CHAPTER 1

INRTODUCTION

Fisheries is one of the major industries found here in the Philippines, given the
location of the country, since it is found in the Pacific Ocean. The fisheries in the
Philippines are mainly composed of fishes, and shellfishes. One of the major marine
life products that is being marketed are crabs. Charybdis natator (Herbst, 1794)
commonly known as the “ridged swimming crab”, belongs to Family Portunidae, a
typical group of marine crabs which is widely distributed across Asia, Australia, and
Africa (Wee and Ng, 1995). In the Philippines, C. natator is locally known as
“kalintugas”. Charybdis natator is characterized by brown to orangish coloration on
the dorsal side of the carapace while the ventral side is found to have a bluish
coloration, mottled with white and pale red. Legs are dark and reddish brown in color.
The carapace is densely covered with short pubescence and is absent on the distinct
transverse ridges in the anterior side. The ridged swimming rock crab has a distinct
pattern from other crab species of having ridges located dorsally on its carapace. The
same pattern could be observed in both sexes of C. natator. Although uncommon and
is not a major commercially-fished species (Atlas of Living Australia) in comparison
to other more abundant and commercially important crabs, such as the mud crabs
(Scylla serrata) and sand crabs (Portunus pelagicus), the ridged swimming rock crab
also contribute to crab fisheries in Asia and Asutralia (Sumpton, 1990).

Crabs live in burrows in sandy beaches, mud in the rivers, and below the rocks
at the bottom of the ocean. But during mating season, large population of mature
crabs migrate to the ocean to copulate and sometimes this can be intervened by the
fisherfolk, and this could be a huge problem because if crabs fail to copulate, there
will be lesser population of crabs in the next generation.

This study aims to determine and describe the reproductive stages of C.

natator from Estancia, Iloilo, Western Philippines. The study also intends to identify
the monthly stages of gonad development of C. natator, through morphological and
histological analysis. Specifically, the study aims to determine: (1) the morphological
appearance of the gonads of C. natator at each stage of development, (2) the
histological appearance of the gonads of C. natator at each stage of development, (3)
size at sexual maturity of the crab, (4) the percent occurrence of each gonadal
maturation stage, and (5) the percent occurrence of male and female crabs from
September 2017 to February 2018.

The information that will be gathered will be significant for the

implementation of regulatory measures for the management and conservation of the
remaining stock in the wild. There were a lot of studies on the reproductive biology of
distinct species of crabs but there is no or little information known about C. natator,
thus, the need for the present study. The results of this present investigation will also
be useful for the brood stock management in the future hatchery culture of this crab
species.
 CHAPTER 2

REVIEW OF RELATED LITERATURE

Description

Charybdis natator (Herbst, 1794) commonly known as the “ridged swimming

crab”, belongs to Family Portunidae, a typical group of marine crabs which is widely
distributed across Asia, Australia, and Africa (Wee and Ng, 1995). The family
Portunidae are groups of crabs which are mainly comprised of swimming crabs
(Bowling, 2012). Swimming crabs can be differentiated from walking crabs because
of their hind limbs, which are flattened, that acts as propellers for them to swim
efficiently. They are called ridged swimming crabs because of the ridges in their
carapace (Samuel, 2014).

