64 log,SL; r - 0.97

3.3.4 Hypophthaimichihys moUtrix

Oro-buccal cavity

Lips: The fHotnisile toothless mouth is bordered by thick lips with a homy edge. The

middle o f the lower lip has a projection (Plate 2c).

Oro'buccai roof. A crescent-sh^>ed respiratory valve projects from the CMal roof at the

inner edge o f the upper jaw (Plate Ic ). The distal part o f the valve is smooth wid taste

buds are almost completely absent, whereas the base o f the valve is piq>iUaied with

taste buds (120^nm^).

69

FIth ttandard ■•ngth (mm)

F ig . 9.3 Th* rd ld t ion sh ip s bdtwddn (a ) g i l l r a k « r length, g i l l rakar apacg and f ia h standard langth o f c a t l a .

( t

70

Oro~buccai Papillae timilar to those on the surface o f the oro-buccal roof extend

inwards from the base o f lower jaw. The density o f taste buds is similar to that o f the

oro- buccal roof. There are some longitudinal folds at the level o f the cheeks which are

deep and run in a caudo-lateral direction towards the first branchial slit (Plate 2c).

These folds allow considerable extension o f the buccal walls. The central area o f the

buccal floM*, often referred to as the tongue, is supported by the mobile glossohyal,

connected to the ventral elements o f the hyoid arch. Many papillae with taste buds are

conspicuous throughout this area.

Pharyngeal cavity

Anterior pharynx: The roof o f the anterior pharynx is covered with a bi-lobed palatal

organ. The surface o f the palatal organ closely fits with the pharyngeal surface o f the

gill rakers. In fresh preparations the palatal organ l^>pea^s as a muscular cushion

suspended from the pharyngeal roof. The muscular cushion contains the hollow spiral

Mgarts, referred to as the suprabranchial cwgan in the literature (Wilamovski. 1972).

The surface o f the palatal organ has four prominent ridges on each side (Plate 3c)

which are heavily papUlated and densely packed with taste buds (530Anm^). The

density o f taste buds between ridges is low (220/ mm’ ), although there are many mucus

cell openings. In the posterior pan o f the suprabranchial organ, the triangular

71

area clofte to the chewing pad it heavily papiUated although with reduced numben o f

ta ite buds (18(Vnim^).

T h e floor o f the anterior pharynx it composed o f the gill arches and their rakers (Plate

4 c ). Each gill arch fcmns a perforated canal with its double row o f gill rakers. The M-

lobed pharyngeal floor is formed by four perforated canals at each side and fits closely

w ith the ridged roof o f this area.

Th e pharyngeal surfaces o f the g ill rakers bear two rows o f conical papillae with taste

buds ( l ^ e 5c). O il! rakers were observed to be fixed due to the fusion o f parts o f

adjacent gill rakers as described by Iwata (1976). N o such fiuion o f gill rakers was

observed among flsh below 30 mm SL. Small fish are thus able to move adjacent gill

arches to collect food particles.

Inter-gill raker spaces were found to be almost constant (25-30 pm) irrespective o f fish

size, whereas gill raker length (O R L ) was found to increase with increasing 0th

standard length (SL). The relationship between gill raker length and fish standard

length is shown in Pig. 3.4 and by the equation:

O RL • -2098.6 1850.5 log.SL; r - 0.98

72

F ig . 3 .4 T h « r e la t i o n s h ip between g i l l raker length and f i s h s tandard l e n g t h o f s i l v e r carp.

73

Suprabranchiai organ: This is a specialized organ, which appears to hang from the

roof o f the pharyngeal surface, and is attached to the base o f the gill arches. Each gill

arch and its canal enters into a hollow tube lined with epithelial tissue beating lots o f

mucus cells, and continues into the palatal tissue block to form the hollow spiml

suprabtanchial organ (Plate 6a, 6b).

3.3,5 Light and scanning electron microscopy o f the oro-pharyngeal wall

Tissue layers

In general, the oro-buccal wall is covered by a stratifled epithelium (25-90 pm thick).

The underlying cotuiective tissue stroma consists of:

i) stratum compactum, a compact layer o f collagen fibers, 3-4 pm thick;

ii) a less dense fibrous layer, 8-20 pm thick, with inteiposed nucleated fibroblasts;

ill) a loose areolar connective tissue layer with blood vessels and nerves.

Palatal organ

The palatal organ has a covering o f mucus epithelium (Plate 7). Taste buds lie along

the flat top o f the papillae. Mucus cells are dominant along the margins o f adjacent

75

papillae. The underlying flbret form mufcular cores for the papillae and »uppoiting

platfomu for the taste buds. The thick muscular tissue backing the p»pill»i- is

composed o f roughly longitudinally and transvenely-otiented striated fibeis.

3.3.6 Specializations o f the oro-pharyngeal epithelium

The stratified oro-phaiyngeal epithelium contains a number o f specialized cell types

including homy cells, mucus cells, club cells, chloride cells, sensory cells and round

cells typical o f fuh epideimis (Whitear, 1971; Sibbing and Uribe, 1983). Their

abundance varies locally and they serve a multitude o f functions. The surface o f the

common epithelial cells is commonly intricately sculptured by microridges (Hawkes,

1974; Hughes, 1979; Ono, 1980; Sibbing and Uribe, 1983). In all the carps studied the

microridges had a similar fingeiprint-type pattern (Plate 8a, 8b and 8c) with minor

variations.

In live fuhes the whole epithelial surface is covered by a slimy, fibrous layer,

approximately 1 pm thick, which is secreted from the underlying epithelial cells as

demonstrated by Whitear (1970) in Phoxtnus and a number o f other teleost fishes. A

similar external coaling appears to cover the aperture o f the taste buds o f the bucco

pharyngeal region and is probably composed o f glycoproteins. The mucus layer o f the

bucco-pharyngeal surface was lost during preparation o f histological sections and

during preparation o f the SEM sample by washing specimens with buffered srUine.

79

Mucus cells

Mucus cells, usually referred to as goblet cells, are the most common unicellular

glands o f the fish epidermis (Henrikson and Maloluy, 1968). According to differences

in size and shape o f mucus cells, it is possible to make a distinction between large

clavate mucus cells and small goblet mucus cells.

The orobuccal surface o f rohu, catla and silver carps consists largely o f small goblet

cells, which fHxxhice small quantities o f mucus, mainly sialomuciires. Few mucus cells

were found on the lips and the anterkM* pan o f mouth in the carps studied. In the

pharyngeal roof both the small goblet and large clavaie cells were pcesenl. goblet cells

containing sialomucines in the anterior, widest area o f the organ. In the

posterity region numerous large sulfomucine-containing clavate cells are |Mesmi. The

distribution pattern o f mucus cells was observed to be the same in all carps examirted.

In the gill raker region o f rohu arKi catla some large clavate cells are fweseiM. Silver

carp, however, have no clavaie mucus cells present in the gill-raker region.

Taste buds

Taste buds are pear-shaped sensory orgaru sometimes extending from the basal

membrane to the free surface o f the epithelium. Light and dark sensory cells run

80

through their length and taper towardi the ftee apical end into a pore (Plate 9a). The

light- coloured receptor cells bear one or two long apical processes, the dark cells

having a large number o f small microvilli. Only recently (Reutter, 1992) has it been

repotted that the dark cells are also sensory in nature instead o f merely providing a

supporting role as had been previously assumed (H ints, 1966).

Taste buds in the carps studied differed somewhat in size (Table- 3.1) although the size

differences were not significant (P > 0.05). The exposed receptor area o f the taste buds

measures 5.5- 12.5 pm in diameter.

Rohu were observed to bear large numbers o f taste buds on their lips, compared with

catla which had few taste buds on the lip surface, and silver carp which had no taste

buds apparent on their lips. The density o f taste buds over the oro-pharyngeal surface

varied, highest densities being on the palatal organ and gill rakers o f all species

studied. Among the carps, however, silver carp had highest densities (530Anm’ ) o f taste

buds on its palatal organ, followed by rohu (320Mim*) and catla (270Anm*). Catla and

silver carp were also observed to bear two rows o f taste buds on the pharyngeal surface

o f each gill raker (Plate 5b and Sc), whereas rohu bore taste buds only along the free

top edge o f the gill rakers (Plate Sa).

T a b i« 3.1 Com parativ« data on ta s ta bud dans ity (No ./bur ) in d l f fa r a n t o ro—pharyngaal ragiona and ta s ta bud s isa (^m) t S.D. o f rohu, o s t ia and s i l v a r carp o f tha sama s isa (90 mm)

Rohu ¿ a t la ------ S ilv a r carp

L ip* Î Ï Î 40 0

O ro-buccal r o o f 40 70 120

Oro-buccal f l o o r 40 110 120

A n te r io r pharyngeal r o o f 250 180 370

P o s ta r io pharyngaal ro o f 130 90 180

Pharyngaal f l o o r 110 150 320

Tasta bud s is a (axposad racap to r d iamatar) 9.1 ± 2 .0 8.8 ± 1.7 8.2 ± 1.7

82

3.4 Discussion

This study provides a description o f the surface stnictun and histological infonnalion

on the bucco-phaiyngeal region o f three o f the most impoitant filter feeding carps,

rohu, catia and silver carp, during the fly and fingerling stages. The distribution pattern

o f mucus cells and taste buds provides some insight into the feeding and food selection

mechanisms.

FUter feeding fish engulf food particles together with water and then collect it by

sieving through the gill rakers. The roles o f lips, the oro-pharyngeal surfaces, gill

rakers and opereula in the feeding o f fish have been well documented (Sinha and

Moitra, 1975; Sibbing, 1982; Sibbing and Uribe, 1983; Northcott and Beveridge, 1988;

Sanderson et al., 1991; Agrawal and Miltal, 1991 and 1992)

The lips o f rohu are densely packed with taste buds whereas catia and silver caip were

observed to have few taste buds on their lips. Sinha and Moitra (1975) stated that the

taste buds on the lips o f tohu helped in the search for suitable food from the water

column as well as from surfaces. Taste buds on the lips may help the catia and silver

carp to delect food particles in the water column. Agrawal and Mittal (1991) also

suggest that the taste buds on the lips o f calla help in searching for food particles

during feeding.

83

Oill n k en were obeerved to have conical papillae with laate budi on their phaiyngeal

surface. The appearance o f papillae with taste buds on the gill rakers o f fish has been

described by various authors, itKluding Iwai (1964); Reutter ec al. (1974); Hossler and

Merchant (1983); Sibbing and Uribe (198S); Sibbing (1986); Notthcott and Beveridge

(1988). Fish may use the conical papillae on the gill rakers to reduce inter-raker space

by interlocking in order to retain food particles, and utilize the uute buds in selecting

food from non-food items.

Most studies o f filter feeding mechanisms in fish have assumed a method o f

mechanical sieving by the gill rakers in which the gill taker apparatus functions merely

as a passive sieve. Measurement o f gill raker length and inter-gill raker space in

determining the efficiency o f filtration has been performed by King and MacLeod

(1976), Durbin (1979), Spatatu et al. (1983). Wright et al. (1983). Oibson (1988),

Smith (1989), MacNeiU and Brandt (1990), Berg et al. (1992).

OUl raker length and inter-raker space among the carps studied indicate that they are

suited for filter feeding. The ratio o f inter-iaker distances in rohu, catia and silver carp

is I : 1.37 : 0.32 respectively, suggesting that calla have the largest mesh size (lowest

retention ability), silver carp the smallest mesh size (highest retention ability) arxl that

rohu ate intermediate. In passive sieving terms silver carp would thus appear to be a

more efficient filter feeder than either rohu or carls. In the natural environment with

smaller food particles silver carp may be able to collect the bulk o f their food by

84

pauive meani. Adamek and Spinier (1984) found dial ailver carp are able to feed on

paiticlee leai than 5 pm in tize. By comraat, the g ill raker spacing! in rohu and catla

suggest that they are unable to feed on small particles i f passive mechanical sieving

is the only means o f filtration.

