13

Chapter 3

REPRODUCTIVE BIOLOGY

3.1 INTRODUCTION

The reproductive biology of crustaceans is a subject that has derived

much attention over centuries. Reproduction and breeding cycles of

crustaceans have been described by Rahman (1967), Ryan (1967),

Haefner (1976) and Zuckner (1978). Pochon-Masson (1983) and Adiyodi

(1985) described the process of spermatogenesis while oogenesis has

been described by Raven (1961), Norrevang (1968) and Adiyodi and

Subramaniom (1983). The effects of hormones on moulting and

reproduction have been described by Passano (1960), Adiyodi and

Adiyodi (1970), Nagabushanam et. ai, (1980), Quackenbush (1986) and

Fingerman (1987). A lot of information on the reproductive biology and

behaviour of lobsters, particularly palinurid and homarid lobsters, has

been documented over the last five decades. Several studies have

been made on fished populations of lobsters. Studies on reproduction in

Jasus lalandii by Fielder (1964) and Heydorn (1965), on fecundity in

Panulirus longipes by Morgan (1972), on the comparison of

spermatophoric masses and mechanisms of fertilization in the Southern

African spiny lobsters by Berry and Heydorn (1970), on the biology of

P. homarus by Berry (1971), on Palinurus gilchrisli by Pollock and

Augustyn (1982) and Jasus edwardsii and J. tristani by Pollock and

Goosen (1991) are some of the most important early studies which

provide primary insight into this complicated biological process. Mac

14

Diarmid (1987) and Mac Diarmid et al. (1991) studied reproductive

patterns and behavior in a population of J. la/andii in a protected

reserve in northern New Zealand. Mac Diarmid (1987) demonstrated the

impact of male aggressive behavior on the selection of males by female

lobsters. Several field studies of tropical spiny lobsters indicate that rapid and

repetitive brood cycles are common, upto five broods per annum (Briones­

Fourzan and Lozano-Alvarez, 1992; Junio, 1987; Mac Diarmid, 1987).

Nelson ef a/., (1988a, b, c) and Waddy and Aiken (1992) monitored the effects

of environmental variables on gonadal maturation in female lobsters and

stressed the importance of monitoring the effects of environmental

variables on gonadal maturation in female lobsters. Junio (1987) carried

out an extensive study of P. penicillafus (Oliver, 1791) in the Philippines.

Lyons ef a/., (1981) summarized the reproductive biology of the Florida

spiny lobster P. argus in populations under light, heavy and no fishing

pressure. Lipcius (1985) and Hernnkind and Lipcius (1989) also studied

reproductive behaviour in P. argus. Briones - Fourzan and Lozano­

Alvarez (1992) studied two populations of closely related palinurids - P.

inflafus and P. gracilis. Quackenbush and Herrnkind (1983) and Lipcius

and Herrnkind (1987) studied the effects of photoperiod on the moult

cycle and reproductive behaviour in P. argus. Gomez et al. (1994)

determined the breeding period of P. longipes longipes in the Philippines

by studying ovarian development and egg-bearing in the fished

population of females. George (1965) has recorded the breeding season

of P. homarus along the south-west coast of India. The maturation cycle

in female P. japonicus has been described in detail by Nakamura (1990) and

15

Minagawa and Sano (1997). Radha and Subramaniom (1985, 1987)

described spermatophore formation in P. homarus. Hussain and Amjad

(1980) have studied the breeding and fecundity of P. polyphagus along

the Pakistan coast. Kagwade (1988a, b) has described reproduction and

fecundity in P. polyphagus from Maharashtra waters.

Several studies on the histological structure and development of the

gonads have been carried out, particularly in palinurid lobsters (Fielder, 1964;

Berry and Heydorn, 1970; Radha and Subramoniam, 1985; Nakamura, 1990;

Minagawa and Sano, 1997 ) and homarid lobsters (Schade and Shivers,

1980; Marcia and Talbot, 1982).

In comparison with spiny lobsters, there is very little information

on the biology and reproductive behaviour of scyllarid lobsters. Fecundity

and spawning seasons have been studied in the Mediterranean Locust

Lobster, Scyl/arides latus (cf. Martins, 1985) and the slipper lobster, S.

nodifer, from the northeastern Gulf of Mexico (Lyons, 1970; Hardwick and

Cline, 1990). In Australia, there has been relatively more documentation

on the reproductive characteristics of T. orientalis (Kneipp, 1974;

Hossain, 1978a, b, 1979; Branford, 1980; Jones, 1988) Stewart et al.,

(1997) have described the size at first maturity and reproductive biology

of Ibacus peronii and Ibacus sp. from Australian waters. Stewart and

Kennelly (1997) have described the fecundity and egg size in I. peronii.

DeMartini and Williams (2001) studied fecundity and egg size in Scyl/arides

squammosus. DeMartini et al. (2005) discussed indicators of sexual maturity

16

in S. squammosus and the spiny lobster P. marginatus. Kagwade and Kabli

(1996) recorded the breeding peaks, fecundity and size at first maturity of

T. orienta/is from the Mumbai coast. Rahman et al. (1987) studied the

egg development in T. orienta/is. Moulting in female T. orienta/is has

been described by Rahman and Subramaniom (1989).

There is not much information on the reproductive biology and

behaviour of P. po/yphagus or T. orienta/is held in captivity. In the present

study, an assessment of the reproductive biology of the two species has

been made through -

• Morphological and histological observations on gonadal structure and

development in lobsters collected from the wild

• Observations on maturation-associated changes in secondary sexual

characters in lobsters collected from the wild and in lobsters held in

captivity

• Observations on mating and spawning behaviour in captivity

• Estimates of Gonadosomatic Index and fecundity in lobsters collected

from the wild

• Estimates of size at maturity in lobsters collected from the wild and in

lobsters held in captivity

Studies on reproductive biology of commercially important lobsters are

ultimately aimed at conservation and management of the resource in their

natural habitat and improving their aquaculture potential. Prediction of

minimum size limits to be observed for protecting spawning populations from

17

being destroyed by fishing activities and awareness of spawning seasons are

direct outcomes of such studies. Any information derived from observations of

reproductive biology and behaviour in captivity will be of use in developing

husbandry practices for the species and thus improve its candidature for

aquaculture. Successful captive propagation has the added advantage of

improving the status of natural stocks through sea ranching.

With growing impetus on establishing lobster aquaculture in India,

studies on reproductive biology and behaviour in the major lobster species

available in Indian waters assumes great significance.

3.2 MATERIALS AND METHODS

All animals for the study were collected from the trawl landings at

Veraval and Mangrol fish landing centres. Decline in catches, high cost and

high export demand for the lobsters greatly restricted the number of animals

available for the study. However, care was taken to ensure that animals

collected represented different size groups and stages of maturity. Animals

obtained live were maintained in the holding systems described in Chapter 2.

3.2.1 Sexual Dimorphism and Secondary Sexual Characters

Descriptions of external morphology for sexual dirnorphism and

secondary sexual characteristics were made from observations on 261 males

and 289 females of P. po/yphagus (35 - 110 mm CL) and 350 males and 221

females of T. orienta/is (35 - 100 mm CL) collected from the wild.

Morphological descriptions were made using standard lobster terminology

18

following Holthuis (1991). Structural variations in different body parts were

studied through direct observation and observation under a stereozoom

trinocular microscope (Carl Zeiss Stemi-2000).

3.2.2 Reproductive System - Structure and Development

Male and female reproductive systems of P. po/yphagus and T.

orienta/is were studied anatomically using 10 animals of either sex in each

species. Animals in the size range of 35 - 90 mm CL were chosen, so as to

get sufficient representation of different maturity stages. Gonadal maturation

was assessed primarily from the anatomy and appearance of the gonads.

Classification of ovarian developmental stages was done through descriptive

macroscopic staging following the methods of Fielder (1964), Jones (1988)

and Nakamura (1990). Testes were described in terms of their size, shape

and colour (Stewart et a/., 1997). Gonadal smears, eggs and spermatophores

were observed under a stereozoom trinocular microscope (Carl Zeiss Stemi-

2000).

Histological observations on gonadal maturity were made from

immature, maturing and mature gonads of male and female P. po/yphagus

and T. orienta/is. The colour and condition of the fresh gonads were

noted and the wet weights were measured to the nearest 0.1 g after

drying the ovaries with blotting paper. The gonads were then preserved

in 10% formalin and kept for standard histological processing. Dissections

were done in crustacean saline between 16:00 - 18:00 hours (to avoid

interference of circadian rhythms in the maturation process). The tissues

19

fixed in Aqueous Bouin's Fixative (ABF) for 24 hours, were treated

through tap water, graded series of tertiary butyl alcohol and

chloroform, before impregnation and embedding in paraffin wax.

Transverse sections of 6 - 8 11 thickness cut from the blocks were spread

on albumen-coated slides, stained in Harris's Haematoxylin and spirit

soluble Eosin, cleared in xylene and mounted in DPX.

Photomicrographs taken with a Canon digital camera attached to a trinocular

dissection microscope (Carl-Zeiss Axiostar) were used to study the

histological variations with maturation.

3.2.3 Mating and Spawning

Observations on mating behaviour, copulation, impregnation,

spawning, incubation and hatching were made on animals maintained live in

the laboratory. 1 tonne FRP tanks holding 900 I of seawater and covered

with light screens were used as brood stock development tanks for P.

po/yphagus. These tanks were operated on a Closed Recirculatory System,

using external biofilters. The tanks used for T. orienta/is were 400 litre FRP

tanks with in situ substrate (fine sea sand) bedfilter. Water temperature was

maintained in the range of 27 - 30'C, water salinity was maintained between

36 and 37 ppt and water pH was maintained between 7.8 and 8.0. Uniform

aeration was provided with a twin lobe air blower. Light exposure was kept

to a minimum. Water quality was monitored daily and water exchange was

carried out @ 30% daily. The animals were fed ad. libitum With fresh meat of

the gastropod, Turbo sp. The tanks were provided with shelters and hiding

20

places and care was taken to keep handling and environmental stress to a

minimum.

Four sets of broodstock development tanks were maintained for either

species. Juveniles of P. polyphagus were raised from 35 mm CL to 80 mm CL

in 250 days. Stocking was done @ 1 male per 3 females and 8 animals were

stocked in each tank. Juveniles of T. orientalis were raised from 30 - 40 mm

CL to 85 mm CL in 180 days. Stocking was done @ 6 animals per tank. The

sex ratio was kept at 1 male for two females.

Unilateral eyestalk ablation was done in early intermoult female P.

polyphagus in which decalcified windows on the fourth ventral sternite had

developed. 18 females were ablated and stocked with males which were used

for the first set of breeding experiments.

