1

I NTRODUCTI ON
Data on the reproductive biology of bivalves form the basis of the
understanding of life history, evolution and population dynamics of these
organisms (Haag and Staton 2003). These also provide information on the
right time to harvest the optimal marketable size of the organism (Rajan et al.
2001) and allow the assessment of the regeneration capacities of the natural
stocks of the population (Baron 1992). Science-based management and
conservation strategies require knowledge on the reproductive biology and
recruitment patterns of the organism.
Bivalves are significant components of the macrobiota of freshwater
systems. Unionaceans (freshwater mussels) are a diverse and conspicuous
element of the benthic fauna in freshwaters of eastern North America (Haag and
Staton 2003). Corbiculid clams in extraordinary high densities have been
reported to be important in freshwater benthic production, nutrient cycling and
water purification (Byrne et al. 2000). The pisiidid clams Musculium partumeian
in small and shallow ponds and M. lacustre in eutrophic lakes contribute
significantly to the secondary production in their habitats (Way 1988). The
mussel, Dresseina polymorpha depletes the food in the water column and so
operates a transfer of energy from the pelagic to the benthic foodwebs (Minchin
and Moriarty 1998). Aside from their ecological importance, freshwater bivalves
are also commercially valuable. Most prevalently harvested are the pearl forming
mussels of the genus Margaritifera. Due to its overexploitation this genus is
2
undergoing great reduction in range and population densities are decreasing
alarmingly (Grande et al. 2001). In Brazil, Paxyodon syrmatophorous, a
freshwater mussel, is collected and the shells are manufactured as buttons
because of their nacreous mother-of-pearl layer. Due to frequent harvesting,
there is a decline in number and sizes of these mussels in the river (Beasley et
al. 2000). Despite the significance of freshwater bivalves little is known about
their reproductive biology especially in tropical and subtropical areas (Byrne
1998).
The status of ecological and commercial value of freshwater bivalves in
the Philippines is even so less documented. There is a danger therefore that
these organisms will start to disappear before having been recognized. In the
first place, there are only two endemic species of freshwater mussels in the
country both belonging to genus Simpsonella
(http://bama.ua.edu/~musselp/proj/grano_salis/indotropica.html). It is,
however, possible that other undocumented species of freshwater mussels in the
country are introduced or not originally freshwater but were only able to adapt to
these systems. Cristaria plicata (Leach), a freshwater mussel, was introduced in
the country through the larvae that were attached to the imported carp species
from Taiwan (Guerrero 1981).
Adaptation of marine bivalves to freshwater habitats is an expression of
advancement of sexuality of bivalves (Mackie 1984). This phenomenon is
consequently accompanied by a number of changes in their life history (Byrne
3
1998). Such changes might include investing in fewer more expensive offspring,
loss or abbreviation of larval stages, nonfeeding larvae, and production of resting
stages (Lee and Bell 1999).
The elongate sunset clam, Gari elongata (Fig. 1), a mollusk belonging to
Family Psammobiidae of Class Pelecypoda, is locally known as bayuyan, in the
Banate Bay area, west central Philippines. It has been reported to be primarily
estuarine (FAO 1998). However, in the Banate Bay area West Central
Philippines, G. elongata thrives in freshwater systems such as rice paddy
irrigation canals and riverine tributaries. Its population density has been shown
to decrease with increase in salinity (del Norte-Campos 2004a). According to
this paper, this distribution pattern indicates a possible upstream transport and
consequent adaptation of this species to lower salinities. Continued flooding from
the upper branches of the riverine system, would have most likely caused the
eventual acclimatization of G. elongata to lower salinity.


Fig.1. The elongate sunset clam, Gari elongata
(Lamarck, 1818) locally known in the study area
as bayuyan
4
Preliminary results on the reproductive biology of G. elongata showed
two spawning peaks: a minor spawning between May to J une and a major
spawning between December to J anuary (del Norte-Campos 2004a). This work,
however, was based solely on Gonadosomatic Index (GSI) and males were not
separated from females. Although GSI estimation is a standard procedure in
describing reproductive activities in bivalves, it often presents a problem
because most species of bivalves (e.g. G. elongata, Donax dentifer, Pinctada
imbricata, etc.) have fused gonads, such that reproductive and non-reproductive
tissues cannot or are difficult to separate (Urban and Riascos 2002). Thus, it is
important that histological assessment of the gonads must be integrated in the
study. Gonad histology examinations give precise results on the first maturity
length and spawning period of bivalves (Sahin et al. 2006). However, gonad
index correlated with histological assessments is the most accurate method in
determining reproductive cycle and gonad maturity.
This study aims to describe some aspects of the reproductive biology of
the elongate sunset clam, G. elongata in the Banate Bay Area, West Central
Philippines. The specific objectives of the study are: a) to determine the sex
ratio, b) to describe the various gonad stages and measure oocyte size, c) to
determine the minimum size at sexual maturity, d) to determine the annual
spawning periodicity of the species and relate it to phytoplankton occurrence and
density. Reproduction of G. elongata will be examined in irrigation canal and
pond sites in the Banate Bay Area, West Central Philippines. Documentation of
5
the reproduction of the elongate sunset clam in these two areas will expand the
knowledge about its reproductive biology and will provide insights on the
reproduction of G. elongata in two relatively different sites. This study will also
note the possible changes in the life history traits of G. elongata considering that
it was reported to have been able to adapt from marine habitat to freshwater
systems.















6
REVI EW OF RELATED LI TERATURE
Taxonomy, Distribution, and Biology
G. elongata, the elongate sunset clam, is a mollusk belonging to Family
Psammobiidae of Class Pelecypoda. Its shells are elongated, moderately inflated,
variable in shape, but sub trapezoidal-ovate in outline. The outer surface of its
shell is often smooth and polished in juveniles and dull with concentric growth
marks in adults. This species reaches a maximum size of 70 mm (FAO 1998).
However, in the Banate Bay Area specimens larger than this reported maximum
size were collected (del Norte-Campos 2004a).
It occurs in the Indo-west Pacific, from East Africa, including Madagascar
and the Red Sea, to the Philippines; north to J apan and south to northern
Queensland. It is found in muddy-sand bottoms occurring in extensive beds in
intertidal and shallow subtidal levels often in bays and near mangroves (FAO
1998). Individuals of G. elongata show a burrowing habit, whereby the anterior
end is sunk into the substrate and the posterior end exposed in the overlying
water (del Norte-Campos 2004a). This particular morphological configuration
exhibits that this species is a suspension-feeder, a characteristic of its family
(Stead et al. 2002).
In the Philippines, G. elongata is actively collected for its prized meat (FAO
1998) and is considered as one of the commercially important invertebrates in
Panay (del Norte-Campos et al. 2000). Studies on G. elongata focused on some
aspects of its distribution and population biology (del Norte-Campos 2004a) and
7
physiology (del Norte-Campos 2004b). These reported that: G. elongata has a
marine ancestry and has been able to acclimatize to freshwater habitats; has a
fast growth rate which is typical of a tropical species; and has the potential as a
biofilter in fresh to brackish water polyculture set-ups.