The capiKity o f a filter, in terms o f volume flltered per unit time, is an important factor

in determining the amount o f food particles that can be retained per unit time. The

capacity o f a filter is determined by the amount o f wiuer prused through the filter ruid

the area o f the filter through which water flows (the filtering area). Using the modified

version o f the formula given by Oibaon (1988) Fa • (L x 1) x 2 (see idxive) in which

the size o f the gill raker elements and inter-gill taker spiKing are both considered in

estimuing the total filtering area, it was found thru the ratio among the three species

was I : 2.78 : 3 for rohu : crula : silver carp. It suggests that silver carp have a

considerably larger filtering area than the other species. This point w ill be considered

further in Oiapter VI.

The involvetneni o f the pharyngeal roof o f fish in food selection has been reported by

Sibbing and Uribe (1983), Sibbing (1987), Hlohowakyj et al. (1989) and Sanderson et

al. (1991). Prom this study it is clear that rohu, catla and silver carp have Urge

numbers o f taste buds (180-S70Anm’ ) in the pharyngeal roof area which may help them

to detect and select food particles. In live carps the pharyngeal roof is closely fitted to

the pharyngeal floor and both surfaces are densely packed with taste buds which

8S

probably help the fuh assess the shape and size o f the food paitkle as well as

determine its olfactcxy fwopeities. In another experiment it was found that silver carp

are ci4Mible o f distinguishing toxic from non-toxic algae in suspension based on cell

surface properties (see chapter IV ). McOlone (1978) rq>Mted that the ¡Mdatal <»gan o f

goldfish responds to tactile stimuli at all stages o f denervation, with local bulging,

possibly caused by proprioceptive reflex loops. Konishi and Zotterman (1963) found

some glossopharyngeal nerve flbers in the palatal organ o f the carp reacting exclusively

to mechanical stimulation. Sibbing (1987) staled that common carp can isolate and

select food particles from non-food particles with the help o f taste buds on the

pharyngeal surface.

AriKMig the carps studied silver carp are unique in possessing a suprabraiKhial organ

on the roof o f the pharyngeal cavity. Wilamovski (1972) stated that the su(MratMaiKhial

organ in silver carp w<m1cs as a simple pump. It draws water into itself by expansion

and accumulates food masses towards the back o f the organ. It then contracts and

pumps out the water, washing accumulated food masses th rou^ the gill raker mesh

to the floor o f the pharynx and the opening o f the gullet. This study appears to confirm

that the suprabraiKhial organ probably works as a pump and in addition, may help in

collection o f small food particles by entrapment by mucus cells.

TTie chemical properties o f mucus were reviewed by Hunt (1970).Two main

componenu are distinct, glycoproteiiu and mucopolysaccharides. Mucous composed

86

mainly o f glycoprotein« ii refeired to a« tialomucine, while mucoiM confuting

principally o f mucopolysaccharides is known as sulfomucine.

Mucus performs a multitude o f roles in a wide spectrum o f biological systems

(Whitear. 1970; Mittal and Baneijee. 1980; Cook and Shirbate. 1983; Sibbing and

Uribe 1983; Northcott and Beveridge 1988), including ( i ) the formation o f a

mechanical barrier to foreign bodies artd pathogens (it has no antibiotic effect in carp

(Hattingh and Van Warmelo, 1973)); ( li ) the formation o f a chemical barrier which

supports osmoregulation and prevenu a flux o f material over sleep chemical or

electrical gradients; (Ui) the reduction o f friction; (iv ) the provision o f a mechanical

buffer which protecu the epithelium from damage and abrasion; (v ) aiding o f

precipitation o f suspended mud by entrapment and cleaning o f the epithelium (often

by cilia); (v i) slicking together particulate food; (v il) prevention o f desiccation by

binding o f water, (v iii) the enhancement o f adhesion in sluggish locomotion; (ix )

serving a mearu o f communication and navigation (e.g. in molluscs); (x ) the feeding

and attachment o f young and (x i) formation o f cocoons and other envelopes for

temporary shelter.

The oro-buccal wall produces mainly tialomucinea. This type o f mucus la common in

the respiratory and alimentary tract o f vertebrates, probably serving principally as

protection Mid lubrication (Hunt, 1970). Sialotnucines may thus well aid in orobuccal

water tnnsport, especially M high suction velocities, when the energy costs increase

87

exponentially.

Obtervations on the origin, development and occurrence o f mucus cells in the bucco

pharyngeal region o f Labeo rohita, Catla catla and tome other fishes have been made

(Saibahi. 1939; Moitra and Sinha. 1972; Sinha and Moitra, 1973, 1975; Sinha, 1973).

Sibbing and Uribe (1985) found that mucus cells producing sialomucinet occurred in

the anterior part o f the oro-pharynx o f the carp and that the tulfomucinet only

appeared in the posteriez part o f the pharynx. Noithcott and Beveridge (1988) also

found two types o f mucus cells in the gill rakers and bucco-pharyngeal region o f O.

niloticus

In this study sulfomucines were found in the posterior pharynx o f all carps studied and

in the g ill raker epithelia o f rohu and catla. This high viscosity mucus may help in

trapping and aggregation o f small food paiticles into boluses and may also assist in the

transportation o f food particles.

The low-viscosity sialomucines in the orobuccal cavity aiKl anterior part o f the pharynx

may serve as lubricants in reducing friction due to water currents during feeding and

protecting the epithelium from damage.

No work has yet been carried out to ascertain the physical properties and secretion

kinetics o f oro-pharyngeal mucus ki fish. It is expected that the secretion o f mucus is

88

somehow regulated in accordance with the feeding activities o f the fish.

MechaiK>- and chemcxeceptcMS are required to detect the qualities o f the ingested food

material during subsequent food processing. M w eover fish also need to be able to

constantly monitor the quality and velocity o f water during respiration and feeding.

Reutter et al., (1974) distinguished three types o f taste buds in the headgut o f the

sword-tail {Xiphophorus helleri Heckel) based on their position with respect to the

surrounding epithelium. A c c ^ in g Reutter's theory, taste buds protruding most (Type

I), which would be most effective in mechanoreception, occur in the gill rakers o f

rohu, catla and silver carp, in the lips o f rohu and in the buccopharyngeal regions o f

all species (Plate 9a). Less protruding taste buds (Type II) line the epithelial surface

(Plate 9b ). Type III taste buds never rise above the normal level o f the epithelium

(Plate 8 c ). H ie other type which was found in the oro-buccal wall in silver carp (Plate

9c), is similar in structure to those found on the inner surface o f the carp operculum

and w hich was suggested as serving a chemoreceptor function (Sibbing and Uribe,

1985).

During filter feeding fish may need information about size, deiuity and olfactory

properties o f food particles. Experiments in Chapter IV indicate that the density and

size o f algae and the toxic and non-toxic properties o f the algae influence the fish's

decision to filter feed. It is proposed that the high density o f taste buds on the gill

92

CHAPTER IV

Filter-feeding o f carps on planktonic algae

4.1. Introduction

The Indian and Chinese caip rohu, Labeo rohila; catla, Caila catia and silver caip,

HypophtHalmichthys molitrix are principally niter feeding planktivores (Khan and

Siddiqui, 1973; Jhingian and PuUin, 198S; Pillay, 1990). There have been increasing

movemenu o f Chinese and Indian major carps for aqurKultuie purposes artd in mruiy

countries, particularly in Asia, the two groups o f carp rue stocked in combinruion in

fish ponds and, bectuise o f escapes, occur together in the wild.

As sirued in the Introduction, filter-feeding Chinese carps appear to out-perform the

Indian m^jor crups in terms o f growth and survival, when stocked together in

polycuhuie. Much work has been done on the food and feeding habits o f carps (e,g.

Khrui tuid Siddiqui, 1973; la in and MusUfa, 1977; Spataru and Oophen, 1983; Dewan

el at., 1991). Although all have been qualitative in nature, they have clearly

demonstrated significruM dietruy overlap among many species, particularly between

silver carp and rohu, bighead carp and crula. Quaruitative studies o f algrU ingestion are

needed in order to establish the basis o f competition and these can only be cruried out

93

in the labofatory.

There have been numeioui laboratoiy based quantitative studies o f filter feeding,

although mainly on zooplankton (McMahon and Rigler, 1963; Frost, 1972; Crowley,

1973; Frost, 1975; Reeve and Walter, 1977; Conover and Mayzaud, 1984) and anuían

larvae (tadpoles) (Seale and Wassersug, 1979; Seale and Beckvar, 1980; Seale el al.,

1982). A little work on quantitative aspects o f filter feeding has been carried out on

some fishes (M oriaity and Moriarty, 1973; Duibin and Duibin, 1975; Dienner et n/.,

1982; Friedland, 1984; McDonald, 1985; Dienner tt at., 1987; Northcolt et at., 1991)

but none has been done on carps.

The lesulu o f quantitative filter-feeding studies have been used to create functional

response models. There are several curves (rectilinear, Ivlev, Michaells-Menten) used

for simplified representation o f their experimental data.

The basis for mathematical models o f filter-feeding in fishes is largely derived from

zoopiMikton studies by Frost (1972). The ingestion rate equation waa ftirther developed

and tested by Conover and Mayzaud (1984), Peters (1984), and Marin et at. (1986) and

most recently has been used by Northcott et at. (1991) for work w ith the tilapia O.

nttoHcus. The advantage o f this form o f equation is that it is based on actual net

chMigea in algal concentrations tai the experimental feeding chamber.

94

There are leveral w ayi to expreu the ingeslion-concentiation curve. Froat (1972) lued

cell number whereas C row ley (1973) used diy weight o f food panicles in the ingestion-

concentration curve. Lehman (1976) proposed an energy optimization theory and

according to his hypothesis, ingestion is related to the energy content o f the suspended

food panicles. Seale and Beckvar (1980) state that tadpoles regulate their food intake

on the basis o f volume o r biomass ingested. Northcott et al. (1991) also reponed that

ingestion by the tilapia O. niloticus is regulated by bio-volume o f the food panicles.

Filter-feeding fishes feed on blue-green algae (Cyanobacteria) along with other algae

and zooplankton (Moriarty, 1973: Kajak, et al., 1977; Spataru and Z o m , 1978; Bowen,

1982). Silver carp are capable o f ingesting small blue-green algal species such as

Microcystis aeruginosa (K^jak, et al., 197S). Many o f the blue-green algae which

proliferate in eutrophic fresh- and brackish wateis, however, produce powerful hepato-

and neurotoxins which can cause mortalities when ingested by animals (Skulbeig, et

al., 1984; Codd, et al., 1989). At present it is not known whether phytoplankton

feeding fishes are capable o f detecting or ingesting toxic cyanobacteria and i f s o , how

they deal with ingested toxins.

It was thus decided to cany out a quantitative filter-feeding study on the fry o f L.

rohlta, C. catla, and H. molitrix. The ingestion rates at various concentrations o f

Pediastrum boryanum and Microcystis aeruginosa were measured in an attempt to

produce functional response curves. In a separate series o f trials sn investigation was

95

carried out to deteimine whether phytoplankton-feeding lilver carp could detect and

ingeit toxk Cyanobacteria.


4.2.1 Introduction

Tw o seto o f experimenu were carried out. The fin t set was designed to evaluate the

effects o f particle size and deiuity on ingestion rates o f the three species o f carps. A

second set o f experinrenta was designed to investigate whether silver carp are able to

delect artd ingest toxic M. aeruginosa.

4.2.1 Algae

The species used in this experiirrent were the green alga Fediastrum boryanum, and

toxic and non toxic strains o f the Cyanobacterium Microcystis aeruginosa. These algae

occur luUutally in tropical freshwater environments and are typical o f those prevailing

in carp culture systems. Culture methods and maintenance o f algae are as described in

Chapter- n. The toxic strain o f M . aeruginosa was obtained from the Department o f

Biology, University o f Durrdee.

96

4.2.2 Fish

AU three fish species, rohu (L. rohiui). c « t a (C . caOa) and sUver caip (H. molitrix)

were used in the quantitative investigation o f algal ingestion. Silver caip only were

used to study whether fish can detect or ingest toxic strains o f M. aeroginosa. Fish

were reared in the Tropical Aquarium. Institute o f Aquaculture (see Chapter III). Two

size o f fish. 1.4 and 7.7 g o f rohu. 1.7 and 8.6 g o f catla. and 1.1 and 7.1 g of

sUver carp were used for the quantitative feeding trial. Silver caip o f S g size were

used in the investigation o f toxic algal ingestion.