3.2.4 Gonadosomatic Index

The Gonadosomatic Index (GSI) was calculated using the formula as

given by Minagawa and Sana (1997) -

where,

1= GSI W = weight of gonad (g) L 3 = carapace length (mm)

The GSI values for individual lobsters were used to arrive at the average GSI

for different size groups (CL, mm) in different stages of maturity.

21

3.2.5 Size at sexual maturity

The size at sexual maturity was assessed from observations on 261

males and 289 females of P. po/yphagus (35 - 110 mm el) and 350 males

and 221 females of T. orienta/is (35 - 100 mm el) collected from the wild and

38 males and 43 females of P. po/yphagus (35 - 110 mm el) and 41 males

and 64 females of T. orienta/is (35 - 100 mm el) held in captivity.

The size at first maturity in both species was estimated by assessing

the size at which the animals become morphologically, physiologically and

functionally mature. The size at physiological maturity was assessed from the

condition of the gonad and its stage of development, based on the colour and

structure of the gonad. Histological observations were also done

simultaneously to ratify the developmental phase judged from the anatomy of

the gonads. All animals in an advanced state of gonadal maturation, those

that had mature or ripe gonads and those that were in a state of rematuration

were collectively grouped as "mature" while immature animals and those in

which gonadal maturation had just begun were grouped as "immature". The

frequency distribution of "immature" and "mature" lobsters, sex-wise, in the

sampled population was analysed, and the size at first (physiological) maturity

was read as the size at which 50% of the lobsters were "mature".

The size at morphological maturity was assessed similarly by following

the development of external indices of maturity like ovigerous setae in

females of both species and changes in the growth of the last three pairs of

pereiopods (walking legs), the ventral sternite length and the maximum width

22

of the carapace relative to carapace length in males and females of both

species. Changes in the length of the penile process in relation to the

carapace length was also studied in male P. po/yphagus. Linear plots of the

somatic lengths against the carapace lengths in size groups <50 mm CL and

> 50 mm CL were used to assess changes in growth patterns from juvenile

phase to sub-adult and adult phases. Comparisons were also made between

growth patterns of males and females of the same species. The sizes at

which deflections (if any) in regression lines between juveniles and sub-adults

or between males and females were studied for indications of the onset of

sexual maturity.

The size at functional or physical maturity was assessed from the

frequency distribution of males and females of either species with respect to

specialised structures which ensure the mating and propagative capabilities of

the animals -

• the formation of decalcified mating windows on the ventral side and the

formation of egg brush on the dactylii of the fifth pair of walking legs in

female P. po/yphagus

• the development and pigmentation of the penile process in male P.

po/yphagus

• the distribution of ovigerous condition in females of both species

3.2.6 Fecundity

Fecundity estimates were made from 47 egg batches (berried

females in the size range of 64 - 119 mm CL in P. po/yphagus and 53 egg

23

batches in the size range of 60 - 102 mm CL in T. orienta/is. The berried

animals were collected from the trawl landings at Veraval and Mangrol fish

landing centres.

The berries (egg masses) were oven dried and cleaned of

adhering material like setae, pleopods etc. The total dry weight (to the

nearest O.Olg) was measured using an electronic balance. The average

number of eggs in three 0.1 g sub-samples was raised to the total dry

weight of the berry to obtain the total number of eggs in that batch.

The relationship between fecundity and carapace length was estimated

by linear regression.

3.3 RESULTS

3.3.1 Sexual Dimorphism and Secondary Sexual Characters

3.3.1.1 Sexual dimorphism

Both P. polypl1agus and T. orienta/is exhibit sexual dimorphism. In

both species, there was considerable size difference between the sexes.

However, in P. polypl1agus, males were larger than their female counterparts

while in T. orientalis, the females were larger than the males. In both species,

the abdomen is considerably narrower in males than in females.

The males and females of either species can be easily identified

by the distinct morphological characteristics. which are summarized in the

following tables -

3.3.1.1A P. po/yphagus

Male

,. Genital opening (gonopore) on

coxa of fifth pair of walking

legs (PI. VII)

,. Gonopore is a complex structure

in the form of a penile process

(PI. VII; Fig. 4c)

,. Dactylus of fifth pereiopod

normal, as in other pereiopods

(PI. VI b; Fig. 4a: b)

j;o Dactylus of fifth pereiopods with

two rows of setae (PI. VI e)

,. Paired pleopods comparatively

small. Endopods vestigeal.

Exopods smaller than in female

24

Female

,. Genital opening (gonopore) on coxa

of third pair of walking legs

,. Gonopore is a simple aperture

,. Dactylus small and curved and

articulates with the small fixed claw

attached to posterior tip of propodus

of fifth pereiopod. (PI. VI a,c; Fig. 4a

a)

r Dactylus of fifth pereiopod with two

rows of longer, dense setae used as

egg brushes during incubation (PI. VI

d)

r Pleopods much larger; pleopodal

endopods of mature females bear

ovigerous setae (PI. VI g; Fig. 4b)

3.3.1.18 T. orientalis

Male

,.. Genital opening on coxa of

fifth pair of pereiopods (PI. X a;

Fig. 6a: b:vi)

r Gonopore is a relatively big

simple aperture (PI. X b)

r Dactylus of fifth pereiopods

bluntly cylindrical and club-

shaped. relatively slender and

sparsely setose. It ends with a

very small inwardly curved spine

(PI. IX c)

:;.. Pleopods comparatively small

(PI. IX d)

3.3.1.2 Secondary sexual characters

3.3.1.2A P. po/yphagus

3.3.1.2.Ai Male

25

Female

,.. Genital opening on coxa of third

pair of pereiopods (PI. X c; Fig. 6a:

avi)

,.. Gonopore is a simple aperture but

much smaller than the gonopore of

males (PI. X d)

,.. Dactylus of fifth pereiopods bluntly

cylindrical, more stout, with a

longitudinal groove on the dorsalr side

against which lies the claw. Dense

setae seen in maturing and mature

females (PI. IX c)

:;.. Pleopods much larger; pleopodal

endopods of mature females bear

ovigerous setae (PI. IX f-g; Fig. 6b)

(a) Pereiopods: (PI. VI b,e; Fig. 4a) The II - V pairs of pereiopods grow

relatively longer in the males as the animals approach sexual maturity. These

legs play an important role in holding the female at the time of courting and

mating.

26

(b) Gonopore and Penile Process: (PI. VII; Fig. 4c) The gonopore at the

end of the ejaculatory duct, on the coxa of the fifth pair of pereiopods, is a

complex structure associated with a pointed and serrated penile process

terminating in a distal tuft of setae. In juvenile male P. po/yphagus, the

gonopores are visible as tiny openings on the coxa of the fifth pair of

pereiopods. As the animal grows, a small knob-like protrusion develops on the

lateral edges of the coxa of the leg. As the animal approaches sexual

maturity, this structure becomes a prominent pointed penile process with a

curved tip bearing a tuft of setae.

The active gonopore in a mature male is membranous, with very strong

elastic folds and is highly sensitive. The pigmentation on the gonopore is also

an indicator of sexual maturity. While the gonopore is translucent white

initially, as the penile process develops, it develops a pinkish pigmentation,

often tending to reddish pink at the time of breeding (PI. VII c).

3,3,1,2.Aii Female

(a) Pereiopods and egg brush: (PI. VI a,c,d; Fig. 4a) The pereiopods in

females play an important role during incubation. The dactyli at the tip of the

pereiopods, particularly the fifth pair of pereiopods, develop long, dense setae

which are used as an egg brush, to clean the eggs carried on the abdominal

pleopods.

27

(b) Ovigerous setae on abdominal pleopods: (PI. VI f,g; Fig. 4b) The

endopods of the abdominal pleopods in females bear ovigerous setae which

are used for attaching spawned eggs until the end of the incubation period. In

juvenile female, the pleopods are devoid of setae. As the female enters into

the sub-adult phase, the pleopods enlarge and the endopods bifurcate and

develop long ovigerous setae. Development of the ovigerous setae marks the

onset of sexual maturity in females. Sometimes the setae are found to persist,

after two or three breeding cycles, even when the animals enter a non­

breeding phase Therefore, while the setae may be indicators of sexual

maturity, they may not always be indicative of the breeding cycle of the

female.

(c) Mating window: (PI. VIII; Fig. 5) As the female enters its breeding

phase, a process of decalcification of the ventral sternal plates commences.

The plates at the base of the fifth, fourth and third pairs of pereiopods develop

soft mating windows which function as the sites of spermatophore deposition

during mating. There are four pairs of windows in p, polyphagus - two at the

base of the fifth ventral sternite and one each at the bases of the fourth and

third ventral sternites. The windows are separated by distinct calcified ridges.

Decalcification takes place from the base of the fifth pair of pereiopods to the

third, and the sizes of the windows increase with the size of the female.

3.3.1.2B T. orientalis

3.3.1.2Bi Male

28

(a) Gonopore: (PI.X a,b; Fig. 6a: b) The gonopore situated in the form of a

simple aperture at the base of the fifth walking leg, is seen to increase in size

as the animal grows.

There are no other distinct secondary sexual characters marking the

onset of sexual maturity in male T. orienta/is.

3.3.1.2.Bii Female

(a) Ovigerous setae on abdominal pleopods: (PI. IX e-g; PI XI; Fig. 6 b)

In T. orienta/is also, the endopods of the abdominal pleopods in females bear

ovigerous setae which are used for attaching spawned eggs until they are

hatched. In juvenile female, the pleopods are devoid of setae. As the female

enters into the sub-adult phase, the pleopods enlarge and the leaf-like

endopods bifurcate and develop long ovigerous setae. Development of the

ovigerous setae marks the onset of sexual maturity in females. There is a

small club-shaped process midway along the inner margin of the endopod of

the first pair of pleopods. The endopods of other three pairs of pleopods are

ovoid at the proximal end and narrow and tubular at the distal end.

(b) Gonopore: (PI. X c,d; Fig 6a: a:vi) The female gonopore, situated in

the form of a simple aperture at the base of the third walking leg, is seen to

increase in size as the animal grows but is relatively much smaller than the

male gonopore.

3.3.2 Reproductive System - Structure and Maturation

3.3.2.1 Reproductive system

3.3.2.1A P. po/yphagus

3.3.2.1Ai Male

(a) Anatomy

29

The male reproductive system (Plate XII; Fig. 7) in P. polyphagus

consists of paired testes and a pair of vas deferens leading to a narrow

ejaculatory duct. The testis is a semitransparent light yellowish organ placed

over the dorsal surface of the midgut gland, beneath the heart. The

mature testis is a H-shaped structure with two anterior lobes and two

posterior lobes. The anterior lobes extend to the gut and the posterior

lobes extend backwards up to the first abdominal segment. Paired vas

deferens arise from the outer side of the posterior lobes and open

through a chitinous sigmOid shaped gonopore on the coxopodites of the

fifth walking leg on either side. Each vas deferens arises as a slender

tube with a highly convoluted proximal end leading into a thicker and

dilated distal end which gradually thins down again to form the

ejaculatory duct. The thicker intermediate portion shows a tendency to

vary in size with the maturation process and contains a mucoid

substance. The proximal coiled region and the intermediate thicker

portion of the vas deferens are the sites of spermatophore production.

