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Department of Biology
College of Arts and Sciences
University of the Philippines – Manila
Padre Faura, Manila
Announcement of
Undergraduate Thesis Presentation
MARIE ANGELIKA EMOCLING BONDOC
JOANNA MAE LUCES CEPCON
Entitled
VARIATIONS IN THE MORPHOLOGY AND HEMOCYTIC RESPONSES OF ASIAN
GREEN-LIPPED MUSSEL, Perna viridis L., IN SELECTED AQUACULTURE SITES IN
MANILA BAY
For the degree of
Bachelor of Science in Biology
May 16, 2018 1100H
(RH 118)
THESIS ADVISER
Samuel M. Go, MSPH
Assistant Professor
Department of Biology
University of the Philippines Manila
THESIS READER THESIS READER
Miriam P. De Vera, Ph.D. Melody Anne B. Ocampo, MSc
Professor Assistant Professor
Department of Biology Department of Biology
University of the Philippines Manila University of the Philippines Manila
Endorsed by:
Miriam P. De Vera, Ph.D. Jay T. Dalet, Ph.D.
Chairperson, Thesis Committee Department Chairperson
Department of Biology University of the Philippines Manila
University of the Philippines Manila
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Department of Biology
College of Arts and Sciences
University of the Philippines – Manila
Padre Faura, Ermita, Manila
ENDORSEMENT
The thesis attached hereto, entitled VARIATIONS IN THE MORPHOLOGY AND
HEMOCYTIC RESPONSES OF ASIAN GREEN-LIPPED MUSSEL, Perna viridis L., IN
SELECTED AQUACULTURE SITES IN MANILA BAY, prepared and submitted by Marie
Angelika Emocling Bondoc and Joanna Mae Luces Cepcon, in partial fulfillment of the
requirements for the degree of Bachelor of Science in Biology was successfully defended on May
16, 2018.
SAMUEL M. GO, MSPH
Thesis Adviser
MIRIAM P. DE VERA, Ph.D. MELODY B. OCAMPO, MSc
Thesis Reader Thesis Reader
This undergraduate thesis is hereby officially accepted as partial fulfillment of the
requirements for the degree of Bachelor of Science in Biology.
JAY T. DALET, Ph.D. LEONARDO R. ESTACIO JR., Ph.D.
Chair Dean
Department of Biology College of Arts and Sciences
University of the Philippines Manila University of the Philippines Manila
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ACKNOWLEDGEMENTS
To the God Almighty, we are grateful for Your guidance and protection in the field, for the
strength and willpower You have given us to push through in adversity, and for the understanding
and patience You have inspired in us that led to the accomplishment of our goals.
We would also like to express our sincerest gratitude to the following people:
Samuel M. Go, MSPH
For your unwavering assistance and encouragement throughout the course of our research.
Thank you for the encouragement and guidance that have helped us better our understanding of
the field and the importance of the study. Your inputs were more than valuable and helpful in
developing our skills in research. We will always be grateful.
Dr. Miriam P. De Vera and Melody B. Ocampo, MSc
For being part of the panel, pointing out areas for improvement, and guiding us through
bettering our data presentation and paper in general. We would like to express our deep
appreciation for your ideas on the study, for your consideration, and for your availability for
consultation. We could not have accomplished our objectives without your involvement.
Irmi Mora, Fernan Zacajusti Cabarles, Roxanne Lagco, and Soc Tuason
For providing the information we needed and the assistance in the collection in Masbate.
Without the help we received from you, we would not have known the proper protocols that were
necessary to ensure our safety and the credibility of our study.
Dr. Aletta Yniguez, Dr. Lilibeth Salvador-Reyes, and Ms. Jhenelyn Mendoza of the Marine
Science Institute
For accommodating our request for the HPLC assay in their busy schedules. Thank you for
extending your services to us and for being available for consultations on the interpretation of the
data.
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Mr. Jortan Tun, MSc and the Biotechnology Core Facilities Laboratory of the Marine Science
Institute
For your assistance in the flow cytometry assay and for being available for data
interpretation beyond work hours. Thank you for being so welcoming, for being considerate of our
schedules, and for the invaluable information you have willingly given.
Ms. Rhea Sulit and Ms. Evelyn Mendoza of the Zoology Division of the National Museum
For accommodating our request despite your busy schedule and for identifying our
specimens on short notice.
Mr. Juan Remigio Abon and Mr. Rico & Ms. Kristy Melo
For your assistance in the collection and genuine concern for our safety. Thank you,
without you we would not be able to complete this study.
Sir Edgar and Sir Max of the Department of Biology
For assisting us in borrowing equipment and getting us reagents that we needed. Thank
you for staying beyond your working hours to allow us to finish our work. Your support and service
have always been inspiring.
John Paul Emman and Steven Ablong
For your involvement in the experiment proper and for aiding in the interpretation of the
results of the statistical tests. For the untiring moral support and encouragements. We are
incredibly lucky to have someone like you for a friend.
Jem Langas, Nico Velasco, and Charyl Diang
For helping in the collection and buying materials that we needed. Your help is greatly
appreciated.
Jose Divinagracia, Michael Falceso, Ian Estrada, Tristan Guerrero, Daryl Dastas, Avel
Bautista, Markus Cuasay, Alex Hapitan, and Evan Baguyo
For the support and reassurances. You have boosted our morale countless times, even more
so in challenging times. Thank you for listening to our frustrations and extending a helping hand
even without asking. You are all blessings to us and we are truly thankful.
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To the Bondoc and Cepcon Families
For the financial and emotional support. Thank you for the love and faith that kept us going.
Thank you for helping without question and for understanding without blame. The study took long
to finish, and now that we have, we realized that we could never have done any of this without
you. And rightfully so, we dedicate this study to you. Thank you for everything. We love you, and
we will always try to make you proud.