4.2J Experimental System

The experimental system consisted o f 16 tanks o f 30 1 capacity incoiporated into a

recirculation system and static water (I.e. no water exchange takes place) system

where the fUter-feeding triata were performed (Fig.4.1). Each tank in the recirculation

system contained two static 2 I feeding chambers. The temperature o f the feeding

chambers was maintained at 26 ± 1*C. A ir was suppUed via separate air supply lubes

to each feeding chamber and the supply regulated by plastic screw clamps. Each

feeding cluanber was fitted with a perspex lid with a small hole In the centre to

facúltate air exchange. An overhead fluorescent lube was fitted to ensure an adequate

light (120 Lux) supply. Each lank was suirounded by black polythene sheet to protect

» 7

98

the fUh from undue exlemal visual distuif>ance.

4.2.4 Experimental Protocol

Ingestion trials:

In all trial« flltered tap water was used. Stocking deniitie« were 6.73 ± 1.74 g fuh. t '

except for the trial« involving toxic algae. The trial« were «ingle end-point experiment«,

f .«. the algal density in the feeding chambers was recorded at time zero and then again

at the end o f the feeding period. A ll feeding trials were o f 4 h duration. Five replicates

for small fish (1.1-1.7 g). three replicates for large fish (7.1 - 8.6 g ) and three controls

were used for each algal concentration (S concentrations, 9 - 193 x 10* pm’ , ml '). A ll

trials were started at 10.00 hours.

Selected fish were moved to the static system and starved for 12-24 hours. Two hours

before the filter-feeding trial fish were transferred to their respective feeding chambers.

Tw o water samples o f 500 pm were taken from each feeding chamber, mixed together

with 20 ml ISOTON II and analyzed by Coulter Muhisizer tai order to deteimine the

background particle count (i.e. sample blank).

Immediately prior to each trial the algal density o f the harvest culture was estimated

by Coulter Muhlsizer. In order to obtain the desired algal density In the feeding

chm ber tha quM ities o f culture that should be added were determined and diluted

99

with filtered tap water prior to adding to the feeding chamber. Actual initial

concentratioiw in the experimental chamberí were determined from lamplet taken S

min after addition o f the algae in order to allow for thorough mixing. Two tamplea

(initial), each cotuiiting o f two SOO pi lamplei were taken from each chamber and

added to 20 ml ISOTON O for counting and another one 2 ml lample waa taken

rarxlondy from each treatment for lize diitribution pattern o f algal colonies. The fish

were allowed to feed on the algae for 4 h in all trials. Final samples were taken at the

end o f this period in the same way as the irritial srunples. On all occasiotu one fish

from each replicrUe was sacrificed and the anterior portion o f the gut dissected out in

order to verify whether the fish had been filter-feeding. The volume o f water and the

totiU weight o f the fishes in each feeding chamber were recorded.

Trials involving toxic algae:

Individual silver carp (S g ) were used in the same experimental system. Toxic and non

toxic strairu o f M. aeruginosa were used as separate treatments. Seven or eight

replicates for each Irealntent and three replicates for control were used in the

experimenu. Sampling waa done as for the previous trials (tee above).

Individual fish o f the same size were slocked in snuUI static flow tanks as detciibed

above, except that no black polytherw waa used around the lank sides in order to

facilitate observation o f opercular beat rates. Three treatmenu were used (control (fish.

100

no Cyanobacteria), fUh ■¥ toxic Cyanobacteria and flab + non-toxic Cyanobacteria) and

three replicatei for each treatment ettabliihed. Fiih were starved for 12 h prior to

transfer to the experimental system, they were allowed 2 h to acclimate. Cyanobacteria

were then introduced to all tanks other than the controls. Initial Cyanobacterial

concentrations were approximately 10* cell, ml '. Observations were made immediately

after the introduction o f Cyanobacteria (0 h) and after 4 h. Observatioru were made for

period o f 1 min using a stopwatch, three fish being observed for each treatment at each

time interval, and fish being selected at random. Care was taken not to disturb fish.

Samples (SOO m l) o f water ftom experimental tanks and from controls were passed

through C|i Sep-Pak cartridges (Water Associates) to concentrate any microcystin-LR

which may have been present. Cartridge eluates were analysed by reverse-phase HPLC

(Water Associates) using purified microcystin-LR as standard at the Department o f

Biological Sciences, University o f Duitdee.

4.2J Sample Analysis

A ll samples were analyzed in ftesh condition immediately after sampling. The Couher

Multisizer was used to determine algal density. At least three counts were made for

each sample. A ll counts were corrected by deducting the blank count value. Pbr the

determination o f algal cell size, a high powered (x 400) light microscope with eyepiece

graticule was used (see Chapter II)

101

4.2.6 Detcrminatkm o f Ingestion rates

Ingestion rates were calculated in terms o f rUgal mean bio-volume rather than the cell

or colony number so that changes in cell or colony sire during the experunent could

be taken into account.

Ingestion nUes were computed using the equation proposed by Cormver A Mayzaud

(1984) and Peters (1984) ;

I > V/W ((CA - C.B,)/ At)

where I • ingestion rate (pm ’g 'h ''): Q> ■■ ■»><■•1 concentration in experimental

chamber (colorties or cells ml'*); C, ■ final algal concentration in experimental chamber

( colonies or cells ml '); B„ - mean algal colony or cell biovolume at start o f feeding

period (pm*); B,* mean rUgal colony or cell biovolume at end o f feeding period (pm*);

At w experimentrU duration (h); V ■ volume o f algal suspension (ml); and W « wot

weight o f fish in experimental container (g).

No correction was made for algal reproduction due to the short duration o f the

experiments (4 h).

Mean algal bio-vohuna during the feeding period for each replicate was calculated

102

uaing the equation from Noithcott et at. (1991):

B. . (C,B, • C ,B ^/ In (C ,B /C A )

Where B, • mean algal biovolume concentration (jim’m l'').

The dependence o f ingettion rate on algal concentration was described by the sigmoid

model used by Kipiboe et al (1983) for zooplaitkton ingestion dau:

I - 1 « . e * *

where 1 ^ ■ maximum ingestion rate; C ■ algal concentration; b ■ a constant. The

model was fitted to experimental daU by linear regression after logarithmic

transfoimation.

Results

4 J .I A lgal Ingestion trials

Five different trealmenu, each consisting o f a different initial concentration o f algae

(9 -193 X l ( f pm’ , m r'), were used for each species o f fish. Fix’ all feeding trial

treatments involving silver carp and rohu a Student’ s Meat on initial versus final algal

103

biovolume concenliatioiu showed a significant decrease (Table-4.1) indicating that

grazing on both M. aeruginosa and P. boryanum had taken place in the experimental

chambers. The ingestion o f algae was verified by the presence o f algae in the stomachs

at end o f the feeding period. In the trials involving catla and Af. aeruginosa no

significani decrease (P > O.OS) at the end o f the feeding trials was apparent, indicating

that no grazing had taken place.

Throughout all feeding trials involving A f. aeruginosa, colony bio-volume was constant

and there was no evidence o f size selection by the fish. Although both A f. aeruginosa

and P. boryanum were harvested at the end o f the log phase o f the culture, it was

found that there was some size variation among the P. boryanum colonies, although

the size distribution panem was similar among replicates. From the data in Tables 4.2,

4.3 and 4.4 it is apparent that there were significant differences in P. boryanum mean

colony diameter among certain treatments. The results also indicate significant

decreasea in mean colony size in some treatments during the feeding trials. For each

species o f fish and size group, ingestion rale I was plotted against the corresponding

algal biovolume concentration B, for each species o f algae (Fig. 4.2 - 4.11). Ingestion

rate versus algal food concentration curves were produced on the basis o f the eqiwtion

I • I .e~*^. The ingestion rate o f rohu (7.7 g ) feeding on Af. aeruginosa and catla (1.7

g ) feeding on P. boryanum showed highest correlation coefficients (r ̂• 0.97 and 0.98

respectively).

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T a b l« 4 .2 R «su lta o f Studant'a t - ta a t c a r r ie d out on P . borynum co lo n y dlamatar b o fo r « and a f t a r g raa ln g . (a ) 1.4 q L. ro h its , (b) 7 .7 g L. ro h ita .

Traatm ant maan b iovolum a Be

Maan colony d la a a ta r ± SD (^m) 1. S ta r t (n)

2. End <n)t P

(a)9.15 X 10* 1. 63.76 ± 9.45 (110) 1.42 >0.1

2.. 61.64 t 6.36 (110)4.62 X 10* 1. 63.06 ± 10.47 (110) 1.01 >0.1

2. 61.65 ± 9.03 (110)9.62 X 10* 1. 62.38 ± 9.35 (115) 1.02 >0.1

2. 61.02 ± 8.14 (112)13.67 X 10’ 1 . 64.18 ± 8.68 (115) 2.01 <0.05

2. 61.76 t 7.91 (115)19.48 X 10’ 1. 61.77 ± 10.69 (110) 2.47 <0.02

2 . 56.52 t 6.62 (110)

(b)

9.05 X 10* 1. 61.27 ± 9.26 (120) 1.14 >0.12 . 59.38 ± 6.56 (120)

4.61 X 10’ 1 . 53.08 ± 10.39 (110 )* 1.24 >0.12. 51.14 ± 9.63 (110)

9.67 X 10’ 1. 64.02 ± 9.59 (140) 2.01 <0.052. 61.76 ± 6.34 (140)

13.77 X 10’ 1. 52.43 ± 10.03 (110 )* 1.01 >0.12. 51.01 ± 9.27 (110)

19.41 X 10’ 1. 64.06 t 9.46 (115) 2.08 <0.05

1----F5CT-X5m5” 7m s2 . 61.41 ±

TO—6.37 (115)

k A A C T u — S E T

add ju a tad by in craaa in g tha co lonay numbar.

T «b l« 4.3 R e s u lts Of S tudont'a ca rr io d out on P. borynum colony d ia m o to r bafora and a f t a r g raa ln g . <a) 1.7 g C, c s tJ s , <b)6.6 9 C. c a t ia

Traatmant maan biovoluma Be

Maan co lony diam atar (pm) ± SD 1. S tart<n )

2. End <n>t P

U )9.14 X 10* 1. 63.98 ± 9.56 (124) 1.31 >0.05

2. 62.04 ± 9.23 (124)4.62 X 10' 1. 64.03 ± 9.21 (110) 1.69 >0.05

2. 62.87 ± 8.63 (110)9.93 x lO ' 1. 63.02 ± 9.55 (120) 1.38 >0.05

2. 61.14 ± 9.02 (120)13.96 X 10' 1. 63.41 ± 9.33 (121) 2.06 <0.05

2. 60.39 ± 8.03 (121)19.49 X 10' 1. 64.19 t 10.9 (115) 2.49 <0.05

2. 60.76 t 9.28 (115)(b)9.06 xlO* 1. 63.92 t 9.74 (146) 1.26 >0.1

2. 62.52 ± 9.19 (146)9.73 X 10' 1. 56.99 ± 9.7 (140) 1.8 >0.05

2. 54.96 ± 9.13 (140)4.64 X 10' 1. 64.2 ± 10.11 (110) 2.67 <0.05

2. 61.72 ± 9.13 (110)13.79 X 10' 1. 62.27 ± 10.11 (112) 1.87 >0.05

2. 59.73 ± 10.02 (112)19.47 X 10' 1. 62.24 ± 9.65 (115)

2. 61.17 * 8.56 11115) 1.06 >0.1

T a b l« 4.4 Rasults o t S tudent's t - t « s t c a r r la d out on P . borynum colony diamatar b a fo ra and a f t a r g rax in g . (a ) 1.1 g H. m o li t r ix , <b) 7.1 g H. m o l i t r ix

Traatmant maan Maan colony diamatar ± SOblovoluma Be (um) 1. S ta rt <n) t P

2. Bnd <n)

(a)9.18 X 10* 1 . 63.86 ± 9.41 (110) 1.43 >0.1

2. 61.47 t 8.59 (110)4.61 X 10* 1. 61.28 t 8.71 (115) 1.03 >0.1

2. 59.62 ± 7.53 (115)9.75 X 10’ 1. 62.53 ± 9.26 (120) 1.17 >0,1

2. 60.81 ± 8.34 (120)13.77 X 10’ 1. 64.03 t 8.64 (110) 1.43 >0.1

2 . 62.01 t 8.01 (110)19.46 X 10’ 1. 62.52 ± 10.26 (115) 1.12 >0.1

2 . 60.17 ± 9.06 (115)

<b)9.02 X 10* 1. 62.36 ± 8.34 (110) 1.01 >0.1

2. 61.03 ± 7.03 (110)4.61 X 10’ 1. 57.26 ± 9.48 (110) 1.42 >0.1

2. 55.81 t 8.65 (110)9.74 X 10’ 1. 63.21 ± 9.06 (110) 1.06 >0.1

2. 61.08 ± 8.45 (110)13.79 X 10’ 1. 61.76 ± 8.64 (100) 1.02 >0.1

2. 59.42 t 8.03 (100)19.44 X 10’ 1. 63.04 t 9.26 (100) 2.03 <0.05

2. 59.23 t 8.04 (100)

108

Fig. 4.2 Ingestion rmte ( I ) as a function o f food concentration (O for L . rohita (1.4 g ) feeding on M. aeruginosa. Fitted curve is I > 2. lE+08.e ’ (r* ■ 0.88) .