The spermatophoric mass (PI. XXI d-e; Fig. 11 f) consists of a highly

convoluted tube containing the sperm mass embedded in a gelatinous matrix

secreted by the inner lining of the proximal vas deferens. The outer covering

of the spermatophore is a layer of thick and hardened gelatinous material

30

secreted in the distal vas deferens. The spermatophores are retained in

the vas deferens until copulation. Following this, the spermatophores

are ejaculated through the ejaculatory duct at the distal portion of the

vas deferens.

(b) Histology of testis and vas deferens

(PI. XIII a-t; Fig 11 a-f)

The wall of the testis is double-layered, consisting of an outer

epithelium and an inner layer of connective tissue. At the onset of maturation,

the testis contains follicles or acini (seminiferous lobules) filled with primary

spermatophores (PI. XIII a-d). A lumen is formed in maturing testes and

development of the spermatids within the follicular cells is aided by the nurse

cells or Sertoli cells. The lumen is placed towards one side of the testis and

empties into the vas deferens.

Transverse sections of the vas deferens reveal a thin outer layer of

muscle and connective tissue and an inner layer of columnar epithelial cells,

enclosing the lumen (PI. XIII g-j). The epithelial cells are glandular in nature

and secrete a gelatinous matrix which is strongly eosinophilic. The sperm

mass, or spermatophore, consisting of spermatids, disintegrating

spermatocytes and Sertoli cells, is embedded in the mucoid matrix secreted

by the glandular epithelial cells. Within the spermatophore, the spermatocytes

and spermatids are embedded in a gelatinous substance. The sperm mass

moves through the lumen of the vas deferens from its proximal part to the

distal part. The glandular epithelium of the proximal vas deferens is folded

31

inwards. Proliferation and invagination of the glandular epithelium form leaf­

like typhlosoles with villi in the distal vas deferens(PIXIlI k-r). The typhlosoles

secrete a strongly eosinophilic granular matrix into the lumen. The

spermatophore mass remains distinct within this matrix. At the time of

copulation, the spermatophoric mass is expelled from the vas deferens by the

muscular contraction of its walls.

3.3.2.1Aii Female

(a) Anatomy

The internal reproductive system of a mature female P.

po/yphagus is shown In PI XIV and Fig 8. The ovary is situated

dorsally, beneath the heart and over the midgut gland. The mature

ovary is H- shaped, with two anterior lobes extending into the cephalic

region and two posterior lobes extending up to the first abdominal

segment. The anterior lobes have curved tips, turning upwards. The two

posterior lobes are of unequal length, with the left lobe being slightly

longer. A pair of semi-transparent slender oviducts arising just below

the transverse connection between the two lobes open to the exterior

through a pore on the coxa of the third walking leg.

(b) Histology of ovary and oviduct

(PI XV a-I; Fig. 9)

The ovarian wall (PIXV h) consists of an outer epithelial layer, a

middle layer of connective tissue supplied with blood vessels and an

inner germinal epithelium. The thickness of the connective tissue

32

changes with the ovarian maturation cycle. The germinal epithelium

forms a series of inward folds as it runs through the length of the

ovary. Ova are formed from the germinal epithelium and are initially

surrounded by a single layer of flat follicular cells. Developed oogonia,

primary and secondary oocytes and developing ova can be seen distributed

from the central germinal epithelium to the peripheral wall of the ovary. The

process of ovarian maturation through oocyte formation and development was

traced through three distinct phases depending on the extent of yolk

deposition -

"y Pre-vitellogenesis phase: (PI. XV a-c) This is the immature phase in

ovarian development, where the outer wall of the ovary is thin. The

developing oocytes are mostly surrounded by follicle cells. The nuclei of the

developing oocytes are large and well-defined, with prominent nucleoli. There

is no indication of yolk deposition. The cytoplasm is basophilic and takes

haematoxylin stain.

"y Primary vitellogenesis phase: The ovary enters into the developing

stage, where the cytoplasm gradually becomes more eosinophilic. This

phase is completed through three stages.

Stage 1 : (PI.XV d) Many oocytes have a few peripheral vacuoles scattered in

the cytoplasm. The cytoplasm takes haematoxylin stain.

Stage 2 : (PI. XV e) There is an increase in the number of peripheral

vacuoles which tend to be distributed uniformly around the nucleus. Yolk

deposition begins in the cytoplasm of the developing oocytes. The cytoplasm

33

turns slightly eosinophilic. The germinal zone is placed centrally and

developing oocytes are seen near to the peripheral region.

Stage 3 The peripheral vacuoles are larger in size. Yolk deposition

becomes more dense. Eosinophilic granules increase in number among the

peripheral vacuoles. The egg membrane is seen between the oocyte

and the follicle cells.

:.- Secondary vitellogenesis phase The ovary is now in a mature

stage, where yolk granules accumulate abundantly in the developed

oocytes. This phase is completed in two stages -

Stage 1: (PI. XV f,i) Mature oocytes are seen in the ovarian cavity. Dense

yolk deposition takes place and nuclei begin to be masked. The cytoplasm is

highly eosin-positive.

Stage 2: (PI. XV g) The size of the oocytes become maximum while the

nuclei decrease greatly in size and can hardly be observed. Yolk deposition

is complete. The oocytes are now in a state of complete maturation. The

ovarian cavity is packed with mature oocytes.

The tubular oviduct (PI. XV j-I) is more compressed than round. The

wall of the oviduct is made up of an outer thin layer of epithelium, a middle

layer of connective tissue and an inner layer of high columnar epithelium. The

lumen of the oviduct in a mature adult is large and bears numerous long villi.

The columnar cells and villi often form semi-closed channels within the lumen

(PI. XV I)

3.3.2.1 B T. orientalis

3.3.2.1 Bi Male

(a) Anatomy

34

The testes (PI. XVI; Fig. 10), situated dorsal to the alimentary tract,

are a pair of highly convoluted white tubular structures joined by a

transverse bridge, giving it an H-shaped appearance. The lobes extend

backwards into the abdominal region. The vas deferens arise posterior

to the transverse bridge. The proximal vas deferens is highly

convoluted while the distal vas deferens is straight and opens through

the genital pore on the coxa of the fifth pair of pereiopods. The vas

deferens in a mature male holds the spermatophoric mass which is seen as a

white gelatinous substance. The spermatophoric mass is expelled from the

terminal tubular part of the vas deferens and through a pair of gonopores on

the coxa of the fifth pair of pereiopods at the time of copulation.

(b) Histology of testis and vas deferens

(PI. XVII a-I; Fig.11 g-h)

The wall of the testis is double-layered, consisting of an outer layer of

membrane and connective tissue and inner layer of epithelium (PI. XVII a-b).

The immature testis contains numerous follicle cells or acini filled with

spermatogonia (PI. XVII c-d). It is within these acini that the spermatogonia

develop into primary spermatocytes, secondary spermatocytes and

spermatids.

3S

Transverse sections of the vas deferens reveal an outer layer of

muscle and connective tissue and an inner layer of glandular epithelial cells,

enclosing the lumen. The spermatophoric mass, consisting of spermatids,

developing spermatocytes, disintegrating spermatocytes and nurse cells, is

embedded in a mucoid gelatinous matrix secreted by the glandular epithelial

cells. The sperm mass moves through the lumen of the vas deferens from its

proximal part to the distal part. The typhlosole (PI. XVII e-g) in the distal vas

deferens in T orienta/is is seen as one big lobe pushing inwards into the

lumen. The typhlosole has only connective tissue lacks villi and columnar

epithelium. The spermatophores (PI. XVII i) are seen arranged along the wall

opposite to the typhlosole, embedded in a gelatinous matrix (PI. XVII h).

Unlike in the case of P. po/yphagus, the final spermatophoric mass in T

orienta/is is not bound by a hardened gelatinous matrix.

3.3.2.1 Bii Female

(a) Anatomy

The ovaries (PI. XVIII & XIX; Fig. 12), like the testes, are a pair of

tubular structures connected by a transverse bridge, giving an H-shaped

appearance. The ovaries are also placed dorsal to the alimentary tract

A pair of thin oviducts arise from the ovary posterior to the transverse

ridge and open through the genital pores on the coxa of the third pair

of pereiopods.

-----------------_ .. _----

36

(b) Histology of ovary and oviduct

(PI. XX a-i)

The ovarian wall consists of an outer connective tissue supplied

with blood vessels and an inner germinal epithelium (PI. XX a-b). The

thickness of the connective tissue changes with the ovarian maturation

cycle, being thin initially and thickest in spenUrecovering ovaries. The

germinal epithelium (PI. XX d) tends to fold inwards and give rise to the ova.

Immature ova are surrounded by follicular cells. The ovary of immature and

maturing individuals lack a lumen; the ovaries of mature individuals were

found to have a central lumen. As in the case of P. po/yphagus, the process of

ovarian maturation in T orienta/is could also be traced through three distinct

phases depending on the extent of yolk deposition -

~ Pre-vitellogenesis phase: The ovary is in an immature state,

with a thin outer wall and immature oocytes. The oocytes have large and

well-defined nuclei and are mostly surrounded by follicle cells. There is no

yolk deposition. The cytoplasm is basophilic and takes haematoxylin stain.

).> Primary vitellogenesis phase The ovary begins developing.

Development of the oocytes progresses through two distinct stages -

Stage 1 : A number of developing oocytes are seen to have a few peripheral

vacuoles scattered in the cytoplasm. The cytoplasm takes haematoxylin

stain.

Stage 2 : (PI. XX c) As development progresses, there is an increase in the

number peripheral vacuoles which grow larger and tend to be distributed

37

uniformly around the nucleus. Yolk deposition begins in the cytoplasm of the

developing oocytes. The cytoplasm turns slightly eosinophilic .

., Secondary vitellogenesis phase In this phase yolk granules

accumulate abundantly in the developed oocytes and the ovary becomes

mature. Two distinct stages can be recognized in this phase -

Stage 1 : Dense yolk deposition takes place and nuclei begin to be masked.

The large vitellogenic oocytes are separated from the follicle cells by the

egg membrane. The cytoplasm takes eosin stain.

Stage 2: (PI. XX a,b,d) The oocytes are large, globular and non-nucleated or

with shrunken nuclei. Yolk deposition is maximum and dense yolk granules

are visible. The ovarian wall is strong and tense.