viii
TABLE OF CONTENTS
PAGE
DEFINITION OF TERMS …………………………………………………………... x
LIST OF TABLES ………………………………………………………………… xi
LIST OF FIGURES ………………………………………………………………… xi
LIST OF APPENDICES ……………………………………………………………. xii
ABSTRACT …………………………………………………………………………... xiii
INTRODUCTION …………………………………………………………………... 1
Background of the Study …………………………………………………... 1
Statement of the Problem …………………………………………………... 3
Objectives of the Study …………………………………………………... 3
Significance of the Study …………………………………………………... 3
Scope and Limitations…………………………………………………............... 4
REVIEW OF RELATED LITERATURE …………………………………………... 5
Description of Manila Bay …………………………………………………... 5
Biology of Perna sp. …………………………………………………………... 6
Morphological Characterization of Perna sp. ………………………………. 8
Algal Blooms and Microalgal Toxins …………………………………... 9
Hemato-immunological Responses of Bivalves …………………………... 10
METHODOLOGY …………………………………………………………………... 13
Collection and Identification of Sample Specimen …………………………... 13
Morphological Description …………………………………………………... 13
Measurement of Saxitoxin Concentration in Bivalve Tissues …………... 13
Quantification of Hemocyte Responses …………………………………... 15
Hemolymph Extraction and Preparation …………………………... 15
Flow Cytometry …………………………………………………... 15
Hemocyte Counts ………………………………………………....... 16
Statistical Analysis …………………………………………………………... 16
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RESULTS ………………………………………………………………………... 17
Morphological Observations ………………………………………………... 17
Presence of Gonyaucoloid Toxins ………………………………………... 18
Concentration of Total Hemocytes ………………………………………... 18
Hyaline and Granular Hemocyte Populations ……….................................... 20
DISCUSSION ………………………………………………………………………... 21
Morphological Characteristics ………………………………………... 21
Absence of Gonyaucoloid Toxins ………………………………………... 22
Total Hemocyte Count in Perna viridis ………………………………... 22
Granular and Hyaline Hemocytes in P. viridis ………………………... 23
Trends and extraneous factors ………………………………………... 25
CONCLUSIONS AND RECOMMENDATIONS ………………………………... 27
SUMMARY ………………………………………………………………... 29
LITERATURE CITED ………………………………………………………... 31
TABLES AND FIGURES ………………………………………………………... 36
APPENDICES ………………………………………………………………... 38
x
DEFINITION OF TERMS
dcStx Decarbomoyl-saxitoxin
DHC Differential Hemocyte Count
DO Dissolved Oxygen
Events Single detected material in flow cytometry; indicated by a single
dot in dot plot analysis
FSC Forward Scatter; describes relative cell size
Gonyaucoloid Toxins Toxins produced by dinoflagellates of the Gonyaulaceae family
Gtx Gonyautoxin
HAB Harmful Algal Bloom
HPLC High Performance Liquid Chromatography
NStx Neosaxitoxin
PSP Paralytic Shellfish Poisoning
PST Paralytic Shellfish Toxin
SSC Side Scatter; describes cell complexity or granularity
Stx Saxitoxin
THC Total Hemocyte Count
GH Granular Hemocytes
HH Hyaline or Agranular Hemocytes
Meat Weight Ratio Ratio of soft tissue or “meat” weight to total organism weight
Total Organism Weight Shell weight and meat weight combined
xi
LIST OF TABLES
Table Description Page
Table 1 Average length and meat weight ratio with standard deviation of
Perna sp. collected from Navotas, Bacoor, and Hagonoy coastal
areas of Manila Bay
36
Table 2 Average total hemocyte count per milliliter of undiluted hemolymph
with standard deviation for the 3 locations
36
LIST OF FIGURES
Figure Description Page
Figure 1 Hyaline hemocytes and granular hemocyte stained with Giemsa-
Wright stain and observed under oil immersion objective (OIO,
x1000 total magnification) of a compound light microscope
37
Figure 2 Percent granular and agranular (hyaline) hemocytes per mL
hemolymph sample from three sampling sites in terms of SSC value
for 20 000 events based on flow cytometric analysis.
37
xii
LIST OF APPENDICES
Appendix Description Page
A Description of Study Sites
41
B Certification for Taxonomic Identification of Perna viridis 43
C Raw Data for Shell Length and Meat Weight Ratio 44
D Stock Charts for Morphological Characteristics 48
E Statistical Tests for Morphological Characteristics
49
F Periodate Oxidation for Determination of Saxitoxin and Gonyautoxin
Concentration
52
G Hemocyte Counting 55
H Statistical Analyses of Total Hemocyte Count (THC) of Perna viridis
From Three Sample Sites
56
I Flow Cytometry Results and Interpretation 58
xiii
ABSTRACT
Many studies about on ecology and hydrodynamics in the Indo-Pacific have utilized Perna viridis
hemato-immunological responses as bioindicators of pollution and environmental stressors due to
its filter-feeding capacities. This study determined the morphology and hemocytic responses of
populations of P. viridis (n=30) obtained from aquaculture sites in Bacoor (Cavite), Hagonoy
(Bulacan), and Navotas (Metro Manila) coastal areas of Manila Bay. Morphology of bivalves was
characterized by measuring shell length and meat weight ratio while hemolymph from the adductor
muscle and the homogenized of shellfish meat were used in the test for hemocytic responses and
presence of gonyaucoloid toxins, respectively. Results showed that average shell length varied
significantly among the three populations; with the Bacoor group having the highest mean in shell
length while Navotas group has the lowest. Whereas the mean meat weight ratios did not vary
between the Hagonoy and Bacoor populations. The Navotas group has the highest average meat
weight values while Hagonoy has the lowest. The total hemocyte counts were observed to be
significant only between Navotas and Bacoor populations. Two hemocyte populations were
determined by flow cytometric analysis and showed that granular hemocytes comprised no more
than 25% of the total circulating hemocytes of the three areas while hyaline hemocytes comprised
76-96%, with highest values of the phagocytic granular hemocytes found samples from Bacoor
and the least in the Navotas population, showing the same trend as shell length and opposite trend
as meat weight ratio. While there are variations in morphology and hemocyte responses in the P.
viridis populations, the samples tested negative for gonyaucoloid toxins (Saxitoxin, decarbamoyl-
Saxitoxin, neoSaxitoxin, and gonyautoxin1and4) for all three locations. According to previous
studies, variations in the morphology and hemocyte responses could be attributed to differences in
temporal establishment of bivalve populations, the periodic hypoxia observed in the bay, and
pollution of heavy metals and other contaminants in aquaculture sites of the coastal bay areas
studied.
Keywords: Perna viridis, Manila Bay, hemocyte, morphology, saxitoxin
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CHAPTER I
Introduction
Manila Bay is a natural harbor that serves as port of Metro Manila and encompasses the
provinces of Cavite on the east, Bulacan on the north, and Bataan on the west in its 190km
coastline. Its strategic geography made Manila Bay a center for commerce and other economic
activities such as inter-island shipping, transport of large oceanic vessels, and as livelihood source
of coastal communities (PEMSEA, 2007). At present, this marine inlet has been classified as
severely polluted due to the anthropogenic activities occurring in and around the bay. Common
pollutants include heavy metals, agricultural runoff, and domestic and industrial wastes disposed
in the bay.
High nutrient loading observed in Manila Bay often leads to eutrophication which may
allow proliferation of algae and dinoflagellates to dangerously high amounts in an event referred
to as harmful algal bloom or HAB (Anderson et. al., 2002). These HABs are usually associated
with blooms of toxic dinoflagellates and algae. In the Philippines, the most detrimental events are
blooms of Pyrodinium bahamense, a member of the Gonyaulacaceae family which produces
saxitoxin (Azanza and Miranda, 2001). Since its first recorded occurrence in 1983, P. bahamense
blooms have affected a total of 22 coastal areas and caused thousands of deaths. Recorded deaths
are usually from ingestion of contaminated seafood, such as fishes, crabs, mussels, oysters, and
clams, that primarily feed on algae and dinoflagellates (BFAR, 2015).
The Asian green-lipped mussel, Perna viridis, is one of the marine resource cultivated in
coastal areas of Manila Bay, more particularly on the province of Cavite and on Navotas. Green
mussels are not costly to cultivate. Aquaculture farms require only hard surfaces such as rocks or
bamboo stakes for attachment (Aypa, 2018). Inherently, it can tolerate a wide range of conditions
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making them ideal products for cultivation and an affordable protein source for residents living
near the coast of the bay. Its ubiquity, sedentary mode of life, filter-feeding behavior, and the
ability to tolerate a variety of conditions makes it suitable for biomonitoring studies (Sellner et al.,
2003).
The filter-feeding mechanism enables it to store and bioaccumulate several organic and
inorganic contaminants, toxin included, which is necessary for studies on water quality. Its ability
to bioaccumulate contaminants in its tissue to levels above what it can tolerate can also serve to
assess bivalve health status such as the number and proportion of the total circulating hemocytes,
which change under stressful conditions (Mitta, 2000; Wang et al., 2012a).
Numerous studies globally have used bivalves as indicator of stressors may it be
environmental (Mello et al., 2010; Wang et al., 2012b) or biological (Montojo et al., 2006; Hegaret
et al., 2011; McFarland, 2015) through the assessment of hemato-immunological parameters. In
the Philippines, bivalves are used by the Bureau of Fisheries and Aquatic Resources to measure
concentration of saxitoxin in bivalve tissue as part of their HAB monitoring procedures.