Reproductive Biology and Management Measures
The decline of the population of freshwater bivalves is a global
phenomenon and current emphasis is on conservation of species vulnerable to
extinction (Byrne 1998). The freshwater mussel, Hyridella depressa in South-
eastern Australia has by now declined in river habitats (Byrne 1998). North
American freshwater mussels (Haag and Staton 2003) and the Margaritifera
species (Grande et al. 2001) were already listed as endangered. Proper
management of the wild populations as well as artificial propagation of the
species can mitigate this problem. However, management and conservation
efforts for freshwater bivalves are hampered by a lack of basic life history
information and determination of life history traits is a key research needed for
their recovery (Haag and Staton 2003). Such efforts require more information
on the species reproductive biology. When in risk of extinction, it is already
unadvisable to sacrifice organisms for researches and thus limiting the
knowledge in their reproductive biology. Thus, it is rational to study these
organisms before they start to decline due to overexploitation and other
anthropogenic causes.
8
Reproductive Studies of Bivalves
The reproductive biology of marine bivalves is well documented through a
long history of research (Sastry 1979). In contrast, reproduction of freshwater
bivalves especially in tropical areas is less understood (Table 1).


Species/
English
name

Site/
Substrate

Type of
Reproductive
System
Size at
Sexual
Maturity
(mm
SL)

Breeding
pattern

Oocyte
Size
(m)

Sex
Ratio
(M:F)

Reference
Anodontia
edentula
(toothless
lucine)
+

Guimaras,
Phil (mud)
Dioecious (w/
occasional
hermaphrodites)
35 Continuous
(peak:
April
May)
53.5 - Samentar,
et al. 2004
Scapharca
inequivalvis
(ark shell)
+

Banate Bay,
Phil (sandy-
muddy flats)
Dioecious 17.3 Continuous
(peak:
Sept-Dec)
1:1.34 Ledesma-
Fernandez
and del
Norte-
Campos
2004
Gari solida
(psammobiid
clam)
Chile (soft-
bottom)
Dioecious - 2 peaks
(Oct and
J an/Feb
and
Mar/Apr
- - Urban and
Campos
1994
Anadara
scapha
(antique ark)
New
Caledonia
(seagrass
beds)
protandric
hermaphrodite
22 continuous - 1:0.47 Baron 1992
Hyridella
depressa
(freshwater
mussel)*
Australia-
Hawkesbury-
Nepean
River System
(river and
lake)
hermaphroditic
(brooding)
40-43 lake:
seasonal
river:
continuous
125 - Byrne 1998
Corbicula
australis
(freshwater
clam)*
Australia-
Nepean
River System
hermaphroditic
(brooding)
10.9 continuous 125 - Byrne et al.
2000
Paxyodon
syrmatophorus
(freshwater
mussel)*
Tocantins
River, Brazil
Dioecious 23 continuous - 1 : 1 Beasley et
al. 2000
Elliptio arca
(freshwater
mussel)*
Alabama,
USA - Sipsey
River
dioecious (rare
hermaphrodites;
brooding)
68.1 - - 0.93:
1
Haag and
Staton
2003
Table 1. Summary of some reproductive studies on different marine, estuarine and
freshwater bivalve species. (mm SL= millimeter Shell Length; M:F = Male: Female)
*Freshwater species, +Tropical species
9
Literatures on bivalve reproduction are mainly concerned with gonad
development and reports on breeding periods. Reproductive periods may be
cyclical or continuous. Reproduction may be divided into three major phases:
gonad development, spawning, and fertilization. The phases of reproductive
activity are functioning continually in coordination with seasonal environmental
changes and may be determined through an interaction between endogenous
and exogenous factors (Sastry 1979).
In temperate areas, temperature is an important triggering factor in
bivalve gametogenesis and spawning (Sastry 1979; Mackie 1984). Because of its
pronounced seasonality, peaks of reproductive activities of temperate bivalves
generally coincide with the drop or increase in the temperature. In tropical
areas, however, temperature variation is minimal over a year. Nonetheless,
short term or daily temperature fluctuations of variable magnitude may trigger
spawning in Gafrarium tumidum, Atactodea striata, and Anadara scapha in areas
of shallow depth (Baron 1992).
In areas particularly affected by monsoons and heavy rains, wide and
periodic salinity variation may occur (Baron 1992) which may serve as signal for
spawning. Low salinity causes intense spawning in the ark shell, Scapharca
inequivalvis, an inhabitant of shallow intertidal mudflats near river mouths
(Ledesma-Fernandez and del Norte-Campos 2004). Salinity per se may not
influence reproductive processes in freshwater bivalves. However, the increase in
the amount of organic matter and the amount of food available caused by
10
precipitation and run off during southwest monsoon months can influence
bivalve reproduction (Ledesma-Fernandez and del Norte-Campos 2004).
The intimate association of the digestive and the reproductive tissues has
a functional significance in bivalve reproduction. As the animal grows, most of
the energy derived from digestion is partitioned directly or indirectly to
reproductive processes (Mackie 1984). The quantity and/or quality of the
available food may influence reproductive processes. In the tellinacean bivalves,
Semele solida and Tagelus (Tagelus) dombeii, gonad index cycles were at
highest values when the density of the micro flagellate and centric diatoms was
at their peaks (Stead et al. 2002). Well-developed and fecund visceral mass of
Hyridella depressa in the river indicates that they were in a good nutritive state
supported by eutrophic, phytoplankton-rich conditions of the area (Byrne 1998).

Colonization of Freshwater Systems by Marine Ancestors of Bivalves
Colonization of freshwater habitats by marine ancestors of the corbiculid
clam, Corbicula australis was fairly a recent event (McMahon 1983). The
dioecious, free spawning and dispersive life history seen in many marine species,
which are representative of ancestral condition, are modified (Byrne et al. 2000).
This modification involved a major change in larval life from a pelagic to a non-
pelagic incubatory one (McMahon 1983). The majority of freshwater bivalves
brood their young in modified portions of their gills called marsupial.
Development with glochidia larvae in these organisms is atypical of bivalves.
11
These larvae evolved from the free-living veligers of their ancestors and are
obligate parasites, usually on fish host, for a variable period of time prior to
metamorphosis (Kat 1984). The modified life history strategy may involve a
short reproductive life, high fecundity and predictable recruitment. Furthermore,
alterations may be exhibited through this suite of life history traits:
hermaphroditism, potential for self-fertilization and brooding progeny to the
crawl-away juvenile stage, which are typical of bivalves that inhabit disturbance -
prone lotic habitats (Byrne et al. 2000).
Transitions to freshwater have occurred not only on macroevolutionary
scales, but also recently, continuing to the present day. Of particular interest is
the marked increase, in historical times, of freshwater invaders not previously
known to occur in freshwater and without obvious mechanisms of tolerating
freshwater conditions (Lee and Bell 1999). These radiations are gaining
importance not only on evolution aspect, but also on ecology, since these
migrations might cause invasion or introduction of new species.







12
MATERI ALS AND METHODS
Study Area and Sampling Protocol
Collection of samples was conducted in Barangay Bugnay, Barotac Viejo,
Iloilo (Fig. 2). Clams were collected manually from an irrigation canal (Fig. 2a)
from September 2004 to February 2005 and J une to August 2005. However,
when the canal dried up on March to May 2005 (summer months), collection was
conducted in a pond approximately 300m away from the irrigation canal (Fig.
2b).











Fifty clams were collected monthly at every last quarter of the lunar
phase. After collection, the clams were packed with ice for transport to the
laboratory. During each sampling, salinity measurements were taken using a
Celebes Sea
Sulu Sea
South
China
Sea
Pacific
Ocean
a b
PANAY
Fig.2. Site of collection of the clam G. elongata: a) irrigation canal (depth
~0.5m), b. pond (depth <0.5m) in Barotac Viejo, Iloilo, Panay Island,
Philippines (modified from del Norte Campos 2004a)

13
refractometer. Phytoplankton was sampled by filtering 15 liters of water with a
phytoplankton net with a mesh size of 25m. Phytoplankton samples were
afterwards preserved in 10% formalin.
To verify and compare reproduction of clams in irrigation canal and pond,
supplementary sampling using the same protocol was conducted in both sites
from March 2006 to December 2006.