Transverse sections of a spent ovary reveal resorbed oocytes and

infiltration of connective tissue (PI XX e). The ovarian wall at this stage is

relaxed. Transverse sections of the oviduct reveal that the wall of the oviduct

is made up of an outer thin layer of epithelium, a middle layer of connective

tissue and an inner layer of columnar epithelium (PI. XX g-i). The tubular

oviduct (PI. XX f) in T. orienta/is is more compressed than round. Unlike in the

case of P. po/yphagus which has a large central lumen in the oviduct, the

lumen of the oviduct in T. orienta/is is smaller, as are the villi. The lumen is

placed towards the peripheral regions on either side of a central layer of

connective and muscular tissues.

3.3.2.2 Maturation

3.3.2.2A P. po/yphagus

3.3.2.2Ai Male

38

The testes in male P. po/yphagus do not show any visible colour

change during maturation (PI XII, Fig. 7) Immature gonads in juveniles

appear as translucent membranes and do not extend into the abdomen.

In sub-adults, the gonads become slightly whitish and the median vas

deferentia in males become opaque. The posterior lobes of the testes

do not reach upto the tip of the carapace at this stage. In adults, the

entire testis becomes milky-white, the distal vas deferens become very

thick (PI XII d) and the external gonopores in the form of penile processes

become pigmented (PI VII c & XII e). The posterior lobes of the testis now

extends beyond the carapace, into the abdomen. A mature male is always

characterized by the presence of spermatozoa in its testis.

Visible changes in the secondary sexual characters are a more reliable

means of assessing the status of sexual maturity. Development and

pigmentation of the penile process are positive indicators of the onset of

sexual maturity in males (PI. VII c & XII e; Fig. 4c).

3.3.2.2Aii Female

Ovaries that are immature ovaries or in the early stages of

development cannot be discerned visually through the dorso-thoracic

musculature. In the advanced stages of development, the ovaries are easily

39

viewed through the musculature. However, such visual assessment can be

misleading in the case of a spent ovary as the yellow colouration and onset of

rematuration can lead to identification as a developed ovary.

The ovary undergoes a series of colour and size variations in

tandem with the maturity cycle (Fig. 8). Immature ovaries in juveniles

appear as translucent membranes and do not extend into the abdomen.

In sub-adults, the ovaries become light yellowish and extend further

anteriorly and posteriorly. Mature ovaries are dark yellow or orange in colour.

The oviducts however, remain translucent. The posterior lobes of the

ovaries extend beyond the carapace, into the abdomen. A spent ovary is

usually light cream or light orange in colour and a number of residual and

resorbing ova are visible through the ovarian wall, lending a patchy

appearance to the ovary (PI. XIV).

Kagwade (1988) classified the ovarian cycle into seven stages. In

the present study the ovarian development was classified into five stages

- Immature, Developing/Redeveloping, Ripe, Spawned and Spent

Immature

Maturing

Thin, flattened,

transparent ova.

(Fig. 8a).

transparent ovary with

Nuclei are distinctly visible

Ovary slightly enlarged and white. There is

differential development of ova and small

transparent immature ova can be seen along

Ripe

Spawning

Spent/Recovery

40

with larger ones in which yolk deposition has

set in (Fig. 8b).

As development progresses, the colour

becomes orange and the ovary is enlarged,

with few transparent immature ova, opaque,

orange mature ova and a number of maturing ova

(Fig. 8c)

Dark orange fully ripe ovary with dark orange

opaque ova (Fig. 8d)

Orange ovary with white patches, retaining

part of the ova. Ova slightly smaller, orange

and opaque.

Flabby ovary white or light orange with

residual opaque ova and a number of

immature ova (PI. XIV).

The average diameter of freshly-laid eggs in P. polyphagus was 0.459

mm.

The onset of maturity in females is marked by the development of

ovigerous setae (PI. VI f-g; Fig. 4b) and decalcification of sternal plates (PI. VII

a-f; Fig. 5a-f). As the female matures, soft, decalcified windows appear on the

ventral sterna at the base of the third, fourth and fifth pairs of pereiopods.

These are the sites of spermatophore reception during copulation, and

storage until fertilization. The size of the windows increases as the female

grows and often the ridges of the sternal plates get rnerged with the windows.

3.3.2.2B T. orientalis

3.3.2.2Bi Male

41

As in the case of P. po/ypl7agus, there is no external indication of

gonadal maturation processes The testis and vas deferens undergo

alterations in size and form (Fig. 7). The colour tends to remain white, but as

maturation progresses, the gonads become less translucent and more

opaque. Immature gonads in juveniles of T. orienta/is also appear as

translucent membranes and do not extend into the abdomen. In sub­

adults, the gonads become slightly whitish and the median vas deferens

become opaque (PI. XVI b). In active adults, the entire testis becomes

milky-white and the median vas deferens becomes very thick (PI. XVI c;

Fig. 10 c). The posterior lobes of the testis extend beyond the carapace,

into the abdomen (PI. XVI a).

The male gonopores, situated on the coxa of the fifth pair of

pereiopods are visible as tiny openings in juveniles, which grow larger in size

as the animals grows and enters sexual maturity. Unlike in the case of P.

po/ypl7agus, there is no distinct protrusion or extension of the gonopores into

penile processes and the gonopores exist as simple apertures (PI. X a-b; Fig.

6a: b).

3.3.2.2Bii Female

Ovarian maturation in T. orienta/is is similar to the process in P.

po/ypl7agus. The ovary undergoes a series of colour and size variations

in tandem with the maturity cycle. The immature ovary (PI. XIX a; Fig. 8a)

42

is translucent and becomes white as the animal enters into the early stages

of maturation (PI. XIX b-c; Fig. 8b). At this stage the posterior lobes of the

ovary do not extend into the abdominal region (PI.XVIII a). As maturation

progresses the ovary becomes creamish to dark yellow (PI.XIX d-e) and

finally dark orange, when it is ready for spawning (PI. XIX f). The oviducts

however, remain translucent. In the mature stage, the posterior lobes of the

ovary extend to the abdominal region (PI. XVIII b).

Kagwade and Kabli (1996) recognized five stages of ovarian

development in Torientalis. In the present study also the ovarian

development was classified into six stages - Immature, Early Maturing, Late

Maturing/Mature, Ripe, Spawning and Spent/Recovery.

Immature

Early Maturing

Late Maturing/Mature

The ovaries are initially seen as a pair of

translucent, white straight, thin structures. There is

no evidence of any individual oocytes (PI. XIX a).

Ovary becomes slightly enlarged and white.

Although individual oocytes are not visible initially,

as the ovary matures, small transparent immature

ova can be seen along with a few larger ones

in which yolk deposition has begun (PI. XIX b­

c).

As development progresses, the colour

becomes orange and the ovary is slightly

enlarged with some transparent immature ova

Ripe

Spawning

Spent/Recovery

43

and opaque, orange maturing ova, which can be

seen through the ovarian wall. Towards the end of

this phase, the ovary is packed with mature ova

(PI. XIX d-e)

Dark orange fully ripe ovary with dark orange

opaque ova. The ovary occupies a major portion

of the cephalothoracic region and is easily visible

through the dorso-thoracic musculature (PI. XIX f).

Orange ovary with white patches, retaining

part of the ova. Ova slightly smaller, orange

and opaque (PI. XIX g).

Flabby ovary white or light orange with

residual opaque ova and a number of

immature ova.

The average diameter of freshly-laid eggs in T. orienta/is was 0.817 mm.

The onset of maturity in females is marked by the development of

ovigerous setae (PI. IX e-g; Fig. 6b). The setae, once developed, lose their

density after the first spate of egg bearing, but regains it during the next phase

of breeding. This continues as cyclic phenomenon, coinciding with the

breeding activity of the female. There are no mating windows in female T.

orienta/is.

3.3.3 Mating and spawning

3.3.3A P. po/yphagus

44

Mating in P. po/yphagus occurs 1 - 12 days prior to spawning (egg

release) and not immediately after the pre-mate moult. Fertilisation is external

and occurs only at the time of spawning. Mating is seen to be associated with

the development of orangish colour in the ventral sinus of the female,

indicating the presence of vitellogenin. The time between the pre-mate moult

and mating, during which the pre-copulatory courtship takes place, ranged

from 20 to 40 days.

Males exhibit aggressive behaviour among themselves and separate

dens should be provided for active males. Usually the largest male

monopolises the attention of all the females in one tank and a single male

successfully im pregnates upto four females. Reproductive success is

maximum between males and females of the same age group wherein the

males are larger than their female counterparts. Older males show poor

response to young females breeding for the first tirne. Males of 70 - 80 mm

CL were found to mate with females of 66 - 75 mm CL and males of 80 - 100

mm CL rnated with females of 75 - 95 rnrn CL. However, males of 100 - 120

mm CL did not show any response to reproductively active females of 66 - 75

mrn CL.

Courtship begins immediately after the female completes the pre-mate

rnoult. The reproductively active female performs stridulation to attract a

45

reproductively active male. Vision and antennal sensitivity also playa major

role in detection of a mating partner by the male. The male tracks the female,

usually from behind, continuously probing with the antennae. The male

chases the female continuously and feeding activity reduces considerably for

some days Courtship behaviour is best observed in the early evening hours.

The chasing activity continues during the day while mating occurs between

late night and early morning hours. There is no aggression between sexes

while mating.

Towards the final phase of courting, the male moves towards the

female and copulation takes place frontally as the male vertically embraces

the female, from head to tail. A diagrammatic representation of courting and

mating behaviour observed in P. po/yplJagus is given in Fig. 13 a.

The duration of copulation is very short and lasts for only two to three

minutes. The extensible cuticular membranes of the active male penile

process stretch and open outwards. As the male moves over the female, the

membranes of the gonopore stick on to the soft window areas on the sternum

at the bases of the fifth, fourth and third pereiopods of the females. The male

deposits the spermatophore along with its gelatinous matrix over the window

areas. Spermatophore extrusion is done continuously and it is deposited as

two layers, the outer layer being the gelatinous matrix which serves to cement

the spernatophore to the female plastron, and the inner core layer being the

convoluted spermatophore chambers. The implantation of the

46

spermatophores of the male lobster on the plastron of the female is

called impregnation and the spermatophore-bearing female is called an

impregnated female. As soon as impregnation is complete, the lobsters

separate immediately. There is no conspicuous post-mating co-habitation or

guarding of the females by the males.

The spermatophoric mass when freshly deposited on the female,

is white and soft. It soon darkens to a lichen-green colour and

becomes hard. It eventually turns blackish in colour, the pigmentation

proceeding inwards, from the margins. In this condition, it is also known

as the "tar spot". The tar spot is butterfly-shaped, broader at the base (over

the windows of the fifth walking leg) and tapering towards the top (over the

windows of the third walking leg) (PI. XXI a-c) It is quite smooth on the

outside and broader than reported in other spiny lobsters. The bigger the

male, the bigger is the spermatophore attachment.