However, there are only few researches dedicated to establishment of normal patterns of
hemato-immunological parameters in bivalves (Barracco et al., 1998), and no documented
morphological and physiological response variations conducted in the Philippines, specifically for
Perna viridis. When Perna viridis is exposed to different levels of gonyaucoloid toxins in
aquaculture sites of Bacoor (Cavite), Hagonoy (Bulacan), and Navotas (Metro Manila), there are
expected variations on the morphology and hemocytic responses.
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Statement of the Problem
Are the variations in the morphology and hemocyte responses attributed to gonyaucoloid
toxins in Perna viridis from selected aquaculture sites in the coastal areas of Manila Bay?
Objectives of the Study
The primary aim of this study was to characterize the morphology and measure hemocyte
responses of Perna viridis collected from three aquaculture sites along the coast of Manila Bay
namely, Bacoor, Cavite; Hagonoy, Bulacan; and Navotas, Metro Manila. Specifically, this study
aimed to estimate the concentration of saxitoxin and its analogs in the mussel soft tissues using
High Performance Liquid Chromatography (HPLC). This study intended to give a characterization
of mussel gross morphology, specifically the average shell length and meat weight ratio. Some
cell-mediated immune responses were also quantified, particularly hemocytic responses, through
quantification of total and differential hemocytes using microscopic cell count with aid of
Neubauer chamber and flow cytometric analysis, respectively.
Significance of the Study
The results of this study may be used as baseline values for the hemocyte responses of
Perna viridis in the absence of saxitoxin and gonyautoxin based on the total hemocyte count and
differential hemocyte count. Findings of this study might be used as reference for further studies
using Perna sp. as biological indicator of environmental contaminants. The measurement of the
shell length and meat weight ratio characterize the Perna viridis specimens cultured in aquaculture
sites in Manila Bay and may provide insight on morphological responses of mussel on bodies of
water with the same physicochemical properties such as water temperature, salinity, and dissolved
oxygen. The hemocyte responses were tested to provide information on the current health status
4
of the mussels, specifically its cell-mediated immune responses to pathogens and toxins found in
Manila Bay, Philippines. This study could also contribute to current literature regarding exposure
of bivalves to stressors in different locations of the same bay area through comparison of their
relative values and with a range of known values observed in the same species by previous studies.
Scope and Limitations
This study includes the determination of the morphology and hemocyte responses of Perna
sp. collected from Manila Bay limited to three aquaculture sites namely, Bacoor, Cavite; Hagonoy,
Bulacan; and Navotas, Metro Manila. Morphological characterization was limited to the shell size
and the meat weight ratio, defined as the ratio of soft tissue weight to total organism weight, and
does not include other morphological observations in individual specimens. This study was also
limited to detecting the following toxins: saxitoxin, decarbamoyl-saxitoxin, neosaxitoxin, and
gonyautoxins 1 and 4. The effects on bivalve immunity were tested based only on total and
differential hemocyte counts does not include other immune responses such as potentially
apoptotic hemocyte counts, phenoloxidase activity, total protein concentration, and
hemagglutinating activity. High performance liquid chromatography by periodate oxidation was
performed to detect the presence of gonyaucoloid toxins. The standard protocol of BFAR to
measure saxitoxin concentration, mouse bioassay, was not performed. This study mainly quantifies
the morphological characteristics and hemocyte responses of Perna viridis in Manila Bay coastal
waters with the natural phytoplankton community and physicochemical properties of the study
areas.
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CHAPTER II
Review of Related Literature
Manila Bay in the Philippines is located at the western coast of Luzon (14°53’ N latitude,
120°76’ E longitude) semi-enclosed by the provinces of Cavite in the southern region, Rizal and
Metro Manila in the eastern region, Bulacan and Pampanga in the northern region, and Bataan on
the western boundary. Water circulation of Manila Bay is described as double gyre, with one gyre
located in Bataan, its western side, and another in Cavite, its eastern side (Azanza and Miranda,
2001).
Studies reveal that eutrophication is rampant in the bay due to excessive nutrient loading,
along with elevated chlorophyll a and inorganic nutrient levels (Jacinto et al., 2011; Sotto et al.,
2014 in Sotto et al., 2015). However, several areas of the bay remain utilized for shellfish farming,
such as Bataan and Cavite. Both areas are red-tide prone; though in Cavite, dinoflagellates are only
a minor portion of the phytoplankton community, with the dominance of diatom Chaetoceros sp.
In Bataan, blooms of Noctiluca scintillans often succeed the Pyrodinium blooms (Azanza and
Miranda, 2001).
The phytoplankton community of Manila Bay is composed mainly of diatoms, which
remain dominant to dinoflagellates year-round. In 2001, there are ten families of dinoflagellates,
nine families of diatoms, and other marine flagellates (such as raphidophytes and chrysophytes).
Of the ten families of dinoflagellates, there are five detrimental species apart from Pyrodinium
bahamense var. compressum identified namely, Gonyaulax spinifera, Alexandrium sp., Dinophysis
caudata, Gymnodinium catenatum, and Prorocentrum micans. Those that occurred in the highest
densities are Noctiluca scintillans, Protoperidinium spp., and Ceratium furca. And of the nine
6
families of diatoms, the species that occurred in the highest densities are Chaetoceros spp.,
Coscinodiscus sp., and Thalassiosira sp. (Azanza and Miranda, 2001).
Manila Bay is a frequent study area due to a vast phytoplankton community in its 19.27 x
103 km2 catch basin and 190km coastline. In addition, it is a major port and source of livelihood
for coastal communities (Sotto et al., 2015) which are estimated to have a population of 31 million
people or 34% of the total population of the country (PEMSEA, 2007). The projected increase in
the populations along the coastline is also expected to worsen the hypoxia in the bay due to nutrient
loading (Azanza and Miranda, 2001). The hypoxia in the bay worsens during wet season with bay-
wide averages falling to 2.10mg/L of dissolved oxygen, a condition that may have effects on the
morphology and hemato-immunological responses of Perna sp. and other bivalves. Moreover,
temperature varies along the bay. The Cavite bay area has higher monthly temperature recorded
than the northern boundaries, Bulacan and Pampanga bay areas (Azanza and Miranda, 2001).
In a study conducted in Brazil, Simoes et al. (2014) noted that upon exposure to HAB, no
individual mortality in bivalves was recorded; however, the mussel Perna perna was most
immunologically affected of the three bivalves namely mussels, oysters, and crabs. The study also
showed Perna having the highest concentration of toxin accumulated in the tissues upon exposure
at any age.
The life cycle of Perna starts at a larval phase found at the pelagic zone which take 8 to 18
days to develop depending on environmental conditions and availability of substrate. It may
exceed this range when a substrate is not available and the metamorphosis is delayed for survival
(McFarland, 2015). However, this is not often observed as a prolonged larval phase increases the
risk of predation. At the planktonic stage, the growth, development, and survival of bivalve larvae
is greatly altered by variations in temperature, salinity, pH, dissolved oxygen, food availability and
7
HAB pollutants (McFarland, 2015; Donaghy and Volety, 2011). To explain the aggregation in
aquaculture farms, it is hypothesized that chemical cues are released by adults to increase
recruitment of other larva to later establish adult populations (McFarland, 2015).