Laboratory Processing of Samples
Clams were measured, weighed and dissected. Shell lengths were
measured to the nearest 0.05 mm using a Vernier caliper. Total weight (TW),
viscera weight (VW), and gonad weight (GW) of each individual were taken to
the nearest 0.01 g using a digital electronic balance.
The reproductive condition of the specimen was assessed by examination
of the visceral mass and the gills for the presence of embryos. Gonads were then
fixed in Bouins solution, dehydrated, embedded in paraffin, cut transversely at a
thickness of 5 m with a microtome, placed on slide, stained with hematoxylin
and counter-stained with eosin (Humason 1972).

Sex, Sexual Maturity and Gonad Development
Determination of sexes of the individuals was done based on the prepared
slides examined under a compound microscope. Monthly and overall sex ratios
were computed after sexes have been determined. A Chi-square test was used
14
to check the hypothesis that males and females are equally represented in the
samples.
Size at sexual maturity was determined based on the smallest shell length
(SL) of the clam staged with a ripe or mature gonad.
Gonads were classified into the following stages of maturity: developing,
mature, redeveloping, partially spawned and spent, based on the morphological
appearance of the predominant gametes present using comparisons with related
literature (e.g. Ledesma-Fernandez 2004; J uhel et al. 2003; Grande et al. 2001;
Byrne et al. 2000; Byrne 1998). Monthly frequencies of gonad stages were
plotted against time.
The quantitative Gonadosomatic Index (GSI %) of each individual was
computed using the equation of Sastry (1979), as follows:

_GW_
GSI (%) = VW X 100

where GW = gonad weight (g)
VW = viscera weight (g)

An index of maturity (= qualitative GSI) was also computed by assigning
values to the gonad stages, as follows: 1 = spent, 2 = developing, 3 = ripe, 2 =
partially spawned, and 2 = redeveloping. These values are so designated to
indicate the degree of deviation from the highest possible gonad stage of
maturity (ripe = 3.0). Mean values of both gonad indices were plotted against
time to assess the pattern of reproductive maturity.
15
Three developing gonads were chosen randomly in each site and all
oocytes in these gonads were measured using a micrometer eyepiece. The same
procedure was also done on mature gonads. Oocytes were stripped from one
mature female collected from the irrigation canal and thirty of these oocytes
were also measured.

Data Analysis
T-test for independent samples was used to establish if there is a
significant difference in the shell lengths of the clams from the pond and the
irrigation canal. For the months when samples were collected at both sites,
factorial ANOVA was used to determine if there is a significant difference and
interactions in the GSI (%) and Index of Sexual Maturity between sexes,
between sites and among months. Homogeneity of variances was tested using
Bartletts test. Statistical analyses were done using the STATISTICA Software.

Phytoplankton Sample Analysis
The volume of the phytoplankton to be examined was standardized to 20
ml before identification of organisms was done. From this volume, a species
cumulative curve was constructed. Based on the results, three 1-ml replicates
were taken for each monthly sample. Fifty random grids of the Sedgewick-
Rafter (SR) were evaluated and based on this and on the standardized 20 ml
volume, a raising factor (RF) was calculated as follows:
16
RF = total # of grids in the SR_ X 20
# of grids analyzed

Examination of subsamples was done using a Sedgewick-Rafter, and not a
hemacytometer since the former allows for a bigger volume of water to be
examined. This is important considering that a bigger volume of water in the
field had to be sampled, compared to laboratory/experimental set-ups, where
use of a hemacytometer is the usual practice. Furthermore, the literature shows
that Sedgewick-Rafters are commonly used in examining phytoplankton densities
sampled from the field (Larsen and Nguyen 2004; Eker et al. 1999; Yamaji
1962).
Phytoplankton organisms in each replicate were identified to the lowest
level possible based on literature (Gran and Angst 1931; Shirota 1966). The
organisms were then grouped together into major taxa and the corresponding %
composition was computed. Mean densities in no. of individuals L
-1
were
computed according to the following equation:
Mean density = RF * (mean count)
total volume filtered
and plotted against time. Mean densities between two sites were tested for
significant variation using T-test for independent samples. Gonad development
was correlated with the phytoplankton densities.



17
RESULTS

G. elongata in the pond and in the irrigation canal formed dense
aggregations on the substrate. With a single manual collection, three-five
individuals can be gathered. Water depth in the irrigation canal is from 0.5 1
meter. In the pond, the banks are shallow while the middle portion is deep
(>1m). Water is turbid at both sites and salinity was consistently zero during the
whole sampling period. Both areas have vegetation at the banks. Substrate in
both sites is mud. The stretch of the irrigation canal opens to the Alacaygan
River in the Banate area. On the other hand, the pond was a tributary of the
same river but was closed when converted for rearing of tilapia.
During the dry months (March to April 2005) water in the irrigation canal
dried out because the area was dammed to control the supply of water to the
canal. However, in summer 2006, water level in the canal though reduced, did
not dry out since it was a relatively wet year. During this sampling year, it was
possible to collect samples at these months in both sites. During low water level
these clams were observed to aestivate, burrowing deeper in the substrate.
A total of 1,600 individuals were collected all throughout the sampling
period, 950 individuals were from the irrigation canal. Of these 466 were female
and 439 were male exhibiting a sex ratio of 1: 1.06 (Table 2). Female to male
ratio based on Chi-square test (
2
= 0.074) does not deviate significantly from
the expected 1: 1 sex ratio.

18

Months Total No. of Samples Hermaphrodite Indeterminate Male Female Sex Ratio (M:F)
Sep '04 50 24 26 1: 1.08
Oct 50 28 22 1: 0.79
Nov 50 26 24 1: 0.92
Dec 50 23 27 1: 1.17
J an '05 50 28 22 1: 0.79
Feb 50 1 23 26 1: 1.13
J un 50 3 21 26 1: 1.24
J ul 50 1 15 19 15 1: 0.80
Aug 50 27 23 1: 0.85
Mar '06 50 1 2 23 24 1: 1.04
Apr 50 1 27 22 1: 0.81
May 50 5 22 23 1: 1.04
J un 50 1 8 15 26 1: 1.73
J ul 50 1 27 22 1: 1.21
Aug 50 23 27 1: 1.17
Sep 50 20 30 1: 1.50
Oct 50 25 25 1: 1.00
Nov 50 2 18 30 1: 1.67
Dec 50 4 20 26 1: 1.30
Total 950 9 36 439 466 1: 1.06
% 0.95 3.79 46.21 49.05

Thirty-six gonads (3.79%) were indeterminate (Fig. 3a) characterized by
indistinct gonad regions and no gamete proliferation. Nine (0.95%) individuals
were hermaphrodites (Fig. 3b) containing both male and female gametes either
organized in separate follicles or muddling in the same follicle.








Table 2. Monthly and overall sex ratios of G. elongata from the irrigation canal
in Brgy. Bugnay, Barotac Viejo, September 2004 February 2005, J une
August 2005 and March December 2006.


a

b

a

a

b

b
Figs. 3a-b. a. Gonad of indeterminate G. elongata, b. Gonad of
hermaphroditic G. elongata showing male and female gametes in
separate follicles

19
From the pond, 650 organisms were collected. Sixteen individuals (2.46%)
were indeterminate and three (0.46%) were hermaphrodites. Male to female
ratio was 1: 1.23 (Table 3) and based on Chi-square test (
2
= 0.000) this ratio
is highly skewed in favor of females.