The impregnated female now prepares itself for spawning, which

takes place 2 - 10 days after mating. Before egg extrusion, the female

scrapes the surface of the tar spot with the chelate part of the fifth leg

to release the sperms. The eggs from the oviduct pass over this sperm

mass and fertilization takes place in the brood chamber. It was observed

that in young females breeding for the first time, in 90% of the cases, the

spermatophore is almost completely scraped off and used up for fertilizing the

first batch of eggs, and only the margins of the attachment remain for a few

47

weeks on the sterna. However, in older females, a considerable part of the

spermatophore attachment is retained after the first egg extrusion. It remains

throughout the incubation period and is used to fertilize the successive batch

of eggs, unless it is tampered during frequent sampling and handling of the

female held in captivity.

The fertilized eggs attach themselves to the ovigerous setae on

the pleopods(PI XI, XXIII & XXIV b) This process is called oviposition.

Three pairs of dark scars mark the remnants of the spermatophoric

mass after it is completely scraped off (PI XXIII). Spawning in the

females is usually complete and the ovaries are very rarely seen to be

in a partially spent state. A mature female in the late intermoult stage

usually spawns twice, and sometimes thrice, during a single intermoult

phase, the gap between two a hatching and the next spawning ranging

from 2 to 5 days. Rematuration takes place after a span of about two

to three months, during which span the animals undergo atleast one

moulting. When mating fails to occur or impregnation does not take place

properly, the mature females release unfertilized eggs, which are pale

pink in colour. These eggs are shed from the pleopods in about two to

five days after spawning. Whenever mating occurs the eggs released

are fertilized and bright orange in colour (PI XXIV a). A single

impregnation usually suffices for two and sometimes even three

successive spawnings within the same maturation cycle of the female.

48

The incubation period in P. polyphagus ranges from 25 to 32 days and

is easily influenced by the water temperature. During this period, the females

maintain their lower abdomen and tail slightly bent inwards and the setose

exopods of pleopods and dactyli of the pereiopods are used to maintain

constant fanning and grooming of the eggs. The female tends to remain in

isolation and shows very little movement. Feed intake is also considerably

reduced during the incubation period.

At the time of hatching, the female straightens its abdomen and

extends the tip of the abdomen to the water surface, and the phyllosoma that

hatch out are fanned away with the pleopods. Hatching takes place only

during the early morning hours and is usually completed the same day. A

considerable number of eggs are prone to be lost during the incubation period

if water quality and tank bottom quality are not maintained well or if the animal

is subjected to increased handling stress.

3.3.38 T. orientalis

Males are smaller than females and are generally more active.

Courtship lasts for a few days prior to mating, when the males actively move

around in the tank often flipping over while swimming. During the courtship

period, the males are very active at night and are often seen swimming even

during the day, chasing the females, which are less active. Mating usually

takes place during late night hours.

49

There is no compulsory premate moult in this species. A female that is

ready to mate has well developed ovigerous setae and exhibits a colour

change in the ventral sinus. The male uses its antennules to sense the

presence of the female during the courtship chase, and as it catches up with

the female, it climbs on to the female and turns it over, holding on to its tail

end. Copulation takes place with the animals facing ventrally in reverse

position so that the ventral sternal tip of the male is slightly in front of the

female's sternum. Fig. 13 b provides a diagrammatic representation of the

courting and mating behaviour observed in T. orienta/is.

Copulation is slightly prolonged in this species, and lasts for nearly five

minutes. As the pair hold on to each other tightly with their legs, the female

probes the inter-tegumental membrane on the last abdominal segments of the

male and nibbles at its uropod. As a result, reproductively active males are

often seen with damaged uropod fans and tail ulcers which invite bacterial

invasion.

When copulation is over, the impregnated female curves its abdomen

inwards and holds it at a slight elevation from the tank bottom. It is not very

active and tends to crouch in some dark corner above the substratum. It

carries the impregnation till the early morning hours. The freshly released

spermatophores are milky white in colour, soft, delicate and embedded in a

gelatinous matrix of mucoid fibrils. The impregnation is seen on the ventral

side of the first abdominal segment as two parallel lines, usually extending

50

from the coxa of the fifth pair of pereiopods to the posterior tip of the second

abdominal segment (PI. XXII a-c). The spermatophore packets are ovoid in

shape and microscopic in size. They holding the active spermatozoa within.

The gelatinous matrix in which the spermatophores are embedded holds a

fibrillar network to which the spermatophores are attached (PI. XXII d-e).

When the spermatozoa are released for fertilization, the empty

spermatophore packets are left attached to the fibrils in the matrix (PI. XXII f).

Egg laying commences in about two hours and the eggs released are

guided from the sternal region to the abdominal brood chamber where they

are attached to the ovigerous setae on the endopod of the pleopods. The

abdomen remains completely curved inwards, forming a 'U'-shaped cup-like

brood chamber laterally sealed by the exopodites and the teeth-like

extensions of the abdominal tergites. The endopodal brances and their setae

spread like a floor on the ventral side of the chamber when all the eggs and

spermatozoa mixed with water have been pushed in.

Almost 90% of the eggs get attached to the pleopodal setae,

irrespective of whether they are fertilized or not. The fertilized eggs (PI. XXVI

a) are dark yellow or orange in colour while the unfertilized eggs turn pale

cream or pinkish. The unfertilized eggs are shed off in 3 - 5 days.

The incubation period in T. orienta/is ranges from 32 to 37 days, during

which embryonic development takes place inside the eggs (PI. XXVI c-f). The

51

abdomen continues to be curved inwards and the eggs are constantly fanned

with the exopods of the pleopods and cleaned with the setal brushes on the

dactyli of the pereiopods. Locomotor activity and feed intake are very much

reduced during the incubation period and the female tends to remain in

isolation.

At the time of hatching, the female holds the inwardly curved abdomen

at a slightly elevated angle and the phyllosoma that hatch out are fanned

away with the pleopods. Hatching (PI. XXVI g) takes place in batches only

during the early morning hours and 'IS usually completed in 1 - 3 days. After

hatching, the empty egg capsules are seen attached to the ovigerous setae.

The capsules are shed, along with a part of the setae, about 48 hours after

hatching. Water quality, tank bottom quality and handling stress, particularly

during the incubation period, greatly influence the success rate of hatching.

During the breeding period, the intermoult phase is highly extended in

the female. After the first brood of eggs have been hatched, the female begins

preparing itself internally for the next breeding, within the same intermoult

phase. Interestingly, the ovigerous setae that developed before the first

breeding continue intact for the next breeding also, in a single intermoult

phase. The entire process takes up to 150 days and in such periods, there is

a considerable delay in growth in these animals. I oj:;r 3 V

3.3.3.1

3.3.3.1A

Breeding in captivity

P. po/yphagus

52

In about 250 days of culture, males grew faster than females. All males

above 70 mm CL were sexually active, with well-developed penile processes

and pigmentation. Females above 66 - 70 mm CL, with full windows, became

active for breeding after a pre-mating moult Breeding success was observed

in 15 numbers of the 24 stocked, out of which 5 were unfertilized (improper

spermatophore attachment). After an incubation period of 25 - 32 days,

leading to hatching, seven lobsters bred again, of which 5 were fertilized and

2 were unfertilized. Post breeding, the females underwent a transition moult

resulting in reduced ovigerous seate and reduced windows. These animals

completed one more moult again, in a span of 150 days from the last

spawning, to enter into a second breeding cycle. A total of 20 animals

survived. 13 animals entered the breeding phase, 4 had unfertilized eggs. Of

these, 10 rematured again and bred.

90% of eyestalk ablated animals bred and spawned in a week's time

after ablation. Further growth progress and maturation in ablated ones was

slightly diminished. Five of the 20 numbers tried gave a second cycle in the

immediate moult itself. There was no delay and therefore continuous egg

production can be induced, compromising the growth. Fecundity and the

quality of eggs however, gets reduced.

53

3.3.3.1A T. orientalis

In 120 days of culture. females became active. Males were

comparatively smaller. Out of the 8 males (61 - 70 mm Cl), all males became

sexually mature while only 3 exhibited sexual activity Of the 16 females tried,

all females developed ovigerous setae beyond 66 - 70 mm CL. Only 6

developed, mated and bred. 5 gave fertilized eggs and 1, unfertilized. After an

incubation period of thirty-five days and a gap of five to ten days, rematuration

and spawning was observed in three lobsters, out of which one gave rise to

unfertilized eggs. Following hatching, these animals moulted into a prolonged

"intermoult" stage. Only ten females survived, completing a second moult in

which a second cycle of breeding was observed only in t3, after a span of

nearly 150 days. Of this two were fertilized and 1, unfertilized. However, the

males did not survive this entire period and new males had to be introduced.

3.3.4 Gonadosomatic Index

3.3.4A P. po/yphagus

3.3.4Ai Male

Average GSI values ranged from 0.48 to 0.77 in immature males of 41 - 55

mm Cl and from 0.84 to 1.61 in early maturing males of 46 - 60 mm CL. In

late maturing and mature males of 56 - 95 mm Cl, the average GSI values

ranged between 2.07 and 3.85. The highest GSI values were observed in

mature males of 66 - 80 mm CL. The average GSI values for different 5 mm

size (Cl) classes among immature, early maturing and late maturing/mature

males are graphically represented in Fig. 14 a. The GSI was seen to increase

with size among immature and early maturing males. In the late

54

maturing/mature group, the GSI increased from 2.07 in the 56 - 60 mm Cl

class to a peak of 3.85 in the 76 - 80 mm Cl class. Thereafter, it varied

between 3.2 and 3.4.

3.3.4Aii Female

Average GSI values ranged from 0.44 to 0.66 in immature females of 41 - 50

mm Cl and from 0.83 to 1.73 in early maturing females of 46 - 60 mm CL. In

late maturing and mature females of 56 - 100 mm Cl, the average GSI

values ranged between 2.97 and 7.73. The highest GSI values were observed

in mature females of 71 - 85 mm CL. In spent/recovery females of 75 - 100

mm Cl, the GSI fluctuated between 0.88 and 2.67. The average GSI values

for different 5 mm size (Cl) classes among immature, early maturing, late

maturing/mature and spent/recovery females are graphically represented in

Fig. 14 b. The GSI was seen to increase with size among immature and early

maturing females. In the late maturing/mature group, the GSI increased from

2.97 in the 56 - 60 mm Cl class and fluctuated between 7.00 and 7.73 in the

size range of 66 - 100 mm Cl. In the spent/recovery females, the GSI

remained at around 1.00 and showed a slight increase only in the highest size

class.