Bivalve mollusks bioaccumulate saxitoxin (Stx) through normal filter-feeding behavior
with little to no mortality. As a consequence, bivalves pose a threat to both human consumers and
natural marine predators. Large quantities of Stx in the tissues of Perna remained even after five
months post-bloom. Specifically, saxitoxin concentrations remained above the regulatory limit of
800µg/g Stx to organism weight equivalent in the mussel tissues. Mussels more rapidly
accumulates high concentrations of toxins produced by HABs compared to many oyster and clam
species (McFarland, 2015).
In Philippines, 1893, the P. viridis showed high toxicity and low survival following P.
bahamense var. compressum bloom with 9620 mouse units (MU)/100g saxitoxin concentration,
leading to an eight-month shellfish ban due to prolonged toxicity. Ban on shellfish consumption is
imposed when the density of Pyrodinium bahamense reaches 500 cells per liter of sea water, and
when the toxin level reaches 60 micrograms per 100 grams of shellfish meat according to the red
tide bulletin uploaded by the Bureau of Fisheries and Aquatic Resources (BFAR) since 2014.
Detoxification is found to be partially dependent upon the peak tissue concentration and
previous exposure history that can affect the uptake kinetics of biotoxins by bivalves, specifically
Perna viridis (McFarland, 2015). The green mussels, Perna viridis, are abundantly found in coastal
waters of the Indo-Pacific and are known to have rapid growth rates which make it an ideal
aquaculture species. It grows into marketable size within six to eight months, serving as food
source (McFarland, 2015). Green mussels grown in aquaculture use bamboo stakes as columns of
8
attachment and these frequently occur in densities of 1000 to 4000 individuals per square meter
(Fajans and Baker, 2005; McFarland, 2015).
The optimum water temperature for P. viridis development ranges from 26°C to 32°C. At
10°C and 35°C, laboratory specimens had 50% survival. Lethal temperatures were identified to be
at 6°C and 37.5°C. Reduced filtration rates, abyssal production, and gonad development have been
observed at temperatures greater than 35°C. Wide ranges reported from short-term laboratory
studies may overstate the boundaries for long term growth and survival. A significant increase in
heat shock proteins (Hsp70) was observed after only 2 hours of exposure to 10°C suggesting that
stress from short-term exposures may have prolonged metabolic effects esp. if other stressors are
involved or repeat exposures occur (McFarland, 2015).
The survival of P. viridis is also dependent upon salinity, since low salinity conditions
reduce the temperature range at which P. viridis can survive. Optimum salinity was identified to
be at 27 to 33 ppt with a tolerance of 19 to 44 ppt, as reflected in the population distribution within
bays and estuaries. At 24 and 80 ppt, there was 50% survival of the specimens. In short-term
exposures to higher salinity than the optimum, physiological changes such as increased valve
closure and inability to osmoregulate and reduced toxin clearance rates were observed (McFarland,
2015).
Upon settling after metamorphosis, juvenile P. viridis showed growth rates of a range of 7
to 15 mm per month which decreases over time (Rivonker et al., 1993). Found in tropical and
subtropical areas, P. viridis grows faster than other mussels found in colder areas (Bayne and
Worrall, 1980), supporting the hypothesis that growth rate increases with increasing temperature
provided food is sufficient for the bivalves (Rajagopal et al., 1998a; McFarland, 2015). Cheung
9
(1991) in McFarland (2015) stated that for P. viridis, growth is capped at 17°C and lower
temperatures.
According to Rajagopal et al. (1998b) in McFarland (2015), one-year-old P. viridis may
grow close to 9.0cm to 11.9cm in shell length. These records vary as the study was at an area
where food and temperature were high. Another species, Perna perna, has a lower recorded shell
length at 6.5cm to 11.7cm (McQuaid and Lindsay, 2000 in McFarland, 2015). These values are
recorded from green mussels collected at Estero Bay, Florida, USA. There are currently no data
on baseline hemato-immunological responses of Perna sp. and variations in these responses when
exposed to algal blooms in the Philippines.
Cases of algal blooms in the Philippines have been documented since 1983, but records of
blooms of Pyrodinium sp. in Manila Bay date back to 1908. The documented blooms of
dinoflagellates Prorocentrum minimum, Cochlodium polykrikoides, and Alexandrium sp. have
been documented in localized regions in the Luzon Island. The unicellular dinoflagellates
mentioned develop algal blooms throughout the world with factors associated with change in
weather, temperature, turbulence, salinity, and transparency of waters they inhibit. However, the
most detrimental phenomenon in terms of public health and economic losses are the blooms of
Pyrodinium bahamense var. compressum, first recorded in 1983. Philippines has the highest record
of PSP cases from Pyrodinium bahamense var. compressum of all countries in the Southeast Asia
(Azanza and Max-Taylor, 2000). In the past three decades, P. bahamense var. compressum blooms
have spread to around 22 coastal areas of the country. Consequently, these have increased the
severity of the cases of paralytic shellfish poisoning and fish kills (Relax et al., 2003).
Saxitoxins (Stx) and gonyautoxins (Gtx) are potent neurotoxins highly involved in
paralytic shellfish poisoning (PSP). Both neurotoxins interfere with the transmission of
10
electrochemical messages by activation of a voltage-sensitive sodium channels and stimulation of
sodium influx into cells (Reynolds et. al., 2005). In the Philippines, Stx and Gtx are the only toxins
monitored, since the dinoflagellates of the family Gonyaulacaceae are the only detrimental species
with documented blooms in the country, specifically Pyrodinium bahamense var. compressum
blooms in Manila Bay. The toxins these dinoflagellates produce are tetrahydropurines, including
saxitoxin, which are heat-stable and acid-stable (Azanza and Miranda, 2001).
For filter-feeding bivalves, the risk lies on the accumulation of toxin exceeding tolerable.
Certain toxins were proven to have sublethal effects on bivalves and several responses against
toxic dinoflagellates including variation in pumping and filtration rate, mucus production, and
cardiac activity (Landsberg et al., 2002).
Bivalves generally possess three discernable hemocyte morphologies, namely, hyaline cells,
which lack conspicuous cytoplasmic granules; granular cells, so described due to the cytoplasmic
granules; and small agranular cells or prohemocytes, which are precursors to hemocytes. In Perna
viridis, the prohemocytes consist 3% to 10% of the total circulating number of hemocytes. The
proportion of hyaline and granular cells varies from each species, but wide fluctuations in the
hemocyte subpopulations may also be an effect of bacterial challenges and/or pollution from the
immediate environment. Hyaline hemocytes have no phagocytic ability, but they play a central
role in coagulation, limiting blood loss, and healing wounds. The different hemocyte types were
identified through monoclonal antibodies and some with identified cell-specific markers (Donaghy
and Volety, 2011). However, these subtypes can easily be differentiated by size and morphology
under light microscopy (Wang et al., 2012).
A study conducted on the impact of Dinophysis acuminata blooms revealed the hemato-
immunological effects of HABs on three bivalve species in Santa Catarina, Brazil. The focus of
11
the said research lies on the adverse effects on bivalve physiology and survival, rather than its
impact on human health. This study hypothesized that bivalves exhibit a certain level of resistance
to toxins; however, exposure to HABs of Dinophysis acuminata presents adverse effects on the
hemato-immunological responses of oysters, mussels, and clams. Moreover, toxin accumulation
is indicative of a health risk to consumers (Simoes et al., 2014).