Months Total No. of Samples Hermaphrodite Indeterminate Male Female Sex Ratio (M:F)
Mar '05 50 24 26 1 : 1.08
Apr 50 23 27 1 : 1.17
May 50 1 22 27 1 : 1.23
Mar '06 50 28 22 1 : 0.79
Apr 50 19 31 1 : 1.63
May 50 1 28 21 1 : 0.75
J un 50 1 24 25 1 : 1.04
J ul 50 23 27 1 : 1.17
Aug 50 8 15 27 1 : 1.80
Sep 50 1 3 14 32 1 : 2.29
Oct 50 1 22 27 1 : 2.23
Nov 50 1 18 31 1 : 1.72
Dec 50 1 1 23 25 1 : 1.09
Total 650 3 16 283 348 1 : 1.23
% 0.46 2.46 43.54 53.54


The mean shell length of the clams in the irrigation canal was 63.36mm
while in the pond was 70.79mm. In the pond, clams were mostly from 60 to
79.99mm while in the irrigation canal, clams were generally from 50 to 79.99mm
(Fig. 4). Based on tests of means (T-test for independent samples) there is a
significant difference (df=1598, p=0.000) between the shell lengths of G.
elongata from the pond and from the irrigation canal indicating that clams from
the irrigation canal are smaller compared to the clams from the pond.


Table 3. Monthly and overall sex ratios of G. elongata from the pond in Brgy.
Bugnay, Barotac Viejo, March May 2005 and March December 2006.
20
0.00
10.00
20.00
30.00
40.00
50.00
0-9.99 10-19.99 20-29.99 30-39.99 40-49.99 50-59.99 60-69.99 70-79.99 80-89.99 90-99.99
SL (mm)
F
r
e
q
u
e
n
c
y

(
%
)
Canal Pond



Size at Sexual Maturity
Sexual maturity of G. elongata in the pond was attained at 44.80mm and
59.10mm in female and male, respectively. On the other hand, those in the
irrigation canal reached maturity at 43.15mm and 43.70mm in female and male,
respectively. This illustrates that individuals in the canal mature early compared
to the individuals from in the pond. At both sites, females mature at smaller
sizes compared to males.

Gonad Morphology and Histology
G. elongata is a dioecious bivalve that exhibits no sexual dimorphism.
Although hermaphroditism was present it is never the predominant state.
Gonads are cream in color regardless of stage, size and sex. Thus, determination
of sex and gonad stage is not possible by macroscopic examination of the
gonads and external morphology of the organism.
Fig. 4. Shell length distributions of G. elongata from irrigation canal and pond
in Brgy. Bugnay, Barotac Viejo, Iloilo
21
G. elongata has a massive gonad comprising a major part of the visceral
mass. In both sexes, reproductive tissues are entangled with visceral mass
which is composed of the stomach and the intestinal loop. Gonad histology
showed that these digestive tissues were diffused within the gonads. The ovarian
follicles and the spermatogenic tubules are dispersed organs surrounded by
connective tissues. In both male and female acini simultaneous presence of
totally mature and developing gametes were observed.
Testicular follicles or tubules are rounded to elongate in developing
stages. In mature gonad the tubules assumed a rosette shape. In developing
ovarian follicle nutritive cells or nurse cells were observed. They were much
smaller compared to oogonia and were numerous. As the ovarian follicle
matures depletion of these cells occurred. A ripe follicle contained three four
mature oocytes. A mature oocyte is free, large, elongated to spherical with a
large prominent nucleus.

Oocyte size
Mean oocyte size in developing female gonads was 61.67m in the canal
and 57.78m in the pond. In mature females, mean oocyte size was 86.36m
and 87.59m in the canal and in the pond, respectively. Smear oocyte
measurement showed that mean oocyte size from a mature female collected
from the irrigation canal was 103.63 m. This shows that there is a considerable
shrinkage in oocyte during histological processing.
22
Reproductive Condition
Examination of the gills of G. elongata showed that they brood their
young in their gills (Figs. 5a-b). Gravid females were observed both in the
irrigation canal and in the pond. In the irrigation canal, 0.46% of females (SL:
55.10 61.10 mm) were observed to brood on J uly 2006. In the pond, 1.58% of
females were gravid: five (SL: 69.50 81.95 mm) were observed on September
2006 and ten (SL: 67.1 82.95 mm) on November 2006. The smallest gravid
female was observed in the pond with the shell length of 55.1mm. Gonads of
gravid females were all in partially spawned stage.



Gonad Stages
Gonad development of G. elongata based on histological examination was
described in Table 4: developing, ripe, partially spawned, redeveloping and spent
(Figs. 6a-e in females and Figs. 7a-e in males).


Figs. 5a-b. a. Brooding gills of gravid female G. elongata; b. embryos
of G. elongata teased off from the gills
a b
23





Stage Male Female
1. Immature/
indeterminate
No definite gonad region or follicle. No gamete
proliferation. Sex cannot be determined.
2.Developing Small and rounded
tubules. Spermatogonia
start to develop at the
edges of the acinus.
Expanded tubules have
smaller cells at the center
and larger cells lining the
edges. Some spermatozoa
with tail start to proliferate
at the center.
Acini filled with nutritive
cells. Oogonia at different
stages of development.
Oocytes are attached to the
edges but starting to fill the
follicles. Nutritive cells start
to disappear at later stages
giving more spaces for
developing oocytes.
3.Ripe Tubules are elongated
assuming rosette
formation and filled with
mature sperm.
Follicles are elongated. Free
oocytes at the center of the
acini, mature oocytes with
prominent nucleus and
nucleolus.
4. Partially
spawned
Tubules show streaky
appearance of streaming
sperms. Spermatozoa still
occupy the lumina but
have numerous gaps, may
have few spermatocytes or
spermatids.

Spacious acini, remaining
mature oocytes at some
acini.
5.Redeveloping Connective tissue present.
Triangle-shaped tubules.
Hemocytes present in the
tubules and outside
generally surrounding
residual spermatozoa.
Connective tissues present.
Oocytes at different stages
are present. Some follicles
are empty.
6.Spent Tubules contain few or no
residual spermatogenic
cells at the follicle wall.
Empty follicles. May contain
residual oocytes at the
connective tissues.
Table 4. Gonad development of stages of the testicular and ovarian regions of G.
elongata
24




































oogonia
nutritive cells
mature oocyte
nucleus
residual oocyte
broken follicle wall
oogonia
connective tissue
oogonia
nutritive cells
mature oocyte
nucleus
residual oocyte
broken follicle wall
oogonia
connective tissue
Figs. 6a-e. Gonad stages of
development of female G.
elongata a. developing, b.
mature, c. partially spawned, d.
redeveloping, e. spent. Scale =
80m
25






















spermatogonia
spermatocytes
spermatozoa
spacious tubule
connective tissue
connective tissue
residual spermatozoa
spermatogonia
spermatocytes
spermatozoa
spacious tubule
connective tissue
connective tissue
residual spermatozoa
Figs. 7a-e. Gonad stages of
development of male G.
elongata a. developing, b.
mature, c. partially spawned,
d. redeveloping, e. spent.
Scale = 150m
26
Gonad Development
In the irrigation canal, during the 2004-2005 sampling, both male and
female clams have particularly high frequency of mature gonads on J uly to
August 2005; partially spawned gonads on November for female and J une for
male; and developing gonads on September 2004 and December 2004 to
February 2005 for female and September 2004 to February 2005 for male (Figs.
8-9). However, during the 2006 sampling period, male and female clams have
highest frequency of partially spawned gonads on September and continued to
be high until December (Figs. 8-9). This is contrary to the results of the previous
sampling period since during these months more individuals were in the
developing stage than in the partially spawned stage.
In the pond, on the other hand, during the March to May 2005 sampling,
high frequency of spawning and mature gonads was observed on March and May
in female clams (Fig. 10) and on March to April in male clams (Fig. 11). During
the 2006 sampling, both male and female clams have highest frequency of
mature and partially spawned gonads on March and April, respectively (Figs. 10-
11) which are consistent with the results during the previous year. Developing
gonads were observed on all months with the highest frequency on J uly in
females (Fig. 10) and on August in males (Fig. 11).