3.3.4B T. orientalis

3.3.4Bi Male

Average GSI values ranged from 0.45 to 0.7 in immature males of 41 - 55

mm Cl and from 1.09 to 1.76 in early maturing males of 46 - 60 mm CL. In

late maturing and mature males of 51 - 85 mm Cl, the average GSI values

55

ranged between 2.17 and 2.77. The highest GSI values were observed in

mature males of 66 - 70 mm CL. The average GSI values for different 5 mm

size (CL) classes among immature, early maturing and late maturing/mature

males are graphically represented in Fig. 15 a. The GSI was seen to increase

with size among immature and early maturing males. In the late

maturing/mature group, the GSI increased from 2.17 in the 51 - 55 mm CL

class to a peak of 2.77 in the 61 - 70 mm CL class. Thereafter, it decreased

slightly and then remained almost steady in animals above 71 mm CL.

3.3.4Bii Female

Average GSI values ranged from 0.6 to 0.88 in immature females of 51 - 65

mm CL and from 1.04 to 2.68 in early maturing females of 56 - 70 mm CL In

late maturing and mature females of 61 - 90 mm CL, the average GSI values

ranged between 4.16 and 4.9. The highest GSI values were observed in

mature females of 85 - 90 mm CL. In spent/recovery females of 75 - 100 mm

CL, the GSI fluctuated between 0.96 and 1.67. The average GSI values for

different 5 mm size (CL) classes among immature, early maturing, late

maturing/mature and spent/recovery females are graphically represented in

Fig. 15 b. The GSI was seen to increase with size among immature and early

maturing males. In the late maturing/mature group, the GSI increased from

4.16 in the 61 - 65 mm CL class and thereafter, did not show much variation

until the spent stage is reached, in the size range of 66 - 100 mm CL. In the

spent/recovery females. the GSI remained close to 1.00 and showed a slight

increase only in the highest size class.

3.3.5 Size at maturity

3.3.5A P. po/yphagus

3.3.5Ai Males

(a) Penile process

56

The male gonopore is visible in juveniles as small as 25 mm CL, but

only in the form of a tiny inconspicuous spot on the coxa of the fifth pair of

pereiopods, ventrally. As the animal grows, the gonopore develops into a

penile process with a hairy tip. The formation of the penile process is first

noticed in male lobsters of 36 - 40 mm CL. The smallest size at which a

penile process was observed was 36.6 mm. Development of the penile

process, is however, slow. About 28% of the animals in the size range of 51 -

55 mm CL, 53% of the animals in the size range of 56 - 60 mm CL and 100%

of the animals above 62 mm CL have well developed penile processes (Fig.

16 a).

Development of the penile process in males held in captivity was quite

similar to the progress seen in the wild. 50% of the males in the size range of

56 - 60 mm CL and 84% of the males in the size range of 61 - 65 mm CL

had well developed penile processes (Fig. 16 b).

(b) Maturing/mature testes

Males in the size range of 36 - 40 mm CL had immature testes. Signs

of gonadal development were observed in some males in the size range of 41

- 45 mm CL In males of 51 - 55 mm CL, the gonads are structurally

completely developed though the vas deferens remains thin without any

swollen ends. 53% of the males in the size range of 56 - 60 mm CL had

57

structurally complete and mature gonads with thickened vas deferens, swollen

at the distal end (Fig. 16 a). At this stage, histological sections revealed the

onset of spermatophore formation. 98% of the males in the size range of 61 -

65 mm CL were functionally mature.

(c) Relationship between carapace length and somatic lengths as

indices of sexual maturity

(i) Carapace length (CL) - Maximum width of carapace (MWC) : (Fig.

17 a)The lines of regression between CL and MWC (at the posterior end of

the carapace) for male lobster <50 mm CL and >50 mm CL did not show any

significant variation in slope or elevation.

(ii) Carapace length (Cl) - Ventral sternite length (VSl) : (Fig. 17 b)

The regression lines of VSL on CL in male P. po/yphagus before 50 mm CL

and after 50 mm CL did not show any marked difference in slope or elevation

(iii) Carapace length (Cl) - length of penile process (PPl) : (Fig. 17 c)

The regression lines of PPL on CL in juvenile and sub-adult/adult male P.

po/yphagus lie on two different elevations and the deflection from one line of

growth to the other lies at 50 mm CL This is the most important external

index of the onset of sexual maturity in the males as the development of the

penile process signifies the functional status of the male's capacity to

impregnate a female.

58

(iv) Carapace length (Cl) - leg lengths (III ll, IV II and V ll) : The

regression lines between CL and length of the third pair of pereiopods in male

P. po/yphagus of <50 mm CL and> 50 mm CL had different slopes and the

point of intersection was estimated to be 55.4 mm CL (Fig. 17 d). In the case

of the fourth (Fig. 17 e) and fifth (Fig. 17 f) pairs of pereiopods in male P.

po/yphagus, the regression lines differed significantly in their elevations,

indicating sharp deflections in growth patterns of the legs during the transition

from juvenile to sub-adult phase.

3.3.5Aii Females

(a) Ovigerous setae

Enlargement of the abdominal pleopods and segmentation of the

endopods begin in females of 46 - 50 rnm CL. More than 50% of the females

of 51 - 55 mm Cl have well developed pleopods. Development of ovigerous

setae begins at this size. The minimum size at which development of

ovigerous setae was noticed was 51.7 mm CL. About 50% of the females in

the CL range of 61 - 65 mm have fully developed ovigerous setae (Fig. 18 a).

In females held in captivity, however, the development of ovigerous setae

appears to begin in slightly higher size class, indicating higher somatic

growth. The size range in which 50% of the females had ovigerous setae was

66 -70 mm CL (Fig. 18 b)

(b) Maturing/mature ovary

Maturation of the ovary was studied in females collected from the

fishery. Females in the size range of 36 - 45 mm CL had immature ovaries.

59

Signs of ovarian development were observed in some females in the size

range of 46 - 50 mm CL. Ovarian development was evident in females of 51 -

55 mm CL, with the development of orangish colour of the ovary, visible

through the dorsal musculature. More than 50% of the females in the size

range of 66- 70 mm CL had ripe ovaries (Fig. 18 a).

(e) Egg brush

The development of the egg brush at the tips of the pereiopods

appears to coincide with the development of ovigerous setae and maturation

of the ovary. Egg brush formation was found in 7% of the sampled females in

the size range of 56 - 60 mm CL. 50% of the females in the size class of 66 -

70 mm CL and 98% of the females in the size class of 71 - 75 mm CL had

well developed egg brushes (Fig. 18 a). In captivity also 50% of the females in

the size range of 66 - 70 mm CL were found to have developed the egg brush

(Fig. 18 b).

(d) Formation of mating windows

Formation of mating windows, i.e. decalcification of the ventral sternal

plates at the base of the third, fourth and fifth pairs of pereiopods, begins in

females in the size range of 66 - 70 mm CL, after ovarian maturation. Almost

50% of the females of 71 - 75 mm CL have well developed pairs of windows

- two pairs each on the fifth and fourth pairs of pereiopods and one pair on

the third (Fig. 18 a). Decalcification begins on the lower most sternal plate,

i.e., at the base of the fifth pair of pereiopods. Window formation signifies that

the female is completely (physiologically and functionally) ready for mating. In

60

captivity also 50% of the females in the size range of 71 - 75 mm Cl were

found to have developed mating windows (Fig. 18 b).

(e) Occurrence of berried females

The minimum size class in which berried females were noticed in samples

from the fishery was 51 - 55 mm Cl and the smallest berried female recorded

measured 54.3 mm Cl and the largest measured 108 mm Cl. However, the

percentage frequency of berried females could not be used as an index to

estimate the size at first maturity as the percentage did not exceed 25% in

any size class from 51 - 55 mm to 106 - 110 mm CL There was a steady

increase in the percentage of berried females upto 81 - 85 mm CL, followed

by a decrease in the higher size classes and only 1 % of the sampled females

of 106 - 110 mm Cl were berried (Fig. 18 a). The peak occurrence of berried

females in the size range of 76 - 85 mm CL follows the completion of ovarian

maturation, development of ovigerous setae, window formation and egg brush

formation. The distribution of berried females thus serves to corroborate the

conclusions drawn from these indices. Occurrence of berried females was

higher in captivity, with more than 50% of the females in the size range of 71

- 80 mm Cl becoming ovigerous (Fig. 18 b)

(f) Relationship between carapace length, maximum width of

carapace, ventral sternite length and leg lengths as indices of sexual

maturity

(i) Carapace length (Cl) - Maximum width of carapace (MWC) : (Fig. 19

a) The lines of regression between Cl and MWC (at the posterior end of the

61

carapace) for female P. po/yphagus <50 mm CL and >50 mm CL differed in

their slopes and the intersection of the two lines was estimated to be at 45.7

mm CL, a size preceding the minimum size at which development of the ovary

was observed in some females.

(ii) Carapace length (Cl) - Ventral sternite length (VSl) : (Fig. 19 b)

The regression lines of VSL on CL in female P. po/yphagus had different

slopes and there was a clear difference in the relationship between the two

parameters before 50 mm CL and after 50 mm CL. The intersection of the two

lines was at 51.1 mm CL, signifying changes due to the animal's entry into

sexual maturity from 51 - 55 mm CL onwards.

(iii) Carapace length (Cl) - leg lengths (Ill ll, IV II and V ll) : The

regression lines between CL and different leg lengths showed variations in the

two size groups with the intersection points lying at 65.2 mm CL for the third

leg (Fig. 19 c), 45.7 mm CL for the fourth leg (Fig. 19 d) and 55.7 mm CL for

the fifth leg (Fig. 19 e). The fifth leg takes a different growth pattern just after

40 rnrn CL. The fourth leg is longer than the third initially but after 55 rnm CL,

the third leg overtakes the fourth. These legs playa role in copulation and

later, in grooming of the eggs during incubation, for which the legs should be

extended up to the brood chamber.

62

3.3.5Aiii Comparison between males and females

A comparison of regression lines of different somatic measurements

mentioned above, relative to CL in male and female P. polyphagus indicate

the following pOints of divergence in relation to sexual maturity -

(a) CL vs MWC : (Fig. 20 a) Initially, males have a higher ratio of MWC upon

CL, but beyond 65 mm CL, females exceed the males in this ratio.

(b) CL vs VSL : (Fig. 20 b) While females have a higher VSL to CL ratio

initially, the males exceed them beyond 50 mm CL, indicating the changes

required for the formation of mating windows. The ventral sternite in females

becomes broader and increase in length is suppressed.