Morphology

According to Wee and Ng (1995) in “The Raffles Bulletin of Zoology”, the

crab species Charybdis natator can be describe through the following: Carapace,
uniformly pilose, sparse granules on anterolateral surface; anterior carapace ridges
present except frontals, epibranchials interrupted by unbroken metagastric ridge,
posterior with one pair of cardiac and three short pairs of mesobranchial ridges; six
frontal lobes, medians on lower plane, projecting beyond egually broad submedians,
laterals acute, separated from sub medians by deeper V-shaped notch; inner
supraorbitallobe broder than frontals, bluntly triangular; six anterolateral teeth, first
tooth truncate, second to fourth subequal with acute tips, last spiniform and least
prominent. Basal antennal segment bearing short granular ridge. Chelipeds unequal,
granular and pilose; anterior border of merus with three to four spines; carpus with
strong spine on inner angle and three spinules at outer angle; manus with four spines
on upper surface and a spinule at distal end of outer border, lower surface with
transverse squamiform ridges; fingers stout, deeply grooved. Propodus of natatory leg
serrated on posterior border. Second to fifth segment of male abdomen keeled,
penultimate segment with lateral borders parallel then converging distally. G1 distal
tip slender and elongate, abdominal surface bears two rows of short terminal bristles
ending proximal to lip region, outer suface with row of longer bristles starting near tip
and extending proximally as widely spaced bristles. Pubescence of dorsal surface
brownish, granules bright red. Ventral surface bluish, mottled with white and pale red
(Wee and Ng, 1995).

The distinct reddish coloration of the anterolateral and frontal teeth, on the
granules and transverse carapace ridges are what make this species easily recognized.
It is similar to that of Charybdis granulata. Leene (1938), but it has since been
considered as a distinct species by Sakai (1976) and Miyake (1983). The under
surface of the manus of the chelipeds vary from having a transverse squamiform
arrangement to that of a surface with scattered granules (Wee and Ng, 1995).
Charybdis natator can grow its carapace ranging from 5 cm to 17 cm (Samuel, 2014).

Habitat and Ecology

Ridged swimming rock crabs, as the name implies, inhabits the bottom of the
ocean on rocky substrates or on coral reefs. They live ranging from 5 to 4 meters
below the sea levels (Samuel, 2014). They can also live on muddy or sandy substrate.
According to Sakai (1976), this species is found on the bottom of rocks, pebbles or
sand at depths of 15-35 meters.

Although uncommon and is not a major commercially-fished species (Atlas of

Living Australia) in comparison to other more abundant and commercially important
crabs, such as the mud crabs (Scylla serrata) and sand crabs (Portunus pelagicus), the
C. natator also contribute to crab fisheries in India and Asutralia (Sumpton, 1990).

Reproductive Biology

In the study conducted by Sumpton (1990), he used trapping methods to

collect samples of Charybdis natator in Moreton Bay, Queensland. Based from his
results, he observed that during the month of June and July there are more females
than males, but in other months the ratio of males to females is two to one. This is
maybe because of the spawning season of the C. natator during June and July. He
also found two major spawning peaks for C. natator. During winter the low
proportion of gravid females and high proportion of females with inactive gonads
indicate that C. natator does not spawn year- round in the subtropical waters of
Moreton Bay. Pillai and Nair (1976) found that the closely related Charybdis feriatus
bred throughout the year in southwestern Indian waters, although gravid females were
more common during January and February. It is thus likely that the cooler conditions
during winter in Moreton Bay limit the spawning activity of C. natator. The ridged
swimming crabs can ovulate up to three times and it was confirmed by ovary
examination that indeed the C. natator are inactive during winter season. Also, he
indicated in his study that the fecundity of a female crab ranges from 181,000 to
976,000. The reproductive biology of C. natator has similarities to other crab species
under the Family Portunidae (Sumpton, 1990).

Morphological characterization

The macroscopic technique in identifying the stage of gonadal development of

both male and female crabs involves only the observation and examination of the
different shapes, sizes, and color of the gonads by means of the naked eye.