27















Fig. 8. Frequency distribution of gonad stages in female G. elongata collected from an irrigation canal in Brgy. Bugnay,
Barotac Viejo, September 2004 - February 2005, J une 2005 August 2005 and March 2006 December 2006.
Fig. 9. Frequency distribution of gonad stages in male G. elongata collected from an irrigation canal in Brgy. Bugnay,
Barotac Viejo from September 2004 - February 2005, J une 2005 August 2005 and March 2006 December 2006.
0.00
20.00
40.00
60.00
80.00
100.00
S
e
p
t

'0
4
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o
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J
a
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F
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b
M
a
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p
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a
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A
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S
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o
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D
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J
a
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'0
6
F
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a
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a
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J
u
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J
u
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A
u
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S
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p
O
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t
N
o
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D
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c
Months
F
r
e
q
u
e
n
c
y

(
%
)developing
mature
partially spawned
redeveloping
spent
0.00
20.00
40.00
60.00
80.00
100.00
S
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p
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'0
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a
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a
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a
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'0
6
F
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a
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a
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J
u
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J
u
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A
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S
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p
O
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t
N
o
v
D
e
c
Months
F
r
e
q
u
e
n
c
y

(
%
)developing
mature
partially spawned
redeveloping
spent
28
F

Fig. 10. Frequency distribution of gonad stages in female G. elongata collected from a pond in Brgy. Bugnay, Barotac
Viejo, March 2005 May 2005 and March 2006 December 2006.
Fig. 11. Frequency distribution of gonad stages in male G. elongata collected from a pond in Brgy. Bugnay, Barotac Viejo,
March 2005 May 2005 and March 2006 December 2006.
0
20
40
60
80
100
S
e
p

'0
4
O
c
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N
o
v
D
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b
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a
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p
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'0
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A
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p
O
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N
o
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D
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c
Months
F
r
e
q
u
e
n
c
y

(
%
)developing
mature
partially spawned
redeveloping
spent
0
20
40
60
80
100
S
e
p

'0
4
O
c
t
N
o
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D
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a
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a
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'0
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F
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A
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N
o
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D
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Months
F
r
e
q
u
e
n
c
y

(
%
)developing
mature
partially spawned
redeveloping
spent
29
In both sites and sexes, redeveloping stage was noted on almost all
months. However, it was observed to be predominantly high on J uly to October
(Figs. 8-11) coinciding with the high frequency of partially spawned gonads. This
shows that redevelopment of gonads occur after spawning. Also, only few spent
stages were observed. This further supports that gonads redevelop after
spawning and that gonad development is protracted or continuous.
There is a monthly variation in gonad stages as supported by the co-
occurrence of gametogenic stages in different frequencies within the sampled
monthly population. This pattern of variability shows that gonad development in
this species is continuous with variation in the intensity of the reproductive
activities. These results show that seasonality has the major effect on the
variation of gonad development in this species in both sites.










30
Gonad I ndices
I. Gonadosomatic Index (GSI)
In the irrigation canal, GSI ranged from 6.98 to 87.71 % in females (Fig.
12a) and 3.13 to 69.14 % in males (Fig. 12b).















Multiple peaks were observed in both female and male clams on the
months of September 2004, November 2004, February 2005, J uly 2005, April
2006, J uly 2006, and September 2006 (Figs. 13-14). Presence of multiple peaks
Fig. 12a-b. Gonadosomatic index (GSI) of a) female and b) male
G. elongata collected from irrigation canal in Brgy. Bugnay,
Barotac Viejo, from September 2004 - February 2005, J une 2005
August 2005 and March 2006 December 2006.

0.00
20.00
40.00
60.00
80.00
100.00
0.00 20.00 40.00 60.00 80.00 100.00
SL (mm)
M
e
a
n

G
S
I

(
%
)
0.00
20.00
40.00
60.00
80.00
100.00
0.00 20.00 40.00 60.00 80.00 100.00
SL (mm)
M
e
a
n

G
S
I

(
%
)
b
a
G
S
I

(
%
)
SL (mm)
0.00
20.00
40.00
60.00
80.00
100.00
0.00 20.00 40.00 60.00 80.00 100.00
SL (mm)
M
e
a
n

G
S
I

(
%
)
0.00
20.00
40.00
60.00
80.00
100.00
0.00 20.00 40.00 60.00 80.00 100.00
SL (mm)
M
e
a
n

G
S
I

(
%
)
b
a
G
S
I

(
%
)
SL (mm)
b
a
G
S
I

(
%
)
SL (mm)
31
suggests continuous gonad development and spawning. On September 2004 to
February 2005, only a minimal fluctuation on the GSI values was observed.
However, during the 2006 sampling, abrupt decrease of GSI was noted on April
to J une and on September to December (Figs. 13-14). This difference in the
pattern of GSI between the sampling periods is also seen on histological
assessment of the gonads showing that on September to December 2004 most
gonads were in the developing stage while on September to December 2006
most were in the partially spawned stage (Figs. 8-9).

32










Fig. 13. Mean gonadosomatic index (GSI) of female G. elongata collected from irrigation canal
(n= 439) and pond (n=348) in Brgy. Bugnay, Barotac Viejo, September 2004 - August 2005
and March 2006 December 2006.
Fig. 14. Mean gonadosomatic index (GSI) of male G. elongata collected from irrigation canal
(n=466) and pond (n=283) in Brgy. Bugnay, Barotac Viejo, September 2004 - August 2005 and
March 2006 December 2006.

10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
60.00
S
e
p

'
0
4
O
c
t
N
o
v
D
e
c
J
a
n

'
0
5
F
e
b
M
a
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A
p
r
M
a
y
J
u
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J
u
l
A
u
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S
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p
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N
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J
a
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'
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6
F
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J
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A
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S
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p
O
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N
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v
D
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c
Months
M
e
a
n

G
S
I

(
%
)
F Canal F Pond

10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
60.00
S
e
p
t

'
0
4
O
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N
o
v
D
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J
a
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'
0
5
F
e
b
M
a
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A
p
r
M
a
y
J
u
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J
u
l
A
u
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S
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p
O
c
t
N
o
v
D
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J
a
n

'
0
6
F
e
b
M
a
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A
p
r
M
a
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J
u
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J
u
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A
u
g
S
e
p
O
c
t
N
o
v
D
e
c
Months
M
e
a
n

G
S
I

(
%
)
M Canal M pond
33
In the pond, GSI ranged from 10.84 to 84.69 % in female (Fig. 15a) and
8.18 to 49.00 % in males (Fig. 15b).
















In both male and female, GSI is at its highest on the months of April and
October. Decrease in GSI was observed from March to May 2005, April to J uly
2006 and October to December 2006 (Figs. 14-15) which suggests spawning
periods. In this site, two peaks were observed: a major peak on April and a
Fig. 15a-b. Gonadosomatic index (GSI ) of a) female and b) male G.
elongata collected from a pond in Brgy. Bugnay, Barotac Viejo, March
2005 May 2005 and March 2006 December 2006.