(c) CL vs Leg lengths: While the third (Fig. 20 c) and the fifth (Fig. 20 e)

pairs of pereiopods are relatively longer in females in the juvenile phase, the

lengths take an upper deflection in males at about 45.1 mm CL and 44 mm

CL respectively. The fourth (Fig. 20 d) pair of pereiopods are almost similar in

length in both the sexes initially, but the length in males takes an upward

deflection at 36 mm CL These are important indicators of the onset of sexual

maturity in males as the pereiopods playa major role in the act of copulation

and impregnation.

From the results obtained, it is evident that while the size at first

physiological maturity is attained at 61 - 65 mm CL in male P. polyphagus

and at 66 - 70 mm CL in female p, polyphagus, the size at morphological

maturity is attained earlier, i.e, at 56 - 60 mm CL in males and at 61 - 65 mm

--.. ----~ -"'- __ \ !n;versity

3havilCi '" ." 63

CL in females. However, the size at functional m:tur~~ i~.~e females '~e s In

a little late, i.e., 71 - 75 mm CL, in tandem with formation of the mating

windows. From these inferences it can be concluded that the critical

maturation phase extends between 56 and 65 mm CL for males and between

66 and 75 mm CL for females. The size at onset of sexual maturity, judged

from the 25% success rate in development of different sexual characters

(Figs. 16 & 18), can be traced to 51 - 55 mm CL for males and 51 - 60 mm

CL for females.

3,3,58 T, orientalis

3,3.58i Males

(a) Maturing/mature testes

Males in the size range of 41 - 45 mm CL had immature testes. Signs

of gonadal development were observed in some males in the size range of 46

- 50 mm CL Almost 50% of the males in the size range of 51 - 55 mm CL

mature gonads (Fig. 21). At this stage, histological sections revealed the

onset of spermatophore formation. 97% of the males in the size range of 61

- 65 mm CL (and all males above 62 mm CL) had mature gonads and fully

developed accessory sexual traits and could be termed adults.

(b) Relationship between carapace length, maximum width of

carapace, ventral sternite length and leg lengths as indices of sexual

maturity

(i) Carapace length (CL) - Maximum width of carapace (MWC) : The

lines of regression between CL and MWC at the anterior end of the carapace

64

(AWC) for male lobster <50 mm CL and >50 mm CL differed in their slopes

and the intersection of the two lines was estimated to be at 52.1 mm CL (Fig.

22 a). The lines of regression between CL and MWC at the posterior end of

the carapace for male lobster <50 mm CL and >50 mm CL differed in their

slopes and the intersection of the two lines was estimated to be at 52.7 mm

CL (Fig. 22 b).

(ii) Carapace length (CL) - Ventral sternite length (VSl) : The

regression lines of VSL on CL in male T. orientalis before 50 mm CL and

after 50 mm CL had different slopes and the intersection point, i.e. the size at

which the growth of the VSL in relation to CL changes, was at 39.4 mm CL

(Fig. 22 c).

(iii) Carapace length (Cl) - leg lengths (III Wl, IV Wl and V Wl) :The

regression lines between CL and lengths of the third and fifth pairs of

pereiopods in male T. orienta/is differed significantly in their elevations,

indicating sharp deflections in growth patterns of the legs during the transition

from juvenile to sub-adult phase (Fig. 22 d & fl. In the case of the fourth pair

of pereiopods, the slopes were found to be different for the two size groups

and the point of intersection was estimated to be 42.4 mm CL (Fig. 22 e).

3.3.5Bii Females

(a) Ovigerous setae

Development of ovigerous setae on the abdominal pleopods begins at

46 - 50 mm CL in the wild. A small proportion (18%) of the females in the

65

size range of 51 - 55 mm CL had developed ovigerous setae. 50% of the

females in the CL range of 61 - 65 mm and all females above 71 - 75 mm CL

have well developed ovigerous setae (Fig. 23 a). The development of

ovigerous setae was quite similar in females held in captivity. The formation of

the setae begin at 46 - 50 mm CL, with about 15% of the females in 51 - 55

mm CL bearing ovigerous setae. (Fig. 23 b). 25% of the females of 55 - 56

mm CL and 50% of the females of 61 - 65 mm CL had ovigerous seate.

(b) Maturing/mature ovary

Among females sampled from the wild, ithe size range of 41 - 45 mm

CL had immature ovaries. Signs of ovarian development were observed in

some females in the size range of 46 - 50 mm CL. 50% of the females in the

size range of 61 - 65 mm CL had ripe ovaries (Fig. 23 a). All females above

76 - 80 mm CL were fully mature.

(c) Occurrence of berried females

Among the females sampled from the wild, the minimum size class in

which berried females were noticed was 61 - 65 mm CL. The smallest berried

female recorded measured 64.1 mm CL and the largest measured 100 mm

CL. However, as in the case of P. po/yphagus, the percentage frequency of

berried females could not be used as an index to estimate the size at first

maturity as the percentage did not exceed 26% in any size class from 61 - 65

mm to 93 - 100 mm CL (Fig. 23 a). The percentage of berried females

increased from 11 % in lobsters of 61 - 65 mm CL to 26% in lobsters of 81 -

85 mm CL, followed by a decrease in the higher size classes. The peak

66

occurrence of berried females in the size range of 71 - 85 mm CL follows the

completion of ovarian maturation and the development of ovigerous setae. As

seen in the case of P. polyphagus, the distribution of berried females thus

serves to corroborate the conclusions drawn from these indices.

Among females held in captivity, the occurrence of ovigerous condition

was first seen in animals of 61 - 65 mm CL. 36% of the females of 71 - 75

mm CL were ovigerous (Fig. 23 b). Maximum occurrence of ovigerious

females was observed in the size range of 71 - 85 mm CL.

(d) Relationship between carapace length, maximum width of

carapace, ventral sternite length and leg lengths as indices of sexual

maturity

(i) Carapace length (CL) - Maximum width of carapace (MWC) : The

lines of regression between CL and MWC at the anterior end of the carapace

(AWC) for female lobster <50 mm CL and >50 mm CL differed in their slopes

and the intersection of the two lines was estimated to be at 47.6 mm CL (Fig.

24 a). The lines of regression between CL and MWC at the posterior end of

the carapace for female lobster <50 mm CL and >50 mm CL differed in their

slopes and the intersection of the two lines was estimated to be at 43.9 mm

CL, a size preceding the minimum size at which development of the ovary

was observed in some females (Fig. 24 b).

(ii) Carapace length (Cl) - Ventral sternite length (VSl) : The

regression lines of VSL on CL in female T. orientalis had different elevations

67

and there was a clear difference in the relationship between the two

parameters before 50 mm CL and after 50 mm CL. In Fig 24 c, the area

between 46 mm and 55 mm CL marks the area of deflection from one line of

growth to the next, signifying changes due to the animal's entry into sexual

maturity.

(iii) Carapace length (CL) - Leg lengths (III WL, IV WL and V WL) : The

regression lines between CL and lengths of the III and IV pereiopods (Figs. 24

d & e) differed in their elevations in the two size groups. The deflection area

lay in the range of 49 - 53 mm CL. The intersection of the regression lines for

the fifth pereiopod was at 47.5 mm CL (Fig. 24 I).

3.3.5Biii Comparison between males and females

A comparison of regression lines of different somatic measurements

mentioned above, relative to CL in male and female T. orienta/is indicate the

following pOints of divergence in relation to sexual maturity -

(a) CL vs AWC : Initially, females have a higher ratio of AWC upon CL,

but beyond 55 mm CL, the males exceed the females in this ratio (Fig. 25 a).

(b) CL vs MWC : Females have a higher ratio of MWC upon CL and the

point of convergence is approached only beyond 90 mm CL (Fig. 25 b). This

indicates the adaptation of the female, even before the onset of sexual

maturity, to accommodate the changes involved with ovarian development.

68

(e) CL vs VSL : While males have a higher VSL to CL ratio initially, the

females exceed them beyond 48 mm CL (Fig. 25 c)

(d) CL vs Leg Lengths: The third and fourth pereiopods remains longer

in females. (Fig. 25 d & e) While the fifth pair of pereiopods is relatively

longer in females in the juvenile phase, the length takes an upper deflection in

males at about 47 mm CL and thereafter, the ratio is higher in males (Fig. 25

f). This is an important indicator of the onset of sexual maturity in males as the

fifth pair of pereiopods (at the base of which is situated the male gonopore)

plays a major role in the act of copulation and impregnation. Although the

relationships between the third and fourth pairs of pereiopods do not show

any significant variation with CL between sexes, it is significant to note the

dominance of these legs in females, since these pereiopods, particularly the

third (which holds the female gonopore), play an important role in holding

onto the male at the time of copulation and in directing the eggs into the brood

chamber at the time of oviposition. Following copulation and OViposition, the

pereiopods remain actively engaged in grooming the eggs during the

incubation period.

From the results obtained, it is evident that while there is not much time

frame between the attainment of morphological, physiological and functional

maturity in T orienta/is. The size at first maturity is attained at 51 - 55 mm CL

in male T orienta/is and at 61 - 65 mm CL in female T orienta/is. It can be

concluded that the critical maturation phase in T orienta/is is not as extended

to several size groups as in P. po/yphagus. The size at onset of sexual

69

maturity, judged from the 25% success rate in development of different sexual

characters (Figs. 21 & 23), can be traced to 46 - 50 mm Cl for males and 51

- 55 mm Cl for females.

3.3.6 Fecundity

3.3.6A P. po/yphagus

The fecundity of P. po/yphagus ranged from 158000 eggs (64.5 mm

Cl) to 931000 eggs (118.4 mm el), with an average fecundity of 455000

eggs for reproductively active females in the size range of 64 - 119 mm CL.

The relationship between fecundity and carapace length (Fig.26 a) was

derived as -

Fecundity ('000) = 12.531 Cl - 636.73 (r2 = 0.9685)

3.3.68 T. orientalis

The fecundity of T orienta/is ranged from 19600 eggs (60 mm Cl) to

59500 eggs (102 mm Cl), with an average fecundity of 39300 eggs for

reproductively active females in the size range of 60 - 102 mm Cl. The

relationship between fecundity and carapace length (Fig. 26 b) was derived as

Fecundity ('000) = 0.7285 CL -19.153 (r2 = 0.9424)

70

3.4 DISCUSSION

3.4.1 Sexual Dimorphism and Secondary Sexual Characters

Lobsters are bisexual animals exhibiting sexual dimorphism, and

conform to the general decapod crustacean reproductive pattern which has

been extensively studied and described (Rahman, 1967; Ryan, 1967;

Haefner, 1976 and Zuckner, 1978). Maturation is a process involving

several physical and physiological changes during the life of an animal,

enabling it to reach a state when it can become biologically productive

and effect propagation of its kind. The process of maturation begins

when the animal reaches a certain optimum size and is exposed to

favourable environments. During the juvenile phases of its life history,

the process of visible growth dominates as the vital physiological

activity of the animal. However, as the animal ages, the growth

processes slow down and there is a diversion of energy for the

development of sexual characteristics, both primary and secondary. The

animal is now in a state of maturation. The end result of maturation is

the formation of an adult completely capable of mating and producing

offspring. The prime aim of maturation is the structural and functional

development of the reproductive organs which are responsible for

production of male and female gametes that can give rise to an

embryo upon fusing. The maturation process is often manifested through

changes in certain externally visible structures, which indicate the internal

changes that occur simultaneously or will occur in close succession.