There are five macroscopic gonadal developmental stages of female crabs

(Minagawa et al., 1992; de Souza et al, 2009; Ikhwanuddin et al., 2012). The five
stages of the female ovaries are: (1) Stage 1 or Immature Stage, (2) Stage 2 or Early
Maturing Stage, (3) Stage 3 or Late Maturing Stage, (4) Stage 4 or Fully Matured
Stage, and lastly (5) Stage 5 or Post-Spawning Stage. The immature stage is described
as a translucent ovary showing a ribbon-like structure, slender and flaccid lobes in a
tubular form, with white-yellow coloration. The next stage, early maturing stage, the
ovaries is compressed dorso-ventrally and the anterior lobes covers almost all
hepatopancreas and the stomach region of the crabs, meaning there is increase in size
of the crabs’ ovaries. The color of this stage is light orange or peach in color. In the
third stage, the late maturing stage, the ovaries change its pigmentation form light
orange or peach into orange, and also the anterior lobes cover the totality of the
hepatopancreas. In a fully matured ovary, the color of the ovary becomes deep orange
or scarlet, the maximum size of ovary is attained, and it covers the whole
hepatopancreas and the stomach area. Lastly, the spent or post-spawning stage, the
ovaries appears in a deep brown color and sometimes it regresses back to its former
color, white-yellow. This is due to the release of mature oocytes and the
previtellogenesis stage can be observed again, in which it indicates a new beginning
in the ovarian cycle. At this stage eggs can be observed in the abdominal flaps of the
female crabs because they have already undergone fertilization.

In the male crabs, there are only three stages of gonadal development for the
sperm (de Souza et al., 2012; Soundarapandian et al., 2013), (1) Immature, (2)
Maturing, and (3) Fully Matured. The gonads of the immature crabs are relatively
small, with a cream color, located lateral to stomach. In a maturing stage, the gonads
possess a creamy white color. The testes appear as a coiled tube placed laterally and
posterior to the stomach, while the vas deferens extends laterally to the heart. The last
stage, fully matured stage, of the male gonads appear as a milky white color. The
male gonads have an enlarged testis, with the vas deferens becomes coiled, and it
completely covers the full body cavity.

Histological examination

In histological examination there is a certain protocol that must be followed on

how to prepare the histological slides of the gonads of each crab. After the
macroscopic analysis of the gonads, portions of each stage of the gonads will be
placed in small vials half-filled with Bouin’s solution and will be stored for 24 hours
at room temperature. The gonads will be dehydrated through a series of increasing
alcohol concentration. Toluene will be added to remove the alcohol. The gonads will
then be placed in soft Paraffin wax for two hours. The gonads will be embedded in
hard Paraffin wax for several hours and the excess wax will be trimmed afterwards.
Thin sections will be prepared using a microtome (5µm), tissues will be stained using
haematoxylin and eosin, and will be left to dry overnight. Each section will be
mounted in slides, will be covered with cover slips and will be labeled with respective
date and crab number.

In female crabs, there are four histological stages that can be observed (de
Souza et al., 2009). The four stages of the female gonad development are: (1)
Previtellogenic stage, (2) Early-Vitellogenic Stage, (3) Late Vitellogenic Stage, and
lastly (4) Post-Spawning Stage. First, the previtellogenesis stage, oogonia may be
observed undergoing preliminary stages of meiotic division. The next stage, early-
stage vitellogenesis, the germinative zone can be seen that it is being compressed by
its surrounding previtellogenic oocytes. The oocytes may be observed as it is
undergoing early vitellogenesis. In the third stage, the mature vitellogenesis, the
ovaries are mostly filled with oocytes undergoing late-stage vitellogenesis. Mature
ovaries appear to be uniform and completely filled with mature oocytes, and lastly the
germinative zone is barely seen. Lastly, in the post-spawning stage, the ovaries can be
observed in a disarrayed arrangement due to the release of the mature oocytes outside
the system. Several previtellogenic oocytes and empty cavities can be observed in the
ovarian stroma along with a few counts of hamatocytesm fibers, and follicular cells.
The lining also assumes a distinct wavy appearance. There is also a restoration of the
ovarian arrangement along with the reduction of empty cavities and hematocytes.

In the male, the three stages of development that were identified histologically
are: (1) presence of spermatogonia in immature stage, (2) Presence of spermatogonia
and spermatocytes in maturing stage, and (3) formation of primary and secondary
spermatocytes, spermatids and spermatozoa in mature stage (Islam et al., 2013).