The findings of this study include elevated number of total circulating hemocytes in
mussels as a response to immune system activation by the phycotoxin, and decreased number of
total circulating hemocytes in clams and oysters as a result of cell migration for dinoflagellate
clearance. Insignificant levels of cell apoptosis for the three bivalves suggest that okadaic acid and
its derivatives do not trigger cell apoptosis in bivalve hemocytes. Phenoloxidase activity was
significantly altered in both bivalve species, and protein concentration increased significantly on
mussels only while, the remaining parameters did not vary in both bivalve species. Data obtained
on the detection of PSP toxin confirms that saxitoxin is indeed present on bivalves exposed to P.
bahamense bloom (Simoes et al., 2014). Hemato-immunological responses of bivalves are lesser
studied compared to other fields of bivalve development as there are no recorded mortalities due
to HABs. However, at the cellular level and at external evaluation, the detrimental effects of HABs
may manifest (Ibrahim, 2007).
Flow cytometric analysis showed that hemocyte found in circulation in Perna perna were
found to be different between male and female mussels. There is a low variation in hyaline and
granular hemocytes from the differential hemocyte count. Hyaline hemocytes are at 64.2% while
granular hemocytes are 35.8% of the total circulating hemocytes per mL of hemolyph for the male,
with 60.1% and 39.9% for the female, respectively (Barracco et al., 1999).
12
Allam et al. (2002) obtained different results with flow cytometry, with hyaline hemocytes
comprising 98 to 99% of the total hemocyte populations. However, these values are for two species
of clams, namely Ruditapes philippinarum and Mercenaria mercenaria, and one species of
oysters, Crassostrea virginica. According to Allam et al. (2002), early reports of flow cytometric
analyses of bivalve hemolymphs were inconsistent, as different numbers of subpopulations were
described for different species studied. The same study assigned a single population of hemocytes
for Crassostrea virginica and Fisher (1988) for finding four subpopulations in the same species.
Three hemocyte groups were found in C. virginica and two in Mytilus galloprovincialis (Allam et
al., 2002).
The total hemocyte count (THC) of P. viridis was averaged at 3.15 x 105 cells per mL of
hemolymph of males with a standard deviation of 1.55 x 105 while it is at 3.67 x 105 cells per mL
of hemolymph with a standard deviation of 1.99 x 105 of female bivalves (Barracco et al., 1999).
However, Barracco et al. (1999) conducted the study in Sta. Catarina, Brazil, with different
environmental conditions as Manila Bay, Philippines. Hence, variations in THC are highly
probable.
13
CHAPTER III
Materials and Methods
Collection and Identification of Specimens
Sampling of Perna viridis using the bucket method was employed in the present study. The
collection at aquaculture farms was done by diving one meter deep to collect a whole stake column
of mussels. From the bucket, every third specimen was selected until a total of 30 specimens were
collected for each location. The specimens, P. viridis, were collected from the aquaculture sites
mapped in Appendix A. Two specimens were selected at random and were brought to the Zoology
Division of the National Museum of the Philippines where they were identified at the species level
(Appendix B).
Morphological Observations
Description of bivalve morphology was defined using two criteria, namely, shell length
and meat weight ratio. The shell size is the length from the anterior to posterior region of the P.
viridis specimens and was measured with a standard and uniform ruler. The meat weight ratio was
computed by getting the ratio of the soft tissue or shellfish meat weight to the total organism
weight. The total organism mass was obtained by weighing each specimen in a triple beam balance.
After recording the organism mass, the hemolymph was first extracted, then the soft tissue was
removed by detaching the adductor muscle from the shell and weighed to get the body mass.
Measurement of Saxitoxin Concentration
The saxitoxin concentration was identified through high-performance liquid
chromatography (HPLC) using the LC-20AT Shimadzu pump at the Marine Science Institute
14
(MSI) laboratory in the University of the Philippines Diliman, Quezon City. To prepare the sample
for liquid chromatography, the soft body tissue including the adductor muscle was removed
entirely from the shell of the P. viridis. The tissue extracts previously washed with ethanol for
surface sterilization were homogenized using an Octus M-12 blender with a speed of 12 rpm. After
homogenization, the sample is referred to as homogenate, which was placed in 15mL falcon tubes
and labeled for each sampling site. Equal volumes of HCl were added to the homogenates and
adjusted to optimum pH 3.0 with 0.1M NaOH and 0.1M HCl. The homogenates for each sample
site were centrifuged using an IEC clinical centrifuge at 3000 rpm for 10 minutes and filtered
through 0.2µL nylon filters. The supernatant was collected and frozen until use.
The saxitoxin and its derivatives (decarbamoyl-saxitoxin and neosaxitoxin) and
gonyautoxins 1 and 4 were quantified by periodate oxidation as described by Lawrence et al.
(1996). The oxidative reagents used were hydrogen peroxide, tert-butylhydroperoxide and
periodate. For loading, 250-µL of the oxidative reaction mix and 100µL of the homogenate were
added to microcentrifuge tubes. The tubes were vortexed for three minutes. Five microliters of
acetic acid were pipetted into the solution and 50µL was finally injected into the machine through
a series-type double plunger. For this method, species and concentration of oxidants, pH,
temperature and reaction time was closely monitored and kept constant. The pre-column oxidation
method used a single reverse-phase column chromatography system. It has been standardized as
European norm EN 14526 (Nuñez et al., 2015). Quantification of the concentrations is down to
one-tenth to one-twentieth of common regulatory guideline levels of 0.8 mg/kg saxitoxin
equivalents. This method is approved Official First Action in June 2005. The limit is around 0.1
mg/kg saxitoxin equivalents, depending on the composition of toxins.
15
Quantification of Hemocyte Responses
A. Hemolymph extraction and preparation
The hemolymph was drawn from the adductor muscle of each specimen using a 21G needle
on a 1mL syringe containing 500 µL of 6% cold formalin in sterile sea water. To prevent the
bivalve from fully closing, a scalpel was used to gently pry open the shell. Sea water contained in
the mantle cavity was drained to ensure that the hemolymph was extracted without other debris.
The hemolymph extracted for thirty specimens per study site was approximately 1.5mL with 12mL
6% formalin in sea water solution. The hemolymph extracted was divided into two portions. One
portion was incubated in ice for cytometric analysis. The other portion is to be used in preparing
total hemolymph (TH) which was used for the total hemocyte count. The cell suspension was
stored at 4 °C temperature to allow cooling without freezing. It was then centrifuged for 8000 rpm
for 30 minutes. The supernatant was obtained and stored in a freezer until use.
B. Flow cytometry analysis
Flow cytometric analysis was performed using the BD FACSCalibur System with an argon
ion laser of 488 nm wavelength at the Biophysiochemical Technology Incubation Core Facility of
MSI in Diliman, Quezon City. The sorting purity of the system is greater than 95% and a threshold
was set to eliminate detection of particles other than hemocytes. The flow cytometry indicated the
relative size and granularity of bivalve hemocytes. The scatter performance for both forward (FSC)
and side scatter (SSC) resolution was optimized at 345 and 380 volts, respectively. The total
number of events was gated at 20000 cells per sample, as is the standard operating procedure of
the flow cytometric system used for hemocytes and according to protocol performed by Lau et al.
(2017) for bivalve hemocytes. The FSC and SSC intervals were transformed with the log function
16
to allow definition of the dispersal of hemocytes on the dot plot. The results were analyzed to
quantify the differential counts of granular and hyaline hemocytes per event per location.
C. Hemocyte Counts
From the fixed hemolymph pools, the total hemocytes were counted with the aid of a
Neubauer chamber, using the protocol found in Appendix G, to give the total hemocyte count
(THC). In addition, the relative percentage of the different hemocyte populations (DHC) was
obtained from the results of the cytometric analysis. Each result was expressed as a relative
percentage of granular hemocytes (GHs) and the remaining percentage corresponds to the
agranulocytes or hyaline hemocytes (HHs).