109

Fig. 4.3 Ingeslion nUe (I) u a function o f food conceninuion (C ) for L. rohita (1.4 g ) feediiw on P. boryanum. Fined curve i i I - 2.1E+08.e * ‘***’ * , (i^ ■ 0.88).

110

Fig. 4.4 Ingestion nUe ( I ) as t function o f food concentration (C ) for L. rohita (7.7 g ) feed ing on M. aeruginosa. Fitted curve ii I a l.lE+08.e ' * * * " * . (r* ■ 0.97).

in

Fig. 4.3 Ingestion m e ( I ) as a function o f food concentmion (C ) for L. rohiia (7.7 g) o "boryanum. Fined curve i i I “ 1.9E+08.e '^ (H ■ 0.901.

112

Algal oonœntratlcn (înW '')

Fig. 4.6 Ingestion rate ( I ) as a function o f food concentration (C ) for C. catla (1.7 g ) feeding on P. boryanum. Fitted curve is I ~ 1.8E+08.e ' ( i * i 0.981.

113

Fig. 4.7 Ingestion fate ( I ) at a function o f food concentration (C ) for C. caila (8.6 g ) feeding on F. boryanum. Fitted curve is I • 2.0E-f08.e ' (r* ■0.88).

114

Fig. 4.8 Ingeition m e ( ! ) ■ ■ • function o f food concentmion (C ) for H. moUtrix (1.1 g) feeding on M. aeruginosa. Fined curve i i I - 2.1E+08.e ’ ‘ •**” * , (H - 0.88) .

115

Fig. 4.9 Ingestion rale (I ) as a function food concentration (C ) for H. molltrtx (1.1 g ) feeding on P. boryanum. Fitted curve is I - 2.7E+08.e ’ (f* m0.89).

116

Fig. 4.10 Ingealkm rale ( I ) ■< a function o f food conceninuion (C ) for H. molitrix (7.1 g ) feeding on M. aeruginosa. Fitted curve i i I • 2.1E+08.e ’ '* ^ * ', (r* ■ 0. 88) .

117

Fig. 4.11 Ingeition rale ( I ) as ■ function o f food concentration ( Q for H. molitrix (7.1 g ) feeding on P. boryanum. Fined curve is I > 2.1E.t08 e (r* _0.88) .

118

4.3.2 Toxic algal ingestion trials

Ingestion o f toxic and non-toxic Cyanobacteria:

In the trial in which silver carp were exposed to both toxic and non-toxic strains o f

the planktonic Cyanobacterium M. aeruginosa the results clearly show that grazing on

toxic M . aeruginosa was significatMly depressed in comparison to grazing rates on the

non toxic strain (A N O V A , d.f. 1. 23, F . 43.6. p < 0.03) (Fig.4.12). Indeed, there was

no signiTicant difference (Tukey M R Test. P < 0.03) in Cyanobacterial cell numbers

at the end o f the trial between the toxic treatment and the control consisting o f toxic

M. aeruginosa with no fish. Out content analysis showed that very few cells (<10*) o f

the toxic stain o f M. aeruginosa had been ingested, compared with fish exposed to the

non-toxic strain (>10’ ).

Behaviouriat response:

The resulu from timed measurements o f opercular beat rates at 0 h ate shown in Fig.

4.13. The behaviours o f Rah held In toxic Cyanobacteria did not differ significantly

from the controls in which fish were held in media without M. aeruginosa whereas

those o f fish held In non-toxic Cyanobacteria were significantly elevated (A N O V A d.f.

2» 26, F " 83.3, P < 0.03; Tiakey MR Test, P < 0.03). The srune statistically significant

reaulu were observed at 4 h.

121

Water analysis:

Analytis o f samples o f water from the experimental chambeis and from the controls

by H P IX : confirmed that no detectable levels o f microcystin (< 250 ng r ‘) were present

in the water.

4.4 Discussion

Functional response models have been used to describe and explain ingestion versus

food coiKentration relationships in many different types o f filter feeders, including fish

(Durbin and Durbin, 1975; Dienner el at., 1987; Northcott el at.. 1991), anuran larvae

(Seale and Wassersug, 1979; Seale and Beckvar 1980) and zooplankton (Frost, 1972;

Crow ley, 1973; Robertson and Frost, 1977; Kiotboe et a!. 1985). Whilst there remains

some disagreement about the exact nature o f the relationship - rectilinear, as suggested

by HoUing (1959, 1965, 1966) or curvilinear as proposed by Ivlev (1961) for fish -

Mullin et al. (1975) have found that for zooplankton at least the ingestion versus

corwentration relationship can be described by both curvilinear and rectilinear models;

in terms o f fit, they are sutistically indistinguishable. Indeed, they also showed that a

Michaelis-Menten type curve can be justifiably fitted to available data. Seale et al.

(1982) also suggested that the fundamental regulatory mechanisms such as satiation or

gut ftiUness are similar between invertebrate and tadpole suspension feeders. The

resulU o f this study appear to confirm this universality tai fundamental regulatory

mechanisms among filter feeders.

122

W hiU l the model applied here doee not leem to fit lome o f the daU sett particularly

weU, oveiaU it fits the dau adequately and other than examination o f residuab after

model fitting there are no appropriate methods for testuig goodness o f fit among

curvilinear data (Sokal and Rohlf, 1973). The ingestion versus food concentration

curves o f small rohu (1.4 g ) and small silver carp (1.1 g ) feeding on M. aeruginosa

(F ig . 4.2 and Fig. 4 .8 ) and small catia feeding on F. boryanum (Fig. 4.6) seem to reach

an asymptotic leve l which is typical for filter feeders and is compatible with a

curvilinear model where filtering surfaces become fully saturated at very high food

concentrations. Ingestion versus food concentration curves for small rohu and silver

carp feeding on P. boryanum (Fig. 4.3 and Fig. 4.9). however, appear not to reach an

aaymptolic level, suggesting that the concentration o f P. boryanum at which saturation

ingestion occurs had not been reached. Similar relationships were also observed for

large rohu and silver carp feeding on both M. aeruginosa and P. boryanum and large

catia feeding on P . boryanum. Durbin aitd Durbin (1975) found that their Atlantic

menhaden data fitted the linear phase o f the feeding curve. Ishii et al. (1985) found

that the ingestion rate o f Euphausia superba Dana mcreased luiearly with increasing

panicle concentration over the range o f food concentrations used. Northcott et al

(1991) also found a linear relationship for the ingestion data o f large O. nlloHcus

feeding on both Microcystis and Anabaena.

Ingestion rates fo r rohu. catia and silver carp feeding on P. boryanum wore higher than

for those feeding on M. aeruginosa (Table 4.5). suggesting that the fish were more

123

efTicient at feeding on larger algae. Picvioui smdiet found that ingestion rate increased

with particle size for Sarotherodon galilatus (Drenner t t oi., 1987) and O. ntioticus

(Noithcolt el al., 1991).

The ingestion rates o f silver carp feeding on both M. aeruginosa aitd F. boryanum

were higher than the ingestion rates o f rohu and catla feeding on the same alga at the

same concentrationa (Table 4.S). This may be because the filtering apparatus o f silver

carp is more efficient than that o f rohu and catla. Seale and Beckvar (1980) repotted

that the tadpole o f one anutan species, Xenopus laevts, had a maximum ingestion rate

significantly higher than the others. Xenopus tadpoles have an extensive area o f mucus

secretory ridges in the buccal region (Seale and Wassertug, 1979), suggesting a mote

efficient mucus entrapment process. Results from Chapter III show that the inter-gill

raker specings o f silver carp are less than those o f rohu and catla. These points are

discussed fiuther in Chapter VI.

In all trials rohu and silver carp were observed to ingest both F. boryanum and M.

aeruginosa. Catla however, were unable to ingest M. aeruginosa. This may be because

gill taker spacings in catla are greater than those o f corresponding sizes o f rohu and

silver carp and ate thus unable to retain M. aeruginosa. Alternatively, catla may reject

M. aeruginosa as food because it may not be digestible. Catla show the ability to

selectively feed on zooplankton tai the natural environment (Jafti and Mustafa, 1977;

JhingtMi Mid PuUin, 198Î; PUlay, 1990). Ingestion o f Af. aeruginosa by rohu and silver

T a b la 4.5 In gas tlo n ra ta fo r d l f tarant spaclas faad lnç on d l f fa r a n t a lgaa a t d lf fa r a n t concan tra tlon s , (a ) L. roh ita , (b) C. c a t ia , (c ) H. m o llt r ix .

A lg a l sp «c l«8 F ishaiza(g )

A lg a l concan- t r a t lo n <|iai*al~‘ ) xlO* ± SE

In ges tion r a t «xlO* ±

SE

M. «•ru g inosa 1.4 8.99 ± 0.08 10.33 ± 2.88P . horymnum 1.4 9.19 ± 0.04 63.93 ± 8.12M. «•ru g inosa 7.7 9.18 ± 0.01 6.61 ± 1.27P . Jboryanuin 7.7 9.13 ± 0.04 37.82 ± 6.10

M. «•ru g inosa 1.4 46.61 ± 0.12 35.43 t 6.38P . borysnum 1.4 45.77 ± 0.30 97.20 ± 8.76M. M T u g in o t* 7.7 46.52 ± 0.21 44.36 ± 13.81P . borysnum 1 a l 46.37 ± 0.10 89.09 t 7.59

M. smruginosa 1.4 95.99 ± 0.22 133.35 ± 6.40P . boryMnum 1.4 97.52 ± 0.36 174.47 ± 16.39M. a^ruginoss 7.7 97.13 ± 0.14 63.69 ± 14.65P . boryanum 7.7 97.79 ± 0.62 133.60 ± 10.98

M. aaruginoaa 1.4 137.45 ± 0.22 154.74 ± 10.31P . boryanum 1.4 137.72 ± 0.24 181.93 t 14.25M. aaruginoaa 7.7 137.49 ± 0.08 100.95 t 10.96P . boryanum 1 a l 137.93 ± 0.15 183.47 ± 8.65

M. aaruginoaa 1.4 193.05 ± 0.13 159.40 t 18.04P . boryanum 1.4 195.07 ± 0.33 273.16 ± 23.51M. aaruginoaa 1 a l 192.98 ± 0.05 199.14 ± 25.05P . boryanum 1 a l 194.18 ± 0.37 235.68 t 31.01

A lg a l spaclas F lsh • isa(g)

A lg a l concantra— t io n (^m’ml’ MslO* ± ES

In gastion ra ta (^ »*g ‘ ^h"*)sl0*± SE

P .P .