0.00
20.00
40.00
60.00
80.00
100.00
0.00 20.00 40.00 60.00 80.00 100.00
SL (mm)
M
e
a
n

G
S
I

(
%
)
0.00
20.00
40.00
60.00
80.00
100.00
0.00 20.00 40.00 60.00 80.00 100.00
SL (mm)
M
e
a
n

G
S
I

(
%
)
G
S
I

(
%
)
SL (mm)
a
b
0.00
20.00
40.00
60.00
80.00
100.00
0.00 20.00 40.00 60.00 80.00 100.00
SL (mm)
M
e
a
n

G
S
I

(
%
)
0.00
20.00
40.00
60.00
80.00
100.00
0.00 20.00 40.00 60.00 80.00 100.00
SL (mm)
M
e
a
n

G
S
I

(
%
)
G
S
I

(
%
)
SL (mm)
a
b
34
minor peak on October. Based on gonad histology highest frequency of partially
spawned gonads was observed on April 2006 (Figs. 10-11).
Based on Bartletts Test ( Bartlett Chi-square = 271.09) variances of mean
GSI were not homogenous so data were normalized using arcsine transformation
and factorial ANOVA was ran on the transformed data. During the March to
December 2006 sampling, a significant fluctuation in the monthly mean GSI (F =
147.84) in both sites (F = 93.73) was observed. This variation was evident in
the multiple peaks of the GSI values in the irrigation canal and in the two
spawning peaks in the pond (Figs. 14-15). However, variation in the mean GSI
between male and female in both sampling sites was not evident (F = 2.89).
Seasonality has the major effect on the variation in the mean GSI between the
two sampling sites (F = 98.48). These effects and interactions computed through
factorial ANOVA are summarized in Table 5.



Effects df F p-level
month 9 147.84 0.00*
site 1 93.73 3.58E-21*
sex 1 2.89 0.09
month x site 9 98.48 0.00*
month x sex 9 0.78 0.63
site x sex 1 3.06 0.08




Table 5. Summary of factorial ANOVA testing
arcsine transformed Mean GSI (%) by
month and sex at two sampling sites (*
Significant difference)

35
II . I ndex of Sexual Maturity

Mean values of index of sexual maturity (qualitative GSI) in the canal for
females ranged from 1.81 to 2.39 (Fig. 16) while in males was from 1.90 to 2.74
(Fig. 17). In both sexes in the irrigation canal, mean qualitative GSI is highest
on August when most clams have mature gonads based on histological
examinations (Figs. 8-9).
In the pond, mean values for females ranged from 1.85 to 2.36 (Fig. 16)
while in males was from 1.91 to 2.54 (Fig. 17). Highest mean qualitative GSI in
the pond in both sexes was observed on March when most of the clams have
mature gonads based on gonad histology (Figs. 10-11).




36







Fig. 16. Index of Sexual Maturity of female G. elongata collected from irrigation canal (439) and
pond (348) in Brgy. Bugnay, Barotac Viejo, September 2004 - August 2005 and March 2006
December 2006.

Fig. 17. Index of Sexual Maturity of male G. elongata collected from irrigation canal (n=466) and
pond (n=283) in Brgy. Bugnay, Barotac Viejo, September 2004 - August 2005 and March 2006
December 2006.
0.00
1.00
2.00
3.00
4.00
S
e
p
t

'
0
4
O
c
t
N
o
v
D
e
c
J
a
n

'
0
5
F
e
b
M
a
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A
p
r
M
a
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J
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J
u
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A
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S
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p
O
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J
a
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'
0
6
F
e
b
M
a
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A
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r
M
a
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J
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J
u
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A
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S
e
p
O
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N
o
v
D
e
c
Months
M
e
a
n

I
n
d
e
x

o
f

S
e
x
u
a
l

M
a
t
u
r
i
t
y
F Canal F pond
0.00
1.00
2.00
3.00
4.00
S
e
p
t

'
0
4
O
c
t
N
o
v
D
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J
a
n

'
0
5
F
e
b
M
a
r
A
p
r
M
a
y
J
u
n
J
u
l
A
u
g
S
e
p
O
c
t
N
o
v
D
e
c
J
a
n

'
0
6
F
e
b
M
a
r
A
p
r
M
a
y
J
u
n
J
u
l
A
u
g
S
e
p
O
c
t
N
o
v
D
e
c
Months
M
e
a
n

I
n
d
e
x

o
f

S
e
x
u
a
l

M
a
t
u
r
i
t
y
M Canal M Pond
37
Bartletts test (Bartlett Chi-square = 33.03) shows that variances in the
mean index of sexuality are homogenous so factorial ANOVA was ran on the
original data. During the March to December 2006 sampling, there was a highly
significant monthly (F= 6.70) variation in the mean index of sexual maturity and
in the pattern of seasonal variability between the two sampling sites (F = 21.65)
as also shown in the quantitative GSI. A significant difference in the mean values
of the index between sexes (F= 16.49) was also noted. This is contrary to the
results of the quantitative GSI because quantitative GSI was based on gonad
weights and in this species the gonad is intertwined with the digestive loop which
can affect the value of the index. On the other hand, qualitative GSI was based
on gametogenic stages as characterized on histological preparations of the
gonads. In Table 6, it can be noted that interactions of month and site do not
cause any significant difference on the quantitative index of male and female
clams. However, there was a seasonal contribution on the difference of index
values between sites similar to what is exhibited by the quantitative GSI.


Effects df F p-level
month 9 6.70 2.43E-09*
site 1 21.65 3.75E-06*
sex 1 16.49 5.32E-05*
month x site 9 7.54 1.06E-10*
month x sex 9 0.93 0.50
site x sex 1 0.40 0.53


Table 6. Summary of factorial ANOVA testing
Index of Sexual Maturity by month and sex
at two sampling sites (* Significant
difference)
38
Major Composition of Phytoplankton
A total of 81 genera were identified in the irrigation canal and in the pond.
These were grouped into five major taxa: blue-green algae, diatoms, euglenoids,
green algae and others (silicoflagellates and dinoflagellates) (Figs. 18-19).
Unidentified group consisted of phytoplankton fragments that were too small to
be identified.
In both sites, diatoms were the most abundant group. However, these
were more abundant in the irrigation canal (56%) than in the pond (41%). On
the other hand, euglenoids, green and blue-green algae had a slightly higher
abundance in the pond than in the irrigation canal.

Green algae
13%
Euglenoids
10%
Others
5%
Unidentified
5%
Blue-green
algae
11%
Diatoms
56%



Fig. 18. Percentage composition of phytoplankton collected from an
irrigation canal in Brgy. Bugnay, Barotac Viejo from September 2004 -
February 2005, J une 2005 August 2005 and March 2006 December
2006.
39
Others
5%
Green algae
22%
Euglenoids
15%
Diatoms
41%
Blue-green
algae
14%
Unidentified
3%





Mean Density of Phytoplankton
Overall mean density of phytoplankton in the irrigation canal was 4181.83
ind L
-1
. Highest density (8610.37 ind.L
-1
) was attained on J une 2006 (Fig. 20).
Euglenoids with a mean density of 8316.44 ind.L
-1
contributed considerably to
this density. Particularly high densities were observed in the months of October
and J anuary. In these months almost all taxa were constantly abundant. In April
2006, there was no collection of phytoplankton because water level in the canal
was very low.
In the pond, overall mean density of phytoplankton was 1126.70 ind L
-1
.
Monthly densities in pond were more or less consistent and no month exhibited
an extraordinary high density although significantly high densities were observed
on March, May, and J uly 2006 (Fig. 21). Blue-green algae contributed
significantly in the March density while, euglenoids in May and J uly 2006. Total
mean density between the two sampling sites is significantly variable (t stat =
3.90; df = 15; p =0.000717).
Fig. 19. Percentage composition of phytoplankton collected from
collected from a pond in Brgy. Bugnay, Barotac Viejo, March 2005
May 2005 and March 2006 December 2006.