Statistical Analyses
The shell length measurement and meat weight ratio of thirty specimens each collected
from the three study sites were tested for normal distribution using Kolmogorov-Smirnov Test and
homogeneity of variances. The shell length measurements and computed meat weight ratio of the
populations from the three study sites were compared using one-way ANOVA with Tukey’s post
hoc test to identify the significant groups for each parameter.
The total hemocyte count (THC) of samples from the three locations were also tested for
normal distribution with Kolmogorov-Smirnov Test and homogeneity of variances. The THC
results were compared via one-way ANOVA with Tukey’s post hoc test. The level of significance
was set at p ≤ 0.05 for all tests.
17
CHAPTER IV
Results
Morphological Observations
The morphology of Perna viridis was characterized for each location using shell length
and meat weight ratio. The records of raw data for 30 specimens on morphological observations
for the three site groups are seen in Appendix D. The distribution of values for the shell length is
shown through stock charts in Appendix E-1, while that of meat weight ratio is shown in Appendix
E-2. The average values and standard deviations were estimated by descriptive statistics for both
parameters (see Table 1).
The Kolmogorov-Smirnov Test was used to establish the normality of the average shell
length and average meat weight ratio values for 30 specimens in each of the three populations of
P. viridis from Bacoor, Hagonoy, and Navotas aquaculture sites, as seen in Appendix F-1. Tested
using the PSPP software, the statistical analyses returned two-tailed asymptotic significance of
0.205 for shell length and 0.497 for meat weight ratio, both indicating normal distribution of data
(p > 0.05).
The Bacoor group had the highest average shell length at 7.03 ± 0.83cm, followed by the
Hagonoy group with 6.30 ± 0.68cm, then the Navotas group with an average of 5.19 ± 0.39cm.
One-way ANOVA with Tukey’s post-hoc test showed three distinct groups for shell length as seen
in Appendix F-3.1 and Appendix F-4.1. Comparing the two parameters for morphology, the trend
of the values in shell length was not paralleled in meat weight ratio.
The Navotas group had the highest average meat weight ratio, with 0.43 ± 0.03, followed
by the Bacoor group with 0.36 ± 0.12, then the Hagonoy group with 0.34 ± 0.04. However, one-
way ANOVA and Tukey’s post hoc test analyses revealed that there are only two significant
18
groups for meat weight ratio as seen in Appendix F-3.2 and Appendix F-4.2, respectively. The
difference between meat weight ratios of Bacoor and Hagonoy populations are not significant.
Statistical tests were performed at 95% confidence (α = 0.05).
Therefore, the Navotas population has the shortest shell length (in cm) and the greatest
meat weight ratio. Bacoor and Hagonoy populations have no difference in meat weight ratio;
however, the average shell length varied between the two locations.
Presence of Gonyaucoloid Toxins
Three trials per location of periodate oxidation through HPLC returned negative for the
presence of Stx, dcStx, NStx, and Gtx 1 and 4. The toxin profiles can be found in Appendix F-1 to
Appendix F-6. The indication of detection of these gonyaucoloid toxins is an overlap in the peak
of the toxins with the sample. The only overlaps were found in Hagonoy and Navotas populations,
both for decarbamoyl-saxitoxin. However, the overlaps were no more than 20,000 µV and are
considered negligible according to protocol. Concentrations are identified at peaks more than
25,000 µV in height. This assay was run and interpreted by the Marine Science Institute laboratory
in University of the Philippines Diliman.
Concentration of total hemocyte count (THC)
The total hemocyte counts in the undiluted hemolymph samples from Bacoor, Hagonoy,
and Navotas were counted on the 0.0025 mm area of the Neubauer chamber according to protocol
and were multiplied by a factor of 104 to acquire hemocyte count per milliliter hemolymph. The
cells were stained with Giemsa-Wright for better distinction of the cells from other debris. Two
cell populations were seen; namely hyaline hemocytes, which contain a densely-stained nucleus
19
and no granules in the cytoplasm as seen in Figure 1a, and granular hemocytes, which contain a
nucleus and numerous granules enclosed by a cell membrane as seen in Figure 1b.
The THC results (see Table 2) show that the Navotas population has the highest average
circulating hemocytes in the hemolymph at 4.48 x 105 per mL, followed by the Hagonoy
population with 3.38 105 per mL, then the Bacoor population with the lowest THC at 2.60 x 105
per mL. The Kolmogorov-Smirnov Test was again performed to establish normality of data and
returned an asymptotic significance (2-tailed) of 0.999, indicating normal distribution of data.
These results can be found in Appendix H-2.
Further statistical analyses using one-way ANOVA (Appendix H-4) and Tukey’s as post-
hoc test (Appendix H-5) showed that there are two significant groups based on mean THC values.
The Bacoor and Navotas populations were shown to vary in THC while no other variations were
found between Navotas and Hagonoy, and Bacoor and Hagonoy groups. THC values from greatest
to least as seen in Table 2, with no significant difference between mean THC values between
Hagonoy and Bacoor populations, mirrors the trend of the meat weight ratio values as seen in
Table 1. The Navotas population has the highest average meat weight ratio and THC, while Bacoor
and Hagonoy populations have no significant difference in average THC and meat weight ratio.
Hence, a direct relationship between the two factors is highly probable; however, it was not
established in this study.
20
Hyaline and Granular Hemocyte Populations
Detected components in the extracted samples were termed “events” in the flow cytometric
analysis. The events were gated at 20, 000 cells according to protocol, controlled in the flow
cytometer.
The distribution of hyaline (agranular) hemocytes to granular hemocytes can be seen
relative to each other in 20,000 events gated by the flow cytometer (Appendix I-2.1 to Appendix
I-4.2). For differential hemocyte count (DHC), percentage of hyaline and granular hemocytes were
distinguished by setting a boundary for two regions at 102 for the side-scatter (SSC) axis or the x-
axis. Those higher than the limit would indicate higher cell complexity or granularity, implicating
that the cells in the specified region contains granular hemocytes, while those lower than the
boundary (SSC < 102) are considered less complex or agranular, which are characteristic of hyaline
hemocytes. The granular and hyaline hemocyte percentages combined account for the percentage
of hemocytes in each hemolymph sample, while the remainder are other particles detected in the
crude hemolymph.
Results show that the Navotas population has the lowest percentage 3.74% of granular
hemocytes, followed by the Hagonoy population with granular hemocyte concentration value of
20.16%, while Bacoor returned the highest at 23.39% of approximately 100% hemocytes in the
crude samples as shown in Figure 2. Incidentally, with regard to meat weight ratio and granular
hemocyte populations, both Bacoor and Hagonoy groups have DHC similar to each other.
Meanwhile, the DHC of the Navotas population is noticeably less than the DHC of the other two
groups. This is observably inverse from the trend of meat weight ratio values of the three
populations.
21
CHAPTER V
Discussion
The average shell length of specimen collected from three different areas was highest in
the Bacoor population and lowest in the Navotas population. In contrast with meat weight ratio,
Navotas group had the highest ratio, followed by Bacoor group, and least in Hagonoy group.
However, no significant difference was found between the average meat weight ratio of the Bacoor
and Hagonoy populations.
A study conducted by Rivonker et al. (1993) in the west coast of India showed that green
mussels peak in shell length and meat weight in the first 164 days of culture. Following the peak,
there was a significant inverse relationship between the shell and meat weight. This implies that
the resource allocation to production of soft tissues decreased while shell size increased. In the
present study, the trend was observed for the average shell length and meat weight ratio.
Comparison of values for the three locations show that groups with large shells have light meat
weight while groups with small shells have heavy meat weight.