boryanumboryanum

1.78.6

9.10 ± 0.06 9.13 t 0.04

37.76 ± 3.34 28.30 t 4.33

P .P .

boryanumboryanum

1.78.6

45.98 t 024 46.26 ± 0.08

93.55 ± 18.58 84.22 ± 8.57

P .P .

boryanumboryanum

1.78.6

97.47 ± 0.65 97.09 t 0.13

167.47 t 14.36 139.34 t 12.29

P .P .

boryanumboryanum

1.78.6

138.70 ± 0,50 137.95 ± 0.06

198.61 ± 28.15 159.44 t 23.81

PaPa

boryanumboryanum

1.78.6

194.47 ± 0.20 194.76 ± 0.03

246.99 t 39.45 195.81 t 36.16

(c)A lg a l apacias Fish

aixa(g)

A lg a l concantra- t lo n (pm3ml*-l)xl06 t SE

Zngastion rata (pm 3g-lh~ l)x l06 ± SE

M. aaruginoaa 1.1 9.03 ± 0.03 22.67 t 5.18P. bpryanuM 1.1 9.08 ± 0.04 69.74 ± 9.98M. smruginosM 7.1 9.09 ± 0.02 16.25 ± 3.85P, boryanum 7.1 9.13 ± 0.06 60.94 t 13.92

M. amruginoaa 1.1 46.52 ± 0.13 76.83 t 19.78P. borymnum 1.1 46.28 ± 0.06 133.58 ± 10.80M. amruginosa 7.1 46.59 ± 0.13 67.78 t 20.59P. boryanum 7.1 46.18 ± 0.03 99.34 t 12.80

M. aaruginoaa 1.1 96.80 ± 0.21 141.12 t 20.94P. boryanum 1.1 96.94 ± 0.18 206.50 t 19.36N. aaruginoaa 7.1 96.95 ± 0.12 146.07 ± 17.55P. boryanum 7.1 97.18 ± 0.14 173.77 ± 22.72

N. aaruginoaa 1.1 137.56 ± 0.16 229.95 ± 23.68P. boryanum 1.1 137.90 ± 0.04 279.07 t 23.95N. aaruginoaa 7.1 137.46 ± 0.15 186.31 ± 22.12P. boryanum 7.1 137.94 ± 0.03 209.39 ± 16.51

M. aaruginoaa 1.1 192.93 ± 0.03 246.54 ± 51.03P. boryanum 1.1 195.01 ± 0.30 353.17 ± 29.54N. aaruginoaa 7.1 192.89 ± 0.01 216.89 ± 18.46P. boryanum 7.1 194.56 ± 0.08 298.54 ± 28.71

126

caip may be by mucus endapmenl as suggested for tUapia by several authors

(Oieenwood, 1953; Northcott and Beveridge. 1988; Beveridge el of., 1989).

Ingestion rates per g body weight o f fish per hour for smaller fish were observed to

be higher than for larger fish in all trials (Table 4.5) although ingestion rates per fish

were observed to be higher in larger than smaller fish. This may be because smaller

fuh need more food per g body weight than larger fish to sustain rapid growth. Energy

loss as heat through the body surface is higher in smaller fishes than among larger

fishes, in part explaining why smaller fishes require more food in per g body weight

terms (Steffens. 1989; Hepher. 1988). Kato el al (1982) observed that the fUtering rale

o f the copepod Euphausia superba Dana increased with body sixe, although filtering

rales per g body weight decreased with increasing body size.

The results also show a significant reduction in mean F. boryanum colony diameter

during the feeding period in some treatments (Table 4.2,4.3 and 4.4). Analysis o f size

frequency distributions indicate slight reductions in trials with higher initial colony

densities during feeding trials with rohu and catla. One explanation is that rohu and

catia selectively feed on F. boryanum colonies > 65 pm diameter. The fry and

fingerlings o f rohu and calla selectively feed on zooplankton and on tom e selected

phytophuikton in natural environment (Khan and Siddiqui, 1973; Jafri aisd Mustafa,

1977; Jhingnui and PuUin, 1985; PUtay, 1990). Size selection in fUter feeding is a

widely reported phenomenon (Porter, 1977; Ishii el al., 1985, Smith, 1989;

127

Hoogenboezem et o/., 1991).

Not only is there evidence o f size selection and species selection but also the abilities

anxmg filter feeding carps to distinguish between toxic and non>toxic straiiu o f M.

aeruginosa. Grazing by silver carp was almost con^>letely suppressed in the presence

o f the toxic stain o f M. aeruginosa. Cyanobacteria are exploited to only a limited

degree by zooplankton because o f problems o f manageability (cell/colony size),

nutritional quality and toxicity <de Bemardi and Oiussani, 1990; DeMott and Moxter.

1991). Concerning manageability, althou^ silver carp is capable o f entrapping and

ingesting particle as small as unattached luicteria (< 1 pm diameter) from the water

column (Chapter IV ), it is undoubtedly more efficient at feeding on larger, colonial

algal species than on small, unicellular organisms (Adamek and Spitler, 1984). Silver

carp, however, can and do ingest M. aeruginosa (Spataru and Oophen. 1985).

Compared with diatoms and other phytoplankton. Cyanobacteria have high levels o f

protein, in excess o f 50% on a dry weight basis (Cummins and Wuychek, 1971; de

M oor and Scott, 1985), although there is some evidence that M. aeruginosa is poorly

digested by the stomachless silver carp and that conversion efficiencies are low

(Hamada et ai., 1983; Bitterlich, 1985). The present resulu suggest that in the presence

o f toxic blooms o f M. aeruginosa, ingestion by silver carp, and hetice growth, are

suppressed.

Although irtcreased opercular beat rates, indicating feeding activity, were observed in

128

flsh expoud to the non-toxic itrain o f M. aeruginosa, general activity leveli o f animala

held in toxic Cyanobacteria were much lower than thoae o f the control or non-toxic

treatmenti. Similar reaponaea have been found in other atudiea o f flah expoaed to

planktonic food (Beveridge et at., 1988, 1991), aa well aa to toxic and non-toxic

iiritanta auch aa formalin and auapended aolida (Roaa et at., 198S). Preaent reaulta, in

which opercular beat ralea o f fiah expoaed to the toxic atrain did not differ aignificantly

from the control (fiah alone), ahow that fiah react to toxic M. aeruginosa in a different

manner from food and irritanu. The feeding mechaniam o f ailver carp haa been little

atudied. Ingeation o f the non-toxic attain o f M. aeruginosa in thia atudy may have been

by mucua entrapment. However , i f , aa haa been auggeated elaewhere (Greenwood,

I9S3; Noithcott and Beveridge, 1988), filter feeding by tilapiaa involvea entrapment

by mucua in the gill apparatua, a mechaniam that abnoat certainly cannot be rapidly

awitched o ff or on, then toxic cella muat alao be retained by the fiah. The implication

here ia that toxic cella can be detected and although peihapa trapped by the bucco

pharyngeal appatatut are rejected aa food itenu.

Analyaia o f water aamplea by HPLC confirmed that no detectable levela o f microcyatin

(<2S0 ng r ' ) were preaent in the water and that the obaerved reaponaea were to whole

cella. ReaulU o f (Chapter n i ihowed that the buccopharyngeal cavity o f ailver carp ia

heavily populated with Mate buda and although the preciae function o f theae remaina

unknown. It la probable that they play an important role in aaaeaaing the palatability

o f food itema. Diactiminarion between toxic and non-toxic M. aeruginosa would a earn

130

CHAPTER V

Filter-feeding o f carps on bacteria

S.l. Introduction

The lole o f bacteria in the diett o f flih and their importance in aquatic production U

not yet ciear (Kuzneliov, 1977; Moriaity and PuUin,1987). Although both free-living

and bacteria attatched to algae, suipended particles and organic ttuuter occur in aquatic

ecosystenu, it is generally presumed that fish such as tilapias and carps can ingest only

the attalched bacteria (Bowen,1976; Schroeder, 1978; Opuzynski, 1981b). In standing

natural freshwaters most o f the bacteria are free-living, toUU numbers o f bacteria varing

with the available suspended particles and season o f the year (Kirchman, 1983; Pedros-

A lio and Brock, 1983). Consumption and assimiUtion o f films o f laboratory cultures

o f methane-oxidizing bacteria by larval silver carp and bighead carp has been reported

(Panov et H/..1969, cited by Kuznetsov, 1977). Filter feeding carps such as silver carp

may also bn able to ingest bacteria associated with algae (Kuznetsov, 1977). However

there is no published information whether silver carp, rohu and catla can detect or

ingest unattatched bacteria. Beveridge et a(.(1989) found that Oreochromis niloricus

try were able to detect utiattatched bacteria and there was a significant, positive

correlation between bacterial ingestion artd their cotKentralion In the trial media.

131

Beveridge et al. (1991) observed thst CypHnia carpio were also able to detect

unattached bacteria.

The present experintent was undertaken to establish whether silver carp, rohu and caria

could detect and ingest unattached suspended bacteria.

5.2 M aterials and Methods:

5.2.1 Fish

H. moUtrix. L. rohita and C. calla fry, weighing 1-2 g were selected from the stocks

maintained in the tropicrd aquarium, as described in Chapter 111.

5.2.2 Bacteria

The bacterium Chromobacterium violaceum was first described by Beigonzini in 1981

(see Sneath, 1984). C. violaceum occurs mainly in soil and water and is common tat

tropical countries. It occasionally causes serious pyogenic or septicemic infections o f

mammab, iiKluding man (Sneath, Ibid). It U a gram-negative, rod-shaped bacterium

and typically meuures 0.6 - 0.9 x 1.5 - 3.5 pm, with rounded ends, sometimes slightly

curved (Sneath, 1984), The species is characterised by the presence o f a violet pigment

called violacein which is soluble in alcohol, and is readily apparent when the colonies

are several days old. The colonies grow well on ordinary media, giving cream or

132

yellowish colonies that him puiple at the edges (Fig. 3.1). C. violaceum is mesophilic,

growing at 37*C and the optimum temperature is 30-33*C. They can grow within 2-3

days in incubators at 22*C (Sneath, 1984). The bacterium has the advantage that

because o f its violet pigmentation it can be readily identified in the presence o f other

bacteria. The bacterial strain used, originated from NCIM B (National CoUection o f

Industrial and Marine Bacteria Ltd. Aberdeen, U.K.), and was obtained from stock

cultures held in the Bacteriology Laboratory at the Institute o f Aquaculture, University

o f Stirling, U.K. The stock cultures were sub-cultured in Ttyptone Soya Agar (TS A )

medium to obtain pure colony growths. Sub-culturing was carried out twice a week to

maintain the bacteria in its log phase o f growth.

5.2J Preparation o f agar media and inoculation

Ttyptone Soya Agar (TS A , Oxiod) was used throughout the experiment as the plating

and counting medium. Standard microbiological methods were used throughout (Collins

and Lyne, 1976). Twenty grammes o f TS A powder were mixed with 300 ml o f

distilled water in a glass bottle and shaken vigorously to dissolve. The solution was

then sterilised by autoclaving at I2 I*C for 13 min and allowed to cool to about 43*C

in a healed water bath in order to keep the agar in a molten phase. Some 13-20 ml o f

the molten agar medium was poured into each sterile petri-dish. the lid replaced, and

the dish left undisturbed until the agar set. Before use, the freshly made agar plates

were dried by placing them In an incubator set at 37*C for 13-20 min, with the lids

133

pwtly open. The streak plate technique was used for the inoculation o f agar pbttes. A ll

inoculated plates were [»operiy labelled and incubated at 22*C fcM* 2>3 days. In all

cases care was taken to avoid contamination.

5.2.4 Experimental system.

Three tanks o f size 44 x 24 x 26 cm. each containing 2 experimental chambers o f 2

1 capacity, were established in an isolated area in the tropical aquarium. The

experimental chambers were cleaned and filled with 1 1 o f sterilised (autoclaved) tap

water and placed in the tanks each o f whose sides was covered with black polythene

to minimise disturbance. Each experimental chamber was fitted with an autoclaved air

hose and air stone. In order to maintain the water temperature in the experimental

chember at 2TX:±I*C. water from a header lank fined with a thermostatically-

controlled heater was passed continuously through the tanks. The lops o f the

experimental chambers were covered with a sheet o f transparent perspex and

Ulumination was by natural, incident light.