40
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
S
e
p
t

'0
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S
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a
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'0
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F
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b
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a
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a
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J
u
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J
u
l
A
u
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S
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p
O
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N
o
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D
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Months
O
v
e
r
a
l
l

M
e
a
n

D
e
n
s
i
t
y

(
i
n
d
.
L
-
1
)


0
1000
2000
3000
4000
5000
6000
7000
8000
9000
S
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'0
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Fig. 20. Phytoplankton overall mean density collected from an irrigation canal in Brgy. Bugnay, Barotac Viejo
from September 2004 - February 2005, J une 2005 August 2005 and March 2006 December 2006.
Fig. 21. Phytoplankton total mean density collected from a pond in Brgy. Bugnay, Barotac Viejo, March 2005
May 2005 and March 2006 December 2006.
41
DI SCUSSI ON
Reproductive Biology
The adaptation of G. elongata to freshwater systems was reported in 2004
(del Norte Campos 2004a). This is the first study reporting that this species
which is supposed to have an estuarine, littoral to sublittoral habitats (FAO 1998)
was able to possibly acclimatize to freshwater systems. This adaptation was
attributed to continued flooding from the upstream area (del Norte Campos
2004a). However, flooding events in a river estuary in Australia have lethal and
sublethal effects on Soletellina alba, a bivalve which also belongs to the same
family as G. elongata. Nonetheless, there are only few ecological studies of
intermittent estuaries thus a very limited knowledge on how organisms persist in
these highly dynamic environments (Matthews and Fairweather 2004).
Interactions between salinity and other physico-chemical factors are important
aspects that are valuable in explaining adaptive mechanisms.
The recent account of transition of habitat of G. elongata (del Norte-
Campos 2004a) adds up to the list of recent invasions of marine species of
freshwater habitats. Depauperate systems like reservoirs and polluted waters are
prone to colonization because competition from indigenous fauna is less (Lee and
Bell 1999). Colonization of these habitats entails morphological and physiological
radiation on the part of the invader in order to survive the new environment.
Among these radiations, life history modifications are essential to ensure
42
reproductive success and consequently survival of the organisms in the newly
colonized system.
In this study, individuals of G. elongata were observed to aestivate in the
canal during dry months (March to May 2005) when water level was reduced.
Drought-tolerant species of freshwater bivalves can aestivate from months to
years (Byrne 1998). Burrowing deeper into the substrate or aestivation of
freshwater bivalves is a response to the erratic water regime of the environment,
which also affects availability of potential food in the water column. Corbicula
luminalis burrows deeper into the substrate when disturbed or availability of
planktonic food becomes limited (J ohnson 1987, as cited in del Norte-Campos
2004a). Furthermore, Macoma balthica (Baltic Macoma) is able to shift from a
suspension feeder to deposit feeder as particle concentration in the water
column is reduced (Stead et al. 2002).
Marine bivalves which are generally dioecious and broadcast spawners
exhibit external fertilization and have planktonic larvae. However, once they
have acclimatized to freshwater systems these characteristics are eventually
modified. Although G. elongata is a dioecious species, sporadic presence of
hermaphroditic gonads was observed. Anodontia edentula (Samentar et al.
2004), Scapharca inequivalvis (Ledesma-Fernandez and del Norte-Campos 2004)
and Paxyodon syrmatophorus (Beasley et al. 2000) are dioecious bivalves but
have also occasional incidences of hermaphrodites. Presence of hermaphrodites
in low percentage does not necessarily mean that an organism deviates from
43
being functionally dioecious. For an organism to be functionally hermaphrodite,
the gonad must contain more than 50% female tissue in order to produce eggs
(Downing et al. 1989). Hermaphroditism occurs due to variable reasons such as
rapid changes in the environmental and nutritional conditions (Baron 1992);
alteration of hormone levels due to developmental errors or parasite infestation
of the gonads (Kat 1983); and most importantly to ensure reproductive success
of individuals found at low population densities (Downing et al. 1989).
Most freshwater bivalves that are functional hermaphrodites have internal
fertilization and are incubatory such as Lamellidens corrianus (Nagabhushanam
and Lohgaonker 1978), H. depressa (Byrne 1998), C. australis (Byrne et al.
2000), Corbicula leana (Park and Chung 2004), and Megalonaias nervosa
(Haggerty et al. 2005). However, G. elongata is a dioecious species but exhibit
incubatory behavior (brood their embryos in their gills). This characteristic is also
exhibited by P. syrmatophorus (Beasley et al. 2000). A study on the reproduction
of G. solida in Chile did not report any brooding activities (Ishiyama and Chavez
1990, as cited by Urban and Campos 1994). This is a first account of brooding
activity in the family Psammobidae. Brooding in this species occurred during wet
months coinciding with the peak in the spawning activities of the clams. Also
during these months phytoplankton densities are particularly high (Figs. 21-22).
High abundance of food available means great benefits in terms of larval and
post-larval growth and survival (Stead et al 2002). However, in C. australis
(Byrne 1998) and P. syrmatophorus (Beasley et al. 2000), spawning and
44
incubation of embryos were limited to warmer months. This reflects that warm
water conditions are needed to optimize juvenile success in these species.
Brooding bivalves have lecitothropic larvae which will develop directly into
a short, non feeding planktonic stage or into a crawl-away juvenile. This kind of
larvae usually develops from large fertilized oocytes (Mackie, 1984) with high
nutritional yolk reserves. These yolk reserves are utilized by the embryos as
they undergo development and metamorphosis. In this study, oocyte size in
both sites is relatively larger compared to other bivalves but are comparable to
C. fluminea (150-170 m) and C. leana (110-130 m), freshwater bivalves that
exhibit direct development (Park and Chung 2004).
Gravid or fully charged gills of female G. elongata are thick compared to
non brooding ones. Seasonal variation in gill structures in relation to
reproductive activities were already studied in some corbiculid clams (Byrne et al
2000; Park and Chung 2004) and hyriid mussels (J upiter and Byrne 1997). Parts
of the gills of incubatory bivalves are modified into marsupial pouch to incubate
ripe eggs and embryos. These parts are thickened and more prominent than non
marsupial regions of the gills (Kat 1983).
Larger size of the clams in the pond compared to the clams in the
irrigation canal may reflect enhanced productivity (Byrne et al. 1998) in the
pond. This is supported by the consistently high phytoplankton density at this
site (Fig. 21). The amount of food available in the environment is a determining
factor of the amount of energy incorporated by the animal, which consequently
45
affects somatic and reproductive growth (Delgado and Perez Camacho 2003).
Thus, individuals in eutrophic sites will grow faster compared to individuals in a
site where food availability and supply is less.
Sex ratio in the pond is skewed in favor of females. Intraspecific variation
in sex ratio among populations of freshwater mussels is often attributed to
phenotypic plasticity in response to local conditions. Skewed sex ratio is an
indicator of isolated and small populations (Haag and Staton 2003) and that
inbreeding and competition for mates occur (Downing et al. 1989). Although,
density of the clams in the pond was not quantified, the area seemed to be
isolated due to its enclosure and stagnant water. In unpredictable environments,
egg production is costly thus, more females is needed to produce more eggs to
increase probability of successful fertilization by the sperm.
Gonad development in male and female clams is asynchronous. Females
mature at a shorter shell length compared to males. This is to ensure that there
is a sufficient production of mature oocytes before mature males spawn.
Delayed maturation of males is nonetheless compensated by the rapid
proliferation of spermatogenic cells.
In the study of del Norte-Campos 2004a, based on GSI, G. elongata
matures at shell length greater than 40mm. This was verified in this study.
Histological analysis showed that mature clams in the irrigation canal and in the
pond have shell lengths greater than 40mm. However, clams in the irrigation
canal mature early compared to the clams in the pond. Early maturation of the
46
clams in the irrigation canal may be an adaptation in response to the water
movement in this site. Fertilization success between mature oocytes and water-
borne sperm is highly sensitive to variations in flow conditions (Downing et al.
1993, as cited by Haag and Staton 2003). In the irrigation canal, it is possible
for the eggs and the sperm to be carried downstream by the current. To
compensate for this, clams must mature early so that they can produce
numerous mature gametes that will be fertilized, will develop and will settle in
the area. However, downstream drifting of juveniles in C. fluminea is a dispersal
mechanism that enhances its colonization of new habitats (Prezant and
Chalermwat 1984, as cited by Byrne et al. 2000).