Fecundity

Fecundity is an index of reproductive capacity and is estimated by the number

of eggs produced by an organism (Baylon and Tito, 2012 “from” Reeby et al. 1990).

The eggs attached to the pleopods of ovigerous females will be scraped off to

determine the mean number of eggs per egg mass. Three replicates of 1 g sub-samples

will be taken and the number of eggs in each replicate will be counted under a

dissecting microscope (Baylon and Tito, 2012). The fecundity of each female crab

will be estimated by multiplying the mean number of eggs in the three replicates by

the total wet weight of the egg mass (Baylon and Tito, 2012 “from” Krajangdara and

Watanabe, 2005). To show the relationship of fecundity versus size of the female

crab, regression analysis will be used (Baylon and Tito, 2012).

 MATERIALS AND METHODS

Collection site and sampling

There will be a six-month survey of crab sampling during September 2017 to

February 2018. The sample will be taken from Estancia Iloilo, and will be delivered

and bought in the wet market of Miagao, Iloilo. Ten samples will be gathered in every

first week of the month. The samples shall have representatives of different sexes with

sizes ranging from the smallest to the largest for the determination of the fecundity

and gonad developmental stages.

Collected crabs is stored in the ice box for preservation, so that the tissues of

the crabs will no deteriorate, and for the crabs to be anesthetized since crabs tend to

have cannibalistic instincts. The samples should not be placed in the freezer for the

gonads will not be of use during the histological analysis because it would be too stiff

to be prepared as thin sections for histological analysis, and the gonads will give off a

different color once it has reacted to the different dyes: haemotoxylin and eosin.

Determination of sexes
 Male and female crabs can be described, and can be differentiated

morphologically through the different shapes of their abdominal flaps. Male crabs

possess a narrow and triangular abdominal flap, while the female crabs have broader

and rounder abdominal flaps which covers the whole sternum of the female crabs

(Nemenzo, 1976). In males, they possess a pair of gonopods which is located in the

anterior part, inside the abdominal flap. Gonopods are long tubules that are used to

facilitate the sperm into the gonopore of the female crabs (Zinski, 2010). Gonopores

are small opening in the female crabs that is located ventrally in the first segment, in

between the first pair of pereiopods (Wilkin, 2002). Another distinct characteristic of

a female crab is that they have hairy appendages that functions as an attachment of the

developing embryos (Nemenzo, 1976). Size of the crabs can also be used to determine

the gender of the crab; female crabs are smaller than male crabs.

Body Measurement

For each crab, the body weight (BW), carapace width (CW), and carapace

length (CL) will be measured. The body weight will be measured by patting the crab

dry using clean cloth or tissue after removing it from the refrigerator. This is to avoid

errors in the results. The crab will be placed on a digital electronic balance and the

weight will be recorded to the nearest tenth of a gram. Carapace width and length will

be measured using a Vernier caliper and will be recorded to the nearest millimeter.

Carapace width (CW) is the distance between anterior lateral spine and the most

posterior lateral spine (Brown, 2009). Carapace length (CL) is the distance between

the centers of the frontal interorbital carapace margin and the posterior margin.

Dissection
 Using a pair of sharp dissecting scissors, the dissection of crab will proceed by

cutting the dorsal carapace from the posterior end of the carapace moving in

counterclockwise direction, making a square shaped incision. Then using a scalpel,

forceps, and dissecting needle, the thin membrane covering the cephalothoracic cavity

will be removed to expose the gonads. A Y-shaped structure of gonads, called the

anterior horn, will be observed. It lies dorsal to the hepatopancreas. The

hepatopancreas has a fingerlike appearance with light yellow to red orange in

coloration. It is located on both sides of the gastric mill on the arterior part of the

cephalothoracic cavity. The hepatopancreas is extended posteriorly, connected to the

gastric mill.