5.2 J Prelim inary Investigation o f carp alimentary canal fo r C. Woloceam

An investigation was made to establish whether C. viotaceum was present in the

alimentary canal o f the experimental fish. At the beginning o f the experimeia, the fish

were tm fe r r e d from holding tanks in the recycle system o f the tropical aquarium iiUo

134

an isolated tank within the system where they were starved for 24 h. A group o f 3

starved fish was then transferred to an experimental chamber. A replicate treatment for

each species o f fish was carried out. A fter 4 h acclimatixation in the experimental

chamber, all flsh were killed in order to examine the intestintd contents.

Having killed the fish, the body cavity was immediately opened, the entire intestine

removed as quickly as possible, chopped into sttudi pieces, and the contents carefttlly

flushed out into individual sterile test tubes and nude up to 10 ml with physiological

saline. They were then mixed for 3 min by a vortex mixer. Using a micropipelte 0.1

ml o f sample was then spread out on the agar plates by a glass spreader and the plates

incubated for 3 days at 22*C in order to test for the growth o f C. violaceum.

3.2.6 Ingestion experiment

Prior to the start o f each trial, single species groups o f five experimental fish were

removed from the stock tanks in the recycle system, weighed and moved to another

tank within the system where they were kept in isolation without feeding for 24 h. The

single-species groups were then tratuferred to the experimental chambers and allowed

4 h to acclinutize.

135

C. Violaceum weie (n iu fened from a culture plate to a iterile bottle containing 2 ml

physiological saline (pH 6.6), agitated for 2 min in a vortex mixer to etuure no clumps

o f bacteria were present, and added to the experimental tank drop by drop thus

minimising disturbance to the Tish. The suspension was rapidly dispersed by aeration.

After 1 min a 5 ml sample was removed from the tank and diluted in the same way

as the intestinal samples (see below) to determine the initial bacterial count.

At the end o f each trial, all five fuh in each tank were sacriflced. The entire intestine

was removed as quickly as possible, chopped into small pieces and the contents

carefully flushed out, transfetied to a sterile test tube and made up to 10 ml with

physiological saline and then mixed thoroughly with a vortex mixture. Using a

micropipette, 1 ml o f the mixed intestinal contents was transfened to a fresh sterile

tube containing 9 ml buffered saline and the contenu again mixed thoroughly. Five

serial I: 10 dilutions were prepared in the same way from each intestinal sample.

Samples o f 0.1 ml were then removed from each dilution and spread on agar plates.

Duplicate plates were made in all cases. Plates were incubated at 2 2 ^ for 72 h.

For couming purposes, only plates containing between 3 and 80 colonies were selected.

By multiplying the number o f colonies by the reciprocal o f the dilution, colony

numbers per unit sample volume were determined.

136

Five triab were carried out for each species o f fish in which groups o f five fish were

exposed to bacteria. In the first series o f experiments the abilities o f fry to ingest

bacteria and the effects o f exposure time (30 min, I h. 2 h and 4 h) on ingestion were

examifted. In a second set o f experiments, the effects o f bacterial concentration on

ingestion rate, in which carp were exposed to different bacterial concentrations fo r a

period o f 1 h, were examined.

Statistical analysis was by Student's t-test (Bailey, 1981).

5.3. Results

5.3.1. Effects o f exposure time on the ingestion o f bacteria

The initial concentrations o f bacteria established in the experimental chambers in the

trials ranged from 3.2 x lO* to 27.3 x lO* cells ml '. During dissection o f the carp

stomachs at the end o f the exposure period, the contents o f some o f the fish were

observed to be stained a deep purple, characteristic o f the bacterial pigment. The

proportion o f fish ingesting bacteria and the mean viable colony counts per fish for

each trial are summarized in Table S.l. The proportion o f fish ingesting bacteria and

the numbers o f viable bacteria isolated fixan the alimentary tracts increased with time.

137

Tabi* 5.1 Xngaatlon o£ baetacia by ailvar carp« robu and catla; numbar of fiah Inçaating bactaria and numbar of ingaatad viabla bactaria par fiah at diffarant axposura timaa.Maanw t. o f f ia h (g )

In i t ia l Bxpoaurabactaria l tima (h) concant ration <calla ml‘M

Maañ------------numbar o f viabla c a li (fia h gut'')

Mumbar o ffiah (%) ingaating

bactaria

ÏTT3------------- Ï73 1300 1 (354)1.25 3.3x10* 1.0 4000 2 (30%)1.15 3.2x10* 2.0 12500 3 (75%)1.25 3.2x10* 4.0 16000 4 (100%)1.25 12.3x10* 0.5 3500 2 (50%)1.15 12.3x10' 1.0 12000 3 (75%)1.15 12.3x10* 2.0 24500 4 (100%)1.25 12.3x10* 4.0 36500 4 (100%)1.25 24.8x10* 0.5 6000 2 (50%)

24.8x10* 1.0 20500 4 (100%)1.25 24.8x10* 2.0 35500 4 (100%)1.15 24.8x10* 4.0 66500 4 (100%)1.5R 3.4x10* 0.5 1200 1 (25%)l .5R 3.4x10* 1.0 3000 2 (50%)1. SR 3.4x10* 2.0 6500 2 (50%)1.6R 3.4x10* 4.0 11500 3 (75%)1 .7R 10.2x10* 0.5 3500 1 (23%)l.SR 10.2x10* 1.0 5000 2 (50%)1. €R 10.2x10* 2.0 12000 3 (73%)1 .6R 10.2x10* 4.0 16500 4 (100%)1.7R 27.3x10* 0.5 7500 I (25%)1 .5R 27.3x10* 1.0 13500 2 (30%)1 .5R 27.3x10* 2.0 26000 2 (30%)l.SR 27.3x10* 4.0 40500 3 (75%)1.5C 3.8x10* 0.5 000 01.3C 3.8x10* 1.0 300 1 (25%)1.5C 3.8x10* 2.0 500 2 (30%)1.5C 3.8x10* 4.0 1100 2 (30%)1.4C 11.3x10* 0.5 200 1 (23%)1.5C 11.3x10* 1.0 500 1 (25%)1 .3C 11.3x10* 2.0 900 2 (50%)1 .4C 11.3x10* 4.0 1600 2 (50%)1 .5C 23.4x10* 0.5 700 1 (23%)1 .3C 23.8x10* 1.0 1400 2 (30%)1 .4C 23.4x10* 2.0 2500 2 (50%)1.4C 23.4x10* 4.0 6500 3 (73%)1.3» 3.3x10* 0.5 1600 2 (50%)1.2» 3.3x10* 1.0 4500 3 (75%)1.2» 3.3x10* 2.0 10500 4 (100%)1.1» 3.3x10* 4.0 16500 4 (100%)1.2» 11.4x10* 0.5 5500 1 (23%)1.1» 11.4x10* 1.0 13000 3 (73%)1.1» 11.4x10* 2.0 23000 4 (100%)1.3» 11.4x10* 4.0 37500 4 (100%)1.1» 23.1x10* 0.5 7500 3 (75%)1.2» 23.1x10* 1.0 22500 4 (100%)

23.1x10* 2.0 40000 4 (100%)1 .2» 23.1x10* 4.0 71000 4 (100%)• » • » i lv a r earp; » • »ohu; C • Catla and » - »ighaad earp.

138

5.3.2.Efr«ct9 o f bacterial concentration on ingestion rale

In the second series o f experiments, groups o f carp fry were exposed to concentrations

o f bacteria nuiging from 4.2 x 10’ to 27.6 x 10’ cells ml '. The numbers o f viable

bacteria isolated from the fish were plotted against bacterial concentration in the

experimental chambers in Fig. 5.1. Highly significanl positive cotrelalions (P < 0.05)

were observed. The ingestion o f bacteria appeared to increase with concentration in the

media and the numbers o f viable bacterial colonies isolated from silver carp and

bighead carp intestinal contents were observed to be the highest whilst those isolated

from catia were lowest (Figure 5.1).

5.4. Discussion

The resulu indicate that planktivorous carp fry are able to ingest unattached bacteria.

Sonkalion o f bacteria ensured that no clumps remained prior to introduction to the

experimental tanks, and although it is posible that mucus sloughed fTOm the fish skin

may have caused clumping o f bacteria, thereby faciliting ingestion, there was no visual

evidence to support this supposition. The propottiora o f fish ingesting bacteria and the

numbers o f bacteria ingested increase with lime and with bacterial concentration in the

media. These findings corroborate those o f Beveridge f t at. (1989, 1991) for common

carp and lilapia fry.

1 3 0

F i g . 5 .1 The r e l a c i o n s h i p be tw een mean v i a b l e b a c t e r i a l counts i s o l a t e d f r om i n t e s t i n a l t r a c t s o f s i l v e r c a rp , rohu and c a t l a p l o t t e d a g a i n s t i n i t i a l b a c t e r i a l c o n c e n t r a t i o n in tank w a te r .

140

'nte mechanisms involved in ingestion o f unattached bacteria by fish remain

unresolved. The mesh sizes o f the branchial sieve o f silver caip, rohu and catla are

around 20-23 pm . 73-90 pm and 110-130 pm respectively (see Chapter m ) and are

clearly unsuited to entrapment o f bacteria i f regarded solely as passive sieving devices.

Studies o f silver carp have demonstrated that it can not ingest paiticle size less than

3 pm by filter feeding (Adamek and Spinier. 1983; Smith. 1989). However, Kuznetsov

(1977) stated that silver caip ingest aggregates o f bacteria associated with algae and

speculated that it may be able to ingest solitary bacteria by entrapment in mucus

secreted by the labyrinthifonn organ. Studies o f filter-feeding in tilapias show that

particles are entrapped among the gill apparatus in a mucus film (Greenwood. 1933;

Drenner et a/.. 1987; Northcon and Beveridge, 1988; Beveridge et al„ 1988a. b). The

mucus is highly negatively-charged and it has been suggested that it may facilitate

flocculation and hence entrapment o f very small particles such as bacteria (Noithcon

A Beveridge, 1988; Beveridge et ai., 1989). Moreover, a recent paper by SaruSerson

et ai. (1991) suggests that mucus-assisted entrapment o f particles on the roof o f the

oral cavity may be important in some suspension-feeding cyprinids.

An altenuUive means o f ingestion o f bacteria may be through drinking. Although

freshwater fishes drink little water (Evans. 1984), since they are continuously faced

with the need to dispose o f water which enters by osmosis across the gills and skin,

the quaiPities that would be needed to explain the bacterial numbers found in the guts

o f small carps are small. Data summarised in Table 3.2 suggests that volumes < 40 pi

141

T a b i« 5.2 Numb«r« o f f is h in g «s tin 9 bsctsria and ratea ot in ges tion observed in groups o f s i lv e r carp, rohu and c a t la held at d i f fe r e n t b a c te r ia l concentrations during an 1 h period.

Species o f f is h

Meanweight o f fish<9)

Mo. o f c e l ls added per ml o f experixie- n ta l tank water

MeannuBiber o f c e l ls ingested per f is h

Number (%) o f f is h ingesting bacteria in each t r i a l

w r s ioJ itr ix — m — 4.3x10' 3,iSS 2L. roh ita 1.6 4.2x10* 2,000 2 (40%)C. 1.4 4.CxlO* 300 1 (20%)A. nobiJ is 1.2 4.3x10* 4,000 2 (40%)

M. m oJitrix 1.1 9.8x10* 6,000 2 (40%)L. 1.6 10.2x10* 5,000 3 (60%)C. 1.4 9.8x10* 700 1 (20%)A. n o b i l i » 1.2 9.7x10* 0,500 4 (00%)

ff. m olitr ix 1.1 17.3x10* 14,500 3 (60%)L. 1.6 15.8x10* 7,500 4 (60%)C. 1.4 16.2x10* 1,000 2 (40%)A. n o b i l i » 1.2 17.3x10* 15,000 5 100%)

M. m olitr ix 1.1 27.6x10* 20,500 4 (60%)L. 1.6 26.3x10* 12,500 4 (60%)C. 1.4 26.8x10* 2,000 2 (40%)A. n ob ili# 1.2 27.5x10* 21,500 5 (100%)

142

h*' may be involved. H iii can be accounted for by estimated drinking rates in

fi«shwater teleosts (1-182 pi h ' ) (Potts et al., 1967; Eddy and Bath, 1979). However,

the number o f bacteria ingested by til^ ias caniK>t be explained by drinking alone.