McFarland (2015) cited that P. viridis may grow to as large as 9.0cm to 11.9cm beyond
their harvest age. The marketable age of Perna is approximately five to six months (Kamal and
Khan, 1998). At this age, P. viridis individuals are 5cm to 6cm in length (Rivonker et al., 1993).
Average shell length of specimens collected from the three areas in Manila Bay are measured at
6.0cm. However, the age of P. viridis samples collected were not taken into consideration in the
present study, and other factors such as food supply and temperature may have played a role in the
variances. Based on projected length of Perna sp., sample specimens are of marketable size which
puts Navotas populations at younger age and Bacoor populations at older age.
22
To explain variations in the gross morphology and hemocyte response of Perna sp. that
may have been induced by a toxin stressor, detection of saxitoxin and its analogs were conducted
using high performance liquid chromatography. Saxitoxin transforms into one of its derivatives,
hence it was essential to maintain the pH at 3.0; however, the presence of the most common Stx
derivatives were also tested in this study.
Sample profiles were compared against commercially available paralytic shellfish toxin
standards namely, neosaxitoxin, decarbomoylsaxitoxin, and gonyautoxins 1 and 4. These toxins
are produced by the gonyaucoloids present in Manila Bay, including the detrimental Pyrodinium
bahamense var. compressum. However, it was found that these toxins were not present at
detectable levels in the bivalve soft tissues. This result establishes that none of the hemocytic
responses have been altered in response to toxins bioaccumulated in the bivalve tissues. However,
there was an observable trend between the morphology and the hemocytic responses recorded.
The increase or decrease in the total circulating hemocytes of P. viridis indicates a cell-
mediated immune response to a stressor. Hence the estimation of the total hemocyte count under
natural, ambient conditions is essential for comparison to THC of other Mytilid species exposed
to varying environmental conditions (Mello et al., 2010). For example, it was established by Mello
et al. (2010) that total hemocyte numbers in mussels exposed to algal blooms were significantly
higher than in reference animals not exposed to algal bloom. It also described granular hemocytes
as the predominant cell type and Perna perna was the only bivalve with altered DHC during the
dinoflagellate bloom.
In the study by Donaghy and Volety (2011), P. viridis total hemocyte concentration was
measured to be 1.30 x 106 cells per mL at average with a range of 7.3 x 105 to 2.2 x 106 cells per
mL. In the present study, the total hemocyte count of undiluted hemolymph samples from Bacoor,
23
Hagonoy, and Navotas range from 1.78 x 105 to 5.75 x 105 cells per mL which is considerably
lower than THC measured in previous study. However, total hemocyte count for specimens from
both Manila Bay, Philippines and Estero Bay, Florida were measured at natural conditions without
the influence of toxin stressors. The differences in physicochemical properties, phytoplankton
communities, and pollutants of Manila Bay, Philippines and Estero Bay, Florida, USA may
account for the large discrepancy in THC values.
For differential hemocyte counts, two distinct cell types, granular and hyaline hemocytes,
were distinguished in the assay based on distribution along the y-axis (SSC) as described in
Appendix I. Flow cytometric analysis showed that hyaline hemocytes are dominant to granular
hemocytes in samples from all three study sites. Specifically, specimens from Bacoor had the
highest percentage of granular hemocytes at 23.39%, followed by those from Hagonoy at 20.16%,
and Navotas specimens at 3.74%.
Despite lack of consensus in hemocytes subpopulations present in bivalves, hyaline
(agranulocytes) and granular hemocytes are consistently observed in the hemolymphatic fluid of
Mytilid species (Donaghy and Volety, 2011). Light microscopy also revealed the presence of the
two hemocyte subpopulations in the undiluted hemolymph of Perna viridis.
In some studies, hyaline hemocytes have been observed to have slight phagocytic
capabilities but is known to play a role in host defense other than phagocytosis. Whereas granular
hemocytes are largely associated with phagocytosis, specifically large granular and large semi-
granular hemocytes (Garcia-Garcia, 2008). The FSC in flow cytometry shows the distribution of
hemocytes by their size relative to each other, which is converted with a logarithmic function for
better definition in the dot plot while the SSC shows cell granularity; however, only granularity
was tested in the present study as beads of known cell diameters were not available for control.
24
Results gathered are different for the same species studied by Donaghy and Volety (2011)
stated that granular hemocytes, the phagocytic population, accounted for 40% of circulating cells.
Meanwhile, hyaline hemocytes dominated the hemocyte population in Perna viridis collected from
Estero Bay, Florida. Results may have varied due to differences in environmental factors such as
temperature, salinity, availability of food, and composition of phytoplankton communities
between Estero Bay in Florida and Manila Bay in the Philippines.
The differences in sampling, handling, and methods may have resulted in disparities among
identified subpopulations of hemocytes and differential hemocyte counts in bivalve hemolymph.
Further, the lack of comprehensive comparative studies such as flow cytometric analyses involving
the different bivalve species in point locations of different environmental conditions may attribute
to the lack of consensus on defining the hemocyte subpopulations.
Most studies use P. viridis as a biological indicator in the tropical and subtropical regions,
especially in the Indo-Pacific regions, detecting changes in their haemolymphatic immune system
in response to environmental contaminants, such as increase in production of reactive oxygen
species, THC, and percentage of granular hemocytes when subjected to heavy metals and
polycyclic carbons (Saenz et al., 2010; Ramos et al., 2017). Hemocytes are vital in cell-mediated
defense against pathogens and hemocyte populations vary in response to environmental
conditions, as observed in the present study.
Physiological and behavioral responses of bivalves are often examined separately,
although both parameters likely affect the other as part of a system (Talkington, 2015). The trend
in variation for meat weight, or soft tissue weight, is paralleled by the trend in differential hemocyte
populations. The Navotas group has the highest meat weight ratio and has the least percentage of
granular, or phagocytic, hemocytes. The Bacoor group follows in meat weight ratio and has the
25
highest percentage of granular hemocytes. Lastly, the Hagonoy group has the least meat weight
ratio and follows Bacoor in the percentage of granular hemocytes. However, both Bacoor and
Hagonoy populations did not vary significantly in mean meat weight ratio while the percentages
of granular hemocytes in their hemolymph did have a difference (p < 0.001), with 23.39% and
20.16%, respectively. However, the variations in morphology and hemocytic responses of Perna
viridis observed in Bacoor, Hagonoy, and Navotas groups are not attributable to gonyaucoloid
toxins as results show that there are no detected concentrations in all of the three study sites.
These findings suggest one or more extraneous factors which could have affected the
morpho-physiology of P. viridis. Such factors may be pollution of nitrogenous wastes, heavy metal
concentrations, and hypoxia. Variations of the quantities of the pollutants and the degree of
eutrophication among aquaculture sites may be reflected in the morphology and hemocyte
responses of P. viridis. Chang et al. (2009) stated that Manila Bay is considered highly
eutrophicated with ammonium. In addition, heavy metal concentrations, specifically Fe, Mn, Zn,
Cu, Pb, Ni, Cd, and Co, were found in Manila Bay (Prudente et al., 1994). Moreover, nitrogenous
wastes such as dissolved inorganic nitrogen, nitrate, and ammonium were found to have average
concentrations of greater than 0.90 µM and 8.00 µM, respectively, in 2009 and were projected to
increase over time (Chang et al. 2009).
All organisms have varied resource allocation strategies, most of which depend on
nutrition. However, environmental conditions often select for different allocation strategies
(Ng’oma et al., 2017). A study by Strong and Luoma (1981) showed variations in body size of
bivalves when exposed to metal contamination. Heavy metal accumulation in soft tissues is known
to decrease the meat weight of the total organisms. Further, it is known that as the bivalve ages, its
growth shifts to the shell instead of the growth of the soft body. Their relation has also been
26
characterized as inversely proportional (Fischer, 1983), and can also be observed in the data on the
morphological observations of P. viridis in the present study.