Gonad analysis

The gonad developmental stages for all sexes of Charybdis natator will be

investigated using two methods: (1) macroscopic technique which will involve the

description of the external morphology of the gonads and (2) microscopic technique

which will involve histological examination of the gonads.

Morphological characterization

The macroscopic technique will involve the examination of the shape, size and

color of the gonads by using the naked eye.

The five gonadal development stages of female C, natator based on

macroscopic observation according to Quinitio et al (2007) were the following: (1)

Immature, (2) Early maturing, (3) Late maturing, (4) Fully Mature, and (5) Spent. The

shape, size and color of the gonads will be analyzed based on this classification. The

male C. natator will be analyzed based on the three stages of gonadal development

according to Silva et al (2012): (1) immature, (2) maturing, and (3) mature.
Histological examination

The entire histological tissue preparation and processing will be done at the

Microtechnique laboratory, College of Fisheries and Ocean Sciences, University of

the Philippines Visayas.

After the macroscopic analysis, portions of the gonads will be placed in small

vials half-filled with Bouin’s solution and will be stored for 24 hours at room

temperature. The gonads will be dehydrated through a series of increasing alcohol

concentration. Toluene will be added to remove the alcohol. The gonads will then be

placed in soft Paraffin wax for two hours. The gonads will be embedded in hard

Paraffin wax for several hours and the excess wax will be trimmed afterwards. Thin

sections will be prepared using a microtome (5µm), tissues will be stained using

haematoxylin and eosin, and will be left to dry overnight. Each section will be

mounted in slides, will be covered with cover slips and will be labeled with respective

date and crab number. The slides will be observed at the Phycology Laboratory,

College of Fisheries and Ocean Sciences, University of the Philippines Visayas.

Using a Motic microscope, the different gonad development stages will be

determined. The sizes of the oocytes and oogonia will be measured in µm. The four

histological stages described by Ikhawanuddin et al (2014) are: (1) immature, (2)

early maturing, (3) late maturing and (4) fully mature. In the male, the three stages of

development that were identified by Silva et al (2012) and Islam and Kurokura (2013)

are: (1) presence of spermatogonia in immature stage, (2) Presence of spermatogonia

and spermatocytes in maturing stage, and (3) formation of primary and secondary

spermatocytes, spermatids and spermatozoa in mature stage

Gonad analysis

Gonad analysis shall include computations of the gonadosomatic indices and

gonad indices. The quantitative gonadosomatic index (GSI) will be calculated using

the equation: GSI = gonad weight (g)/body weight (g) x 1000 (Krajangdara and

Watanabe, 2005). Pre-weighted values (WV) will be assigned to the female gonad

stages as: undeveloped and recovering - 1, developing - 2, ripe and spawning – 3. For

the male testes, the values to assign will be: immature - 1, and mature - 2. The mean

value of gonad indices (GI) or the index of sexual maturity will then be calculated

using the equation: GI = Σ(n x WV)/N, where n is the number of individuals in a

given developmental stage; WV is the pre-weighted value for the developmental

stage; and N is the total number of crabs per monthly sample. Values of mean gonad

indices per month will then be plotted against time (months) to determine the

spawning pattern or reproductive cycle (Baylon and Tito, 2012).

Fecundity

Fecundity is an index of reproductive capacity and is estimated by the number

of eggs produced by an organism (Baylon and Tito, 2012 “from” Reeby et al. 1990).

The eggs attached to the pleopods of ovigerous females will be scraped off to

determine the mean number of eggs per egg mass. Three replicates of 1 g sub-samples

will be taken and the number of eggs in each replicate will be counted under a

dissecting microscope (Baylon and Tito, 2012). The fecundity of each female crab

will be estimated by multiplying the mean number of eggs in the three replicates by

the total wet weight of the egg mass (Baylon and Tito, 2012 “from” Krajangdara and

Watanabe, 2005). To show the relationship of fecundity versus size of the female

crab, regression analysis will be used (Baylon and Tito, 2012).