In this experiment the highest number o f viable bacteria were isolated from silver carp

arMj the lowest from the catla. Silver carp may indeed ingest more bacteria than other

carps although the results may to some extent reflect interspecific differences in

conditions in the intestinal tract or differertces in environment in the buccopharyngeal

region. It is clear from published data on C. vtoiaceum ingestion by tilapia (Beveridge

€t a/., 1989) that many fewer bacteria have been isolated from carp than till^>ia guts

although bacterial concentrations in the water and exposure times were similar.

However, because o f interspecific differences discussed above, it is not possible to

conclude if carps ingest less unattached bacteria than tilapia. On the basis o f numbers

isolated from gut contents and low mass o f individual bactrial cells, however, it is

unlikely that unattached bacteria play a significant role in frsh nutrition in either gross

m ergy or trace elmient terms.

143

CHAPTER VI

General Discussion

In the present study an attempt was made to investigate the possible filter feeding

mechanisms with respect to the feeding ecology o f three Asian caips. L. rohita, C.

catla and H. molitrix through detailed study o f the bucco-phatyngeal apparatus and

experimental studies o f quantitative feeding behaviour on algae and bacteria. Due to

limited facilities it was not possible to measure the extent o f gill raker adjustment in

relation to the size o f food panicles. It was also impossible to study the flow o f water

through the mouth, the movement o f water iiuide the bucco-pharyngeal cavity,

intensity o f mucus cell production from the bucco-pharyngeal cavity, or the movement

o f the surface o f the palatal organ during feeding. Such studies are desirable in order

to complete our understanding o f how filter-feeding is achieved.

Neveilheleas, on the basis o f observations made during the present study a tentative

description o f filter-feeding is possible. Rohu, calls and silver caip may be described

as pump filler-feeders (renru stricto Lazzaro, 1987), taking water in through their

mouths and passing it through the branchial sieve and out through the gill opercular

openings during both feeding and respiration. The sequence o f events is as follows;

during feeding, fish were observed to take water in through the mouth whilst keeping

the opercular opening closed. Water was drawn in by expanding the bucco-pharyngeal

144

region as ■ retail o f lowering o f the floor o f the region and widening o f the lateral

bucco-pharyngeal region tides. The mouth was then cloted. the opetculae opened and

the bucco-pharyngeal region compretted in order to pump the water through the

branchial sieve into the opercular cavity and from there out through the opercular

openings. The respiratory valve at the bate o f the upper lip (Plate la-c) prevented the

water flowing back out o f the mouth. The pretence o f folds on the buccal floor (Plate

2a-c) and lateral sides o f the region o f these three species lends support to the

hypothesis o f water inhalation by expaiuion o f the region. However, although the basic

principle o f water intake and ejection appears to be the same for all three species, the

mechanism o f particle retention may well differ.

The retention o f food putkles in the branchial sieve depends on several factors: i) the

mesh-size mid homogeneity o f the branchial sieve (Wright et al., 1983; Drenner et n/..

1984; Oibson, 1988); ii) the size and shape o f the food pruticles (Wright ei al., 1983;

Oibson, 1988); iii) the orienution o f the food particle when approaching the mesh o f

the briHichial sieve (Hoogenboezem, 1991); iv ) the surface chemistry o f the braiKhial

sieve (Northcott aivl Beveridge, 1988).

In the present study it was found that the branchial sieves o f rohu, calla and silver carp

were more-or-less homogeneous except for the posterior region o f the gill luch

channels. Moreover, the gill arch channels o f rohu and calla rue occluded by the piUatal

organ whilst those o f silver carp penetrated into the suprabranchiol organ. The

14S

impUcalioni o f these differences are discussed below.

It is viitually impossible to determine the orientation o f approaching panicles. Thus,

it is generally accepted that a particle has a retention probability rather than an absolute

retention value. The concept o f retention probability is related to particle size and

shape ar>d possible orientation. Nevertheless, Wright el at. (1983) and Oibson (1988)

both expressed difficulty in determining an appropriate measure o f prey items which

would reliably estimate retention probability because o f questioiu o f prey orientation

in the bucco-pharyngeal cavity.

Results on algal feeding indicale that all three species were able to ingest P. boryanum

colonies o f 60 pm in diameter although only rohu and silver carp were able to ingest

the much smaller M. aeruginosa cells ( - 4 pm diameter). The inter-gill raker spaces

o f rohu, catia and silver carp are 80-90, 110-120 and 2S-30 pm respectively. It is thus

clear that passive branchial sieving is not the only mechanism o f particle retention in

fiber feeding among these carps.

The role o f mucus tai the filter feeding process o f fishes has been proposed by several

authors and most coiuider mucus is important for retention o f food particles.

Greenwood (1933) observed a discrepancy between the coarse architecture o f the

branchial sieve and the tiny phytoplankton Ingested by Ttlapla esculenta and suggested

that these small particles are entangled fat a copious mucus supply secreted by cells in

146

the epithelium o f the bucco-pheryngeal cavity. Nofthcott and Beveridge (1988) and

N<xthcott et ai.t (1991) ataumed that in the t i l^ ia Oreochromis nitoticus, mucus fonns

a net’ like structure between the arches and this net helps in retention o f M.

aeruginosa. Beveridge et ai.t (1989) proposed that mucus was responsible for

entrapment o f the bacterium Chromobacterium viotaceum in O. niloHcus. Mucus

entrapment o f food particles in cyprinids has also been reported by Sibbing and Uribe

(1985) and Hoogenboezem (1991). The results o f histological and ultrastructural

examination in the fuesent study show the presence o f very large numbers o f two types

o f mucus cells in the bucco-pharyngeal region o f the carps studied. It is thus possible

that mucus plays an important role in the retention o f M. aeruginosa by rohu aikl silver

carp.

The results from the experiments conducted on bacterial ingestion showed bacteria are

ingested in comparatively small numbers (Table 6.1). There is no need to invoke the

involvement o f only mucus-based mechanisms, as ingestion by drinking would account

for the small numbers found in the fore-gut. In view o f the numbers involved

Beveridge et ai., (1991) also suggested that the benthic-feeding common carp Cyfînus

carpio ingest unattached susperuJed bacteria along with the drinking water, despite the

fact that Sibbing and Uribe (1983) have proposed that the fish ingesu small food

particles by nwcus entrapment. By contrast the t il^ ia O. niloticus ingests unattached

suspended bacteria in considerable nuntbers (Table 6.1) and Beveridge et at (1989)

proposed that mucus entrapment must be involved. Thus, from the present study and

147

Table 6.1 Summary o f laboratory studies on ingestion o f the bacterium C.

violactum by planktivorous fishes. Duration o f trials in all cases is 0.5 h, except

for O. niioticua (0.26 h).

Species Size (g> Bacterial

concentration

in media (x

10“ ml '

Bacterial

concentration

in g u t (x

10*)

Reference

O.niioticua 2.6 - 3.4 2.2 • 14.0 600 - 17,000 Beveridge et al 1989

C. carpio 8.0 - lfi.9 6.4 ■ 40.3 0.6 • 90 Beveridge et ai 1991

63.6 • 69.S 2.8 - 46.6 0.1 • 0.8 - - " •*

H. moiUrix t . l ■ 1.2 3.2 - 24.8 1.6 - 8.0 Chapter V

L. nhUa 1.6 - 1.8 3.4 - 27.3 1.2 - 7.6

C. catta 1.3 - 1.6 3.8 - 26.6 0 - 0.7

148

the review o f literature on bacterial ingeition by flih , it may be concluded that tilapia

ingest unattached suspended bacteria in significant numbers whilst carps do not.

The results from trials investigating ingestion o f toxic algae showed that silver carp do

not ingest toxic M. aeruginosa. Fish may delect the toxic algae in the environment by

the numerous taste buds on their bucco-pharyngeal surface and either avoid ingestion

it or reject them as a food item. Cyanobacteria (blue-green algae) often dominate the

phytoplankton communities o f eulrophic water bodies (Reynolds, 1984), iiKluding

tradilkmal and modem fisheries. Although many o f these Cyanobacteria can produce

powerful hepato- and neuroloxins which can kill i f ingested in sufficienl quantity

(Skulberg el a l„ 1984; Codd el al., 1989), they are apparently an impoitruit component

o f the diet o f a number o f tropical cichlids and cyprinids (Bowen, 1982; Hofer and

Scheimer, 1983; Adamek and Spinier, 1984; Spalaru and Gophen, 1983). In the light

o f the present study, it must be concluded that fish may face food scarcity in many o f

these eutrophic environments where toxic algae may predominate, hampering fish

growth and, i f the toxins are liberated as the result o f a bloom dying off, even causing

mortality.

The results o f the feeding experiments on algae at different densities indicate that

ingestion late changed with algal density. Similar results have also been reported by

other authors (Seale and Wassersug, 1979: KI4>iboe rrof., 1985; NorthcoM er a/., 1991).

149

From the present study and the above review o f small food particle ingestion by carps

and tilapias we may coiKur with the views expressed by other researchers (see above)

and conclude that retention o f small food particles depends on several factors: i)

particle size; i i) particle density; iii) surface chemistry; iv ) digestibility arKl v ) energy

content. Considering all these factcMi filter feeding mechanisms among carps are

proposed as follows:

Rohu take in water along with particles into the bucco> pharyngeal cavity through the

mouth. There are numerous taste buds on the bucco-pharyngeal surface which help the

fish to detect arMl assess the particle and decide how it should be handled. W ith regard

to haiKlling food particles the animal may have three options:

i) i f the food particle is sufficiently large to be passively entrapped by the branchial

sieve fish can ingest it easily. This would be the least experuive mearu o f particle

retention in terms o f energy expenditure.

U) i f a food particle is insufficiently large for passive retention by the branchial sieve

it might still be retained by adjustment o f the bnmchial sieve. Pish could thus ingest

particles somewhat smaller than gill raker spacings might suggest, although this method

is likely to be more expensive than the previously proposed mechanism in terms o f net

energy expenditure. Ingestion o f F. horyanum by rohu can be explained by branchial

sieve adjustsnem.

ISO

Ui) i f the food panicle U insufficiently large to be trapped by passive means by the

branchial sieve and even insufficienlly large to be actively caught by readjustment o f

the branchial sieve members, it could still be utilized as food if the fish were able to

use a mucus entrapment mechanism. In this mode o f food panicle retention fish use

the gill arches not as a sieve, but as a barrier in order to create turbulent flow

coirditions inside the bticco-phatyngeal cavity. Under such conditions small food

panicles may then be entrapped by mucus produced from cells on the bucco

pharyngeal surface. Similar feeding mechanisms have also been proposed by Sanderson

el ai. (1991) for the cyprinid Orthodon microlepidotus. Ingestion o f small M.

aeruginosa cells ( - 4 pm diameter) by rohu with 80-90 pm gill raker spacings may

thus be possible by mucus entrapment. Mucus entrapment, however, is likely to be the

most expensive feeding mechanism for fish in view o f costs o f synthesis. However,

fish ingest moat o f the mucus used for panicle entrapment and, provided that it may

be efficiently digested, this would compensate to some extent for the energy costs

associated with this method o f feeding.

Catla are likely to have somewlut diHerent feeding methods and abilities from rohu.

They have long free gill takers with large inter-taker spacings (110-120 pm). Calla

feed on both zooplankton and phytoplankton although they show positive selection

preference for zooplankton (Jafri and Mustafa, 1977; Jhingtan and PuUin, 1983; Pillay,

1990). Caila may use similar branchial sieving mechanisms to rohu for particle

rslention although tot the prasant trials calla did not itigast M. aeruginosa. Than are

151

• number o f explanations: catla may reject the alga as a food item, or it may be unable

to digest it or it may be suitable as a food but unprofitable to feed on such small

phytoplankton in view o f energy cottt (see above).