Reproductive cycle
G. elongata has a continuous or protracted breeding season. Qualitative
and quantitative indices of gonad development confirm the prolonged
reproductive period of this species. Indices significantly vary from month to
month (Figs. 14-17). This indicates that gonad development and spawning are
continuously occurring with variation in the intensity of the reproductive activities
resulting to the peaks of the breeding period. Histological examination of the
gonads also shows co-occurring gonad stages within the monthly sampled
population (Figs. 8-11). Extended gametogenesis is also a characteristic of the
bivalves S. inequivalvis (Ledesma-Fernandez and del Norte-Campos 2004), A.
edentula (Samentar et al. 2004), A. scapha, Gafrarium tumidum and Atactodea
47
striata (Baron 1992), C. fluminea (Byrne et al. 2000), H. depressa (Byrne 1998),
P. syrmatophorus (Beasley et al. 2000) and L. corrianus (Nagabhushanam and
Lohgoanker 1978). Seasonal patterns of gametogenesis result from a response
to the existing environmental factors (Baron 1992). This is why continuous
gametogenesis is particularly exhibited by organisms in areas where seasonal
variation is very minimal such as in the tropics and in the deep sea (Stead et al.
2002). G. solida, a temperate species but belongs to the same genus as G.
elongata, exhibits a biannual cycle corresponding to spring and late summer
seasons in Chile (Ishiyama and Chavez 1990, as cited by Urban and Campos
1994). The discrepancy in the reproductive periods of these two species further
shows that gonad development and spawning is influenced by exogenous or
environmental factors.
Based on the indices, reproductive cycle of the clams in the irrigation
canal has multiple peaks with the highest peaks on the months of September
and April (Figs. 13-14). However, in the study on the population biology of this
species from this site, spawning peaks were observed on the months of
December to J anuary (minor peak) and May to J une (major peak) (del Norte-
Campos 2004a). GSI peaks in the present study, however, are supported by
gonad histology which shows that massive maturation and spawning of gonads
are taking place during wet months. During southwest monsoon (May to
October), rain and strong winds may increase terrigenous input in the irrigation
canal consequently increasing nutrients in the site. Also during these months that
48
phytoplankton density is at its peak (Fig. 20) which contributes to the food
available for the clams. In the pond, two distinct peaks on the reproductive cycle
of clams were observed in the months of April and October (Figs. 16-17). Based
on gonad histology, a more massive spawning occurred on April than on October
(Figs. 10-11). This shows that gonad maturation and spawning peaks are during
dry months. This pattern is opposite to what is exhibited by the clams in the
irrigation canal. During dry months phytoplankton density in the pond is at its
highest (Fig. 21). High availability of food corresponds to breeding season since
most of the energy derived from digestion is partitioned directly or indirectly to
reproduction (Mackie 1984). In Ruditapes decussatus, the energy from ingestion
was primarily channeled to reproductive processes and the available food was
directly related to the rate of gonad development (Delgado and Camacho 2005).
The functional correlation between food availability and reproductive
cycles in bivalves has been illustrated (Baron 1992; Byrne 1998; Stead et al.
2002; Delgado and Camacho 2005; Sahin et al. 2006). Coecella chinensis, an
intertidal clam in Korea, has a single spawning peak associated with intense
phytoplankton bloom (Kang et al. 2007). In T. dombei and S. solida the gonadic
index cycles were at their highest value when microflagellate and centric diatom
densities were at their peak (Stead et al. 2002). The significant variation in the
reproductive cycles of G. elongata in the irrigation canal and in the pond might
be due to the quantity and quality of the available food present in each site.
49
Differences in reproduction of river and lake populations of H. depressa were
also due to the variation in nutritional status between the two sites (Byrne 1998).
Difference in water dynamics between the two sites was observed. In the
irrigation canal, water is continuously flowing producing a current. It is possible
that phytoplankton is carried constantly downstream by this current, thus the
lower density of phytoplankton in this site. This might be the reason why
nutritional input from terrestrial areas during rainy months is very substantial in
augmenting food supply in the irrigation canal. Furthermore, irrigation canal is
regulated by dam and more prone to human disturbances and control. This
might also explain the more irregular pattern of reproductive cycle in the
irrigation canal. On the other hand, in the pond water is quite stagnant and this
allows concentration of phytoplankton in the water column causing the density in
this area to be higher than in the irrigation canal. This is why densities are
particularly high on dry months since water level is low and the effect of the sun
is strongest. This variation in water flow might be the cause of the differences in
phytoplankton density and composition between the two sites which
consequently affect the differences in the reproductive cycle of the clams.
However, this study was conducted in the natural habitat and it is difficult to
isolate the specific factors that influence reproductive phenomenon in this
species.


50
SUMMARY AND CONCLUSIONS
Gari elongata is a dioecious bivalve with occasional incidences of
hermaphrodites. Although it is not a functional hermaphrodite, it exhibits an
incubatory behavior. It has large mature oocytes (103m smear and 86-88m in
prepared slides) which are typical of larvae that have a lecitothrophic or direct
development.
Sex ratio in the canal does not significantly depart from the expected ratio
while in the pond sex ratio is highly skewed to females. In isolated and stagnant
populations more females are necessary to ensure that sufficient eggs are
produced and fertilized by the sperm.
Sexual maturity of G. elongata in the pond is attained at 44.80 mm and
59.10 mm in female and male, respectively. On the other hand, those in the
irrigation canal reached maturity at 43.15 mm and 43.70 mm in female and
male, respectively. This shows that female matures firsts compared to male and
that gonad development is asynchronous between sexes. This is also to ensure
sufficient production of eggs. Delayed maturation of males is, however,
compensated by the rapid proliferation of spermatogenic cells.
Five determinate gonad stages based on histological examination were
described in male and female clams: developing, mature, partially spawned,
redeveloping and spent. Indeterminate or immature gonads were also observed.
Reproductive cycle of G. elongata is protracted or continuous. A difference in
this cycle was observed between irrigation canal and pond. In the irrigation
51
canal, multiple peaks were observed in the reproductive cycle with the highest
on wet months. On the other hand, in the pond, maturation and spawning
occurred on dry months and development on wet months. This variation may be
due to the differences in nutritional status and water dynamics in both sites.
Intraspecific variation in the reproduction of this species between the two sites
might be a manifestation of phenotypic plasticity in response to the differences in
the local conditions.
In terms of management measures, it is important to leave a proportion
of reproductively mature individuals in the population to allow breeding and
recruitment. Thus, in this species collection of individuals smaller than 43mm
must not be conducted and collection must also be regulated during maturation
and spawning months.
In summary, G. elongata exhibits a mosaic of marine and freshwater
reproductive characteristics which allows the survival of this species in an
environment where water regime is unpredictable and prone to disturbance.














52
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