Presence of pollutants in the environment might result to accumulation of toxic wastes in
their tissues and increased risk of inducing physiological changes in marine bivalves (Matthew
and Menon, 1983). Another study in Sta. Catarina found that Perna perna transplanted to a site
contaminated by domestic sewage had increased levels of DNA damage (Saenz et al., 2010). DNA
damage may cause both observable and behavioral changes in the organism. It can be hypothesized
that the variations in the morphology and hemocytic responses of Perna viridis are due to different
environmental conditions in the three aquaculture sites, particularly the water pollutants in the area
such as heavy metals and nitrogenous wastes.
In addition, previous studies indicate that phagocytosis is significantly affected by
dissolved oxygen, as well as interactions between DO and duration of exposure (Wang et al.,
2012b). The same study stated that there are lower values of phagocytosis, implicating a lower
percentage of granular hemocytes, under hypoxic conditions than normoxic conditions. Given the
periodic hypoxia in Manila Bay (Sotto et al., 2015), it is hypothesized that the DO levels in the
respective study sites were varied.
Specifically, the Navotas population may have had the least granular hemocyte population
due to its hypoxic state. The area of collection in Navotas, which is the Navotas Fish Port Complex,
is the largest fish port and fish market complex in the Philippines. Anthropogenic activities such
as widescale cultivation of marine organisms and disposal of domestic and industrial wastes
possibly contributed to hypoxia and increased nutrient loading in the area (PEMSEA, 2007).
However, there is a possibility that the effect of natural environmental variability on the
morphology and physiology of bivalves masks the sublethal effect of pollutants (Jorgensen, 1990).
27
CHAPTER VI
Conclusion and Recommendations
The findings of the present study showed that there were variations in the morphology and
hemocyte responses of Perna viridis populations from aquaculture sites of Bacoor, Hagonoy, and
Navotas. However, these variations were not attributed to toxin stressors since there were no
gonyaucoloid toxin levels detected in the P. viridis tissues of samples from all locations.
It is highly suggested that same day and on-site testing of the physicochemical properties
be performed to minimize the discrepancy in measurements. Moreover, follow up studies on the
water quality of these areas (Bacoor, Hagonoy, and Navotas) and possibly all other coastal areas
within Manila Bay is recommended to provide information as to why such inconsistency exists.
This will also provide more recent and more comprehensive physicochemical analysis of water
quality in said bay areas.
It is also recommended to conduct a time-series study on the morphology and hemocytic
responses of bivalves during different seasons, as it is established that water temperature, dissolved
oxygen, and heavy metal concentration, among other factors, affect the aforementioned
parameters. This will establish baseline values for different seasons, depending upon time of
collection in Manila Bay.
Despite lack of toxin stressors in the chosen study sites, variations on the morphology and
hemocytic responses of Perna viridis was still observed. Hence, further studies on morphological
and physiological responses of P. viridis in Manila Bay against other possible stressors, such as
hypoxia, heavy metals, organic and inorganic contaminants, and temperature are also
recommended. For hemocytic responses, sizes of hemocytes given by the forward scatter only
described relative size of the hemocytes. Hence, it is advised to include beads of known size to
28
clearly demarcate other subpopulations of bivalve hemocytes. Further studies need to be conducted
to characterize the relationship between the morphology and the hemocytic responses of P. viridis.
Lastly, it is advised that other hemato-immunological parameters such as hemagglutinating
activity, production of reactive oxygen species and nitric oxide, and percent apoptotic hemocytes
be included in further studies for a complete assessment of bivalve behavior in Manila Bay.
29
CHAPTER VII
Summary
Trends were observed in the average shell length and meat weight ratios of Perna viridis
populations collected in Bacoor, Hagonoy, and Navotas. Average shell length was recorded
highest in Bacoor and least in Navotas. However, the inverse was observed for the meat weight
ratio wherein the highest was measured in Navotas and least in Hagonoy. It was noted that Navotas
had the smallest shell length at 5cm but the heaviest meat weight at 43% of the total organism
weight indicative of higher biomass allocation to soft tissues.
The measured hemocytic responses for all sample groups were not caused by exposure to
gonyaucoloid toxins and their most common analogs as indicated by HPLC analysis which
recorded negative presence of toxins in bivalve tissues obtained from the three sampling sites.
However, variation in hemocytic responses were still observed for two sampling groups.
The total hemocyte count was recorded highest in the Navotas population and lowest in the Bacoor
population. One-way ANOVA, assessed at 95% confidence level, for THC indicated that statistical
difference exists. Tukey’s post-hoc test established that the significant difference in mean THC
was between Bacoor and Navotas. Lowest THC was recorded in Bacoor samples with 2.6 x 105
cells per mL and highest in Navotas samples with approximately 4.5 x 105 cells per mL.
Normal proportion of granular to agranular hemocytes is 40:60. However, quantified
proportions were vastly different from normal. The highest granular to agranular proportion was
recorded in the Bacoor population with 23.39% granular to 76.84% agranular hemocytes. This is
followed by the Hagonoy population with 20.16% granular hemocytes to 79.64% agranular
hemocytes. The Navotas population had the lowest granular hemocyte concentration in the
30
hemolymph with only 3.74% granular to 96.05% agranular hemocytes. These values are out of 20,
000 cells detected in the flow cytometric analysis.
It is hypothesized that the morphology and hemocyte responses of P. viridis are closely
linked. Previous studies in Manila Bay and bivalve behavior, indicated that variations in the
morphology and hemocyte responses of the samples from Bacoor, Hagonoy, and Navotas may be
due to heavy metal contamination, periodic hypoxia, and nitrogenous wastes deposition in the
aquaculture sites of the study areas and not necessarily attributed to saxitoxin contaminants.
31
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36
TABLES AND FIGURES
Morphological Characterization of Perna viridis
Table 1. Average length and meat weight ratio with standard deviation of thirty Perna sp. each collected from
Navotas, Bacoor, and Hagonoy coastal areas of Manila Bay.
LOCATION AVERAGE SHELL LENGTH
(cm)
AVERAGE MEAT WEIGHT
RATIO
Navotas 5.19 ± 0.39C 0.43 ± 0.03A
Bacoor 7.03 ± 0.83A 0.36 ± 0.12B
Hagonoy 6.30 ± 0.68 B 0.34 ± 0.04B
Note: Values with a common letter notation per column are not significantly different from one another.
Concentration of total hemocyte count (THC)
Table 2. Average total hemocyte count per milliliter of undiluted hemolymph with standard deviation for the 3
locations.
Study Sites Average THC per mL
Bacoor 260 000 ± 61 543B
Hagonoy 337 500 ± 63 048AB
Navotas 447 500 ± 88 553A
Note: Values with the same letter notation are not significantly different from one another.
37
Measurement of Hyaline and Granular Hemocyte Populations
Figure 1. a) Hyaline hemocytes (left; arrow indicates nucleus; high nucleocytoplasmic ratio) and b)granular hemocyte
(right; cytoplasmic granules; low nucleocytoplasmic ratio) stained with Giemsa-Wright stain observed under oil
immersion objective (OIO, x1000 total magnification) of a compound light microscope.
Figure 2. Percent granular and agranular (hyaline) hemocytes per mL hemolymph sample from three sampling sites
in terms of SSC value for 20 000 events based on flow cytometric analysis.
Bacoor Hagonoy Navotas
Granular 23.39 20.16 3.74
Agranular 76.84 79.64 96.05
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