In v iew o f the comparatively small gill raker spacings (25-30 pm) passive braiKhial

sieving is likely to be the princqMd mechanism used by silver carp fcK retention o f

particles larger than 20 pm although mucus is probably involved in entrapment o f

smaller food particles. Unlike the other two species silver carp have a suprabranchial

organ on the roof o f the frftaryngeal cavity inside the palatal organ. The results o f the

histological study indicate the presence o f large numbers o f mucus cells on the walls

o f the suprabraiKhial <Ngan. Each gill arch bears two rows o f gill rakers which form

charmels. the posteriez section o f which enter into the suprabrarKhial c^gan. After

drawing water containing food particles into the bucco-pharyngeal cavity, pressure is

created which forces the water out through the branchial sieve. Silver carp have

numerous taste buds on the bucco-pharyngeal surface which help the fish detect aiKl

assess the foexi particle. On the basis o f particle size and other factors, perhaps related

to olfactory properties, fish may adopt different strategies to retain those particles, such

1) i f the food particles are large enough to be retained by the branchial sieve ( > 20

pm), the fish may arrange die g ill arch channels in such a way that the bulk o f the

water passes through the branchial sieve. The remaining water containing food particles

1S2

would enter the tuprabranchUl organ where food paiticles could be entraped by the

poeterior part o f the organ. A bolus o f food paitklet and mucus from several such

pumping actions would then be passed back irMo the posteriez part o f the pharyngeal

cavity and from there into the oesophagus by reversing the flow o f water during

contraction o f the suprabranchial <Mgan. Ingestion o f large colonial P. boryanum by

silver carp is likely to take place by passive brattchial sieving.

ii) i f the food particle is insufficiently large enough to be retained by the taanchial

sieve, the animal may arrange the gill arch channels in such a way that the water

containing food particles is driven throu^ the gill arch channels directly into the

suprabranchial organ where the particles are trapped by the mucus cells and formed

into a bolus. As a result o f the punu> action boluses o f food plus mucus are passed

back again into the pharyngeal cavity and into the oesophagus. It is proposed here that

ingestion o f small algal cells, such as M, aeruginosa, by silver carp occurs by this

mechanism. The net energy expenditure by the fish is likely to be greater than the

previous passive method o f entrapment.

E>ominance o f larger food particles in the diet o f silver carp has been repmted by

several authors (BMuckij, 1973 cited by Lazzaro 1987; Kajak et al., 1977; Adamek and

Spinier, 1984). Borucl^j (1973) also reported that silver carp feed selectively on

particles larger than 20 pm. In the laboratory trials conducted in the present study the

153

ingeition rate o f lilver carp wa< higher when feeding on P. boryanum in comparison

to M. aeruginosa.

Prom the present study o f algal ingestion rates it was found that in all cases silver caip

ingest highest amounts o f algal biomass, followed by rohu and then by catla, although

the ratio o f total filtering area o f the branchial sieve o f rohu, catla and silver caip o f

same size is 1 : 2.78 : 3. Thus, despite having a greater filtering area than rohu, catla

ingest less interms o f algal biomass, indicating that the filtering apparatus o f catla is

less efficent in comparison with that o f fohu or silver caip. In composite culture o f

Chinese and Indian major caip at higher densities, growth o f Indian major carp such

as catla and rohu are adversely affected (Lakshmanan et at., 1971; Dey el at. 1979).

Dewan el at. (1991) repotted that the degree o f dietary overlap among the fiy and

fingerlings o f Chinese and Indian major carps is more prominent between catla and

silver caip. In v iew o f the infonnation from the present studies it is recommended that

Chinese caip should not be slocked together with Indian major carp, particularly with

calls and paiticularly during the fry-fingerling stages.

On the basis o f the resulu o f Chapter IV, we may conclude that small (1-2 g ) silver

carp ingest highest amounts o f algal bio-volume (in leima o f g ' body weight h ') when

feeding at highest (193 x 10* pm’ ) algal concentrations which are 9.32 x 10̂ - 6.56 x

10 ̂pm’ algae per day (calculated on the basis o f 24 x the maximum hourly ingestion

rale o f P. boryanum detennined from the feeding trials). Reynolds (1984) repotted the

■nasi o f M. aeruginosa u 32(23-44) pg cell '. Mean energy content o f aquatic algae

if 13.3 KJ g ' diy weight ((Tuininini and Wuycheck, 1971). Hence it can be calculated

that a imall silver carp (1.1 g ) might be able to ingest 0.11 • 0.13 KJ energy per day

when only phytoplankton is avilable as food item. There is some evidence that Af.

aeruginosa and other phytoplankton are poorly digested by the stomachless silver caip

and that conversion efficiencies ate low (Kqjak et at., 1977; Hamada et al.. 1983;

Bitterlich, 1983).

Brett and Oroves (1979) derived a general energy budget for young, well-fed

herbivorous fish :

1001 • 37M + 20(i * 43E

where, I - rate o f ingestion, M - metabolic rate, O - growth rate, E • total excretion

rate and E is further divided into fecal - 41 and non fecal - 2. A ll values are

expressed in terms o f percentage o f total calories consumed (I). Chakraborty (1992)

reported that Cyprinus carpio need 33.43 KJ/Kg/day energy to fulfill routine metabolic

requirements. It is therefore clear that carp fry and fingetiings could not grow solely

on a diet o f planktonic algae, thereby explaining the observations that try and

fingerlings o f rohu, calla and silver carp selectively feed on sooplankton (Khan and

Slddlqui, 1973; JaffH and Mustafa, 1977; Jhlngran and PuUln, 1983; PUUy, 1990).

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A P P E N D IX - 1.

C o m p o s it io n o f B G I I a lga l g ro w th m ed iu m (m o d if ie d f r o m S tan ier el at. 1 971 )

C o n s t itu en t c o m p o u n d C o n cen tra t io n in stock s o lu tio n

In c lu tio n in g ro w th m ed iu m

N a N O , 7 5 .0g . 5 0 0 m l ' lO m l . l '

M g S O , . 7 H ,0 IS .O g. 2 0 0 m l ' I m l . l '

K jH P O , IS .O g. 2 0 0 m l ' **

C a C I , . 2 H ,0 IS .O g. 2 0 0 m l ' -

N a ,C O , IS .O g. 2 0 0 m l '

C i t r ic ac id IS .O g. 2 0 0 m l '

F e S O ,. 7 H )0 IS .O g. 2 0 0 m l ' -

E D T A IS .O g. 2 0 0 m l ' "

T r a c e e lem en ts c o p r is in g

H ,B O , 2 .6 8 0 g .l '

M n C I , . 4 H ,0 I .S IO g . l '

N a ^ M o O «. 2 H ,0 0 .3 9 0 g . r ‘ I m l . l '

Z n S O , . 7 H ,0 0 .2 2 .g .l '

C u S O ,. 5 H ,0 0 .0 7 9 g . r ‘

C o (N O ) , . 6 H ,0 0 .0 4 9 g .l '

A PPE N D IX - 2

C o m p o s it io n o f J M (J a w o rs k i 's M e d iu m ) a lg a l g ro w th m ed iu m

C on s titu en t com p ou n d C on cen tra tion in s to c k so lu tion

Inclusion in g ro w th

m ed ium

C a (N O , ) j . 4 H jO 4 .0 g . 200 m l ’ 1 m l. 1 '

K H ,P O , 2 .48 g . 200 m l '

M g S 0 . . 7 H j0 10.0 g . 200 m l ' -

N a H C O , 3 .18 g . 2 00 m l ' -

E D T A F e N A

E D T A N a ,0 .4S g . 200 m l '

0 .4S g . 200 m l '

••

H ,B O , 0 .4 9 6 g 2 00 m l ' -

M n C I „ 4 H jO 0 .2 7 8 g 2 00 m l '( N H , ) .M o ,0 2 . . 4 H ,0 0 .2 g 200 m l ' "

C ya n o co b a la m in 0 .0 0 8 g 2 00 m l ' "

T h ia m in e H C I 0 .0 0 8 g 2 00 m l ' "

B io tin 0 .0 0 8 g 2 00 m l '

N a N O , 16.0 g 200 m l ' -

N a ,H P O .. I 2 H ,0 7 .2 g 200 m l ' -

A P P E N D I X - 3

C o m p o s it io n o f B B (B o ld 's Basa l M e d iu m ) a lg a l g r o w th m ed iu m

C o n titu e n i com p ou n d C o n cen ira tio n in S tock

so lu tion

In c lu s io n in g row th

m ed iu m

N a N O , 10.0 g / 4 00 m l 10 ml/I

M g S O „ 7 H jO 3 .0 g / 4 00 m l

N a C I 1.0 g / 4 00 m l *•

K ,H P O , 3 .0 g / 40 0 m l *•

K H jP O , 7 .0 g / 40 0 m l *•

C a C lj . 2 H , 0 1.0 g / 40 0 m l *•

T ra c e e le m e n t

Z n S O „ 7 H ,0 K.82 g / 1 1 m l / 1

M n C lj , 4 H , 0 1.44 g / 1

M o O , 0 .71 g / l

C u S O ., S H jO 1.57 g / l

C o N O j , 6 H jO 0 .4 9 g / 1

H ,B O , 11.42 g / l ••

E D T A 5 0 .0 g / 1

K O H 3 l . 0 g / l

Peso,. 7 H , 0 4 .9 8 g /1 ••

H ,S 04 (c o n e . ) 1.0 m l /1

A P P E N D IX - 6

P e r io d ic A c id • S c h i fT i ( P A S ) R ea c tion

(1 ) S ec tion s to w a t e r

(2 ) 1 % P e r io d ic a c id 10 m in

(3 ) W a s li in la p w a te r S m in

(4 ) S ch ifT s r e a g e n t 20 m in

(S ) W ash in la p w a te r

(6 ) l la e n ta lo x y le n 5 m in

(7 ) W ash in t a p w a te r

(8 ) D ir re re n lia le In 1% acid a lc o h o l

(9 ) B lue in S c o t t 's la p w ater su b s tllu e

(1 0 ) W ash in l a p w a te r

( I I ) M e th y la ted s p ir it 30 s

(1 2 ) A b so lu te a lc o h o l 1 m in

(1 3 ) 0 .3 % la r t r a z in e in celloaive 3 m in

(1 4 ) A b so lu t a lc o h o l 1.5 m in

(1 5 ) X y len e 5 m in

(1 6 ) M ou n t in s y n th e t ic resin

R e s u lt s ; P A S posit v e red o r n tegen ta

Nuciei I b lu e

A PPE N D IX - 7

C o m p a ra tiv e g i l l m o rp h o m a tr ic data o f g i l l a rch II and total

f ilte r in g area o f s a m e s ize rohu, ca tla and s ilv e r carp.

rohu ca tla s i lv e r c a rp

S tan dard L en g th (m m ) 91 91 92

W e ig h t ( g ) 17.32 19.53 15.33

G i l l R a k e r L en g th ( ) im ) 833 1575 1720

G i l l A r c h L en g th ( ) tm ) 13220 2 3 4 3 0 24150

G i l l R a k e r Space (p m ) 93 130 30

T o ta l G i l l R ak er no. 120 246 720

T o ta l Ta s te Buds on G il l A r c h I I > 1 8 3 0 > 7 0 0 0 > 1 6 0 0 0

T o ta l n ite r in area (p m ’ ) x 10’ 3.83 16.22 17.63

A P P E N D IX • 8

Routine automatic proceu

1. 70 per cent alcohol three hours

2. 90 per cent alcohol three hours

3. Absolute alcohol I one hour

4. Absolute alcohol n one hour

3. Absolute alcohol III two hours

6. Absolute alcohol IV two hours

7. Toluene 1 one and half hours

8. Toluene II two and half hours

9. Wax bath I three hours

10. Wax bath II three hours

22 hours

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Abstract - dspace.stir.ac.uk · concentntiona caused cell shrinkage in C. vuigaris and A#, aeruginosa after 24 h of preservation. The abilities of carp fry and fingerlings to ingest