These structures are referred to as secondary sexual characters.

These traits serve the purpose of indicating the maturity state of the

71

animal and its sex. Many such traits also serve different purposes

leading to mating and brooding.

Sexual dimorphism and the secondary sexual characters identified for

the two species in the present study, and their usefulness as indices of sexual

maturity conform to earlier findings in other lobster species (Berry, 1970;

George and Morgan, 1979; Lipcius ef a/., 1983; Bertelsen and Horn, 2000;

Chubb, 2000; DeMartini et a/. , 2005). George (2005) observed the interesting

trend from simple to complex mating structures in both males and females,

reflecting patterns of speciation in the spiny lobster genus Panu/irus. Unlike

the simple gonopore seen in the primitive species, P. cygnus, the structure of

the male gonopore in P. po/yphagus is similar to the complex gonopore

described for the recently evolved species, P. homarus (MacDiarmid and

Sainte-Marie, 2006), indicating the evolutionary status of this species.

Hossain (1978) discussed sexual dimorphism in T. orienta/is based on the

telson , which, in sexually mature females, bore elongated plumose setae at

the end, presumably to protect the berried eggs and also to enable water

circulation among the berried eggs.

3.4.2 Reproductive System - Structure and Development

Lobsters conform to the generalized decapod reproductive pattern

(MacDiarmid and Sainte-Marie, 2006) with paired ovaries or testes lying

dorsally in the body cavity leading via paired oviducts in females or vasa

deferentia in males, to reproductive apertures or gonopores on the coxa of the

third pair of pereiopods in females and the fifth pair in males (Meglitsch,

72

1967). The reproductive anatomies of P po/yphagus and T. orientalis, as

observed in the present study, follow this general pattern. The maturation

process and developmental changes in the gonads observed in the present

study also conform to the general pattern described for other lobsters and

crustaceans (Fielder, 1964; Yano, 1988; Nakamura, 1990; Minagawa and

Sano, 1997).

Observations in the present study on histological changes in the

gonads during the maturation cycle conform to descriptions given for other

crustaceans and lobsters ((Fielder, 1964; Yano, 1988; Nakamura, 1990;

Demestre and Fortuno, 1992; Minagawa and Sano, 1997; Balasubramanian

and Suseelan. 2000). The production of spermatocytes inside seminiferous

lobules or acini. and oogenesis through different stages of vitellogenesis

observed in the two species are similar to the observation made on other

crustacean and lobster species.

A clear difference between P. po/yphagus and T. orienta/is is seen in

the histological development of the male gonads. While the villiform typhlosole

in P. po/yphagus conforms to the descriptions given for other palinurid

lobsters (Berry and Heydorn, 1970). the typhlosole in T. orienta/is is not

villiform and is more ovoid in shape. The typhlosole in T. oriental is was found

to lack a glandular epithelium, and was instead found to be with connective

tissue. The structure and nature of the typhlosole may be directly related to

the nature of the spermatophore. The spermatophore in T. orienta/is is not like

the complex one seen in P. po/yphagus and there is only a singe type of

73

gelatinous fibrillar matrix (which remain mucoid even after impregnation) in

which the spermatophore masses are embedded.

The structure of the oviduct too is different in the two species. The

oviduct is seen to be more flattened in T orienta/is and the lumen appears to

be pushed to the lateral peripheral sides by a mass of connective tissue which

is placed centrally. This structure completely differs from the oviduct in P.

po/yphagus, which has a large central lumen, with villi and a lining of high

columnar epithelial cells.

3.4.3 Mating and Spawning

Mate selection, courting and copulation in a lobster species are

intricately related to the distinctive morphology of the sexes. Olfactory, visual,

auditory and tactile stimuli have been known to playa role in the attraction,

recognition and choice of mates in different lobster species (MacDiarmid and

Sainte-Marie, 2006). The visual sense in spiny lobsters is well developed

especially in tropical lobsters of the genus Panulirus (Meyer-Rochow, 1975,

1988). MacDiarmid and Sainte-Marie (2006) stressed that while olfaction

plays the critical role in determining mate attraction, recognition and choice in

clawed lobsters, vision is likely to play an important role in spiny lobsters,

increasing from least critical in Jasus to most critical in the recently evolved

species such as P. ornatus. Spiny lobsters of the Stridentes group also

possess a stridulating organ (Patek, 2001). MacDiarmid and Sainte-Marie

(2006) in a review of lobster reproduction mention that in Pa/inurus e/ephas,

stridulation by a reproductive female attracts mature males from a radius of at

74

least 15 m, and in the laboratory, males were found to move towards an

underwater speaker broadcasting female stridulation. Berry, 1970 reported

similar stridulation and male reaction in P. homarus. In the present study

mature females of P. po/yphagus were also found to use the stridulating organ

to attract males.

Atema and Voigt (1995) and MacDiarmid and Kittaka (2000) have

reviewed courting and cohabitation in spiny and clawed lobsters. The courting

frontal approach in spiny lobsters has been reported to continue from several

minutes to 11 days prior to copulation (Lipcius and Herrnkind, 1985;

MacDiarmid, 1989b). Copulation in lobsters is usually very brief, typically

lasting less than a minute in nephropid (Framer, 1974; Talbot and Helluy,

1995) and in palinurid lobsters (MacDiarmid and Kittaka, 2000). In the present

study, copulation was found to last for about two to three minutes In P.

po/yphagus. There is not much information on the mating behaviour of

scyllarid lobsters in captivity. In the present study, copulation in T. orienta/is

was found to be slightly prolonged, lasting for five minutes. Interestingly, the

courting approach is also different in this species, with the male crawling on

top of the female and overturning it. Copulation takes place with the animals

in reverse positions and they swim away in opposite directions after the

process.

Sperm transfer from male to female during mating in crustaceans

is effected by means of a spermatophore, which is a specialized sperm

75

packet serving as a vehicle for sperm transport. The spermatophore

essentially contains the sperms surrounded by protective layers of

cellular secretions produced in the vas deferens (Aiken and Waddy,

1980, Kooda-Cisco and Talbot, 1982) Berry and Heydorn (1970)

described the spermatophoric mass in macrurans as being generally

composed of three components - a sperrnatophoric tube, a basal

adhesive matrix and a protective gelatinous matrix. The nature of the

sperrnatophoric masses in P. po/yphagus and T. orienta/is are also seen to be

completely different. The matrix of the spermatophoric mass in P. po/yphagus

changes from its gelatinous form to a hardened stiff form when exposed to

seawater, which is similar to the description given for the spermatophoric

mass of Pa/inurus gi/christi (Berry and Heydorn, 1970). In species exhibiting

external fertilization, the soft mucoid spermatophore extruded and fixed

onto the female thelycum usually hardens, following chemical changes

induced by exposure to seawater. (Radha and Subramoniam, 1985). In

most decapod groups, this hardening of the spermatophore has been

shown to be due to chitin (Spalding, 1942, King, 1948, Uma and

Subramoniam, 1979) and phenolic tanning (Malek and Bawab, 1974,

Subramoniam, 1984). However, this is not found to be the case in T.

orientalis. The spermatophoric mass of T. orientalis lacks an external

gelatinous matrix. The spermatophores remain embedded in a fibrillar mucoid

matrix, which does not harden on exposure to sea water. It breaks open in a

few hours after exposure to seawater. Berry and Heydorn (1970) have given a

similar description for the nature of the spermatophoric mass and its matrix in

J. lalandii.

76

3.3.3.1 Breeding in captivity

Maturation and breeding in captivity remain to be major

challenges in the evolution of husbandry packages for lobsters. The

establishment of culture conditions in which reproduction can be controlled to

produce larvae all year round is one of the requirements for successful larval

rearing of spiny lobsters (Vijayakumaran et al., 2005). Captive breeding,

mating behaviour, mate selection, pre-mating courtship and copulation have

been well studied in the American lobster H. american us (eg. Atema and

Voigt, 1990; Waddy et ai, 1995). Over the last 20 years, there have been an

increasing number of in situ experimental studies that have expanded our

understanding of the complexities of lobster reproduction (MacDiarmid and

Sainte-Marie, 2006) Laboratory experiments have determined that long days

enhance female gonadal development and spawning frequencies in

Panulirus argus and P. japonicus, while warmer temperatures significantly

accelerate these processes in these species (Lipcius and Herrnkind, 1987;

Matsuda et al., 2002) as well as in P. cygnus (Chittleborough, 1976). In

captive breeding experiments in P. homarus, Vijayakumaran et al. (2005),

reported production of four broods in a year by a single female. The number

of spawnings by individual lobsters also varied from one to seven within the

same year. Senthilmurugan et al. (2005) reported repetitive breeding of P.

ornatus. As in the observations made on P. polyphagus in the present study,

there was a considerable time gap (25 to 63 days) between spawning and the

pre-mating moult in female P. ornatus. The growth rate of male P. ornatus

was higher than that of females, as seen in the case of P. polyphagus. Three

77

spawnings in six months just after attaining sexual maturity were reported in

P. ornatus. These observations and the observations made in the present

study in P. polyphagus reiterate the fact that spiny lobsters are amenable to

captive breeding, given the right environmental cues, as suggested by

sachlikidis et al., (2005). Under highly favourable conditions, adult females

are capable of spawning more than once within the same breeding

cycle, thereby exhibiting higher total fecundity. Ino (1950) suggested

that a considerable number of female P. japonicus spawn twice in a

season. Sutcliffe (1953), Williams (1965) and Buesa (1969) recorded

two spawnings in some female P. argus in a single season, without a

moult between two breeding cycles. Berry (1971) observed upto four

repetitive breeding cycles in P. homarus In a year. Chittleborough (1976)

observed two spawnings in a season In about 10.5% of the female

population of P. longipes cygnus George in the wild while about 66.7%

of the females stocked in an aquaria at lower ambient temperature but

with an abundance of food bred twice in a season.

Compared to spiny lobsters, captive breeding of scyllarid lobsters is an

area less explored. The observations made in the present study point to the

fact that T orientalis has a faster growing regime and can be easily

maintained in captivity. However, the breeding responses were not as high as

seen in the case of P. polyphagus, suggesting the need for identifying the

critical environmental cue that may trigger the right physiological response for

captive breeding. Kizhakudan et al. (2004) in further experiments on captive

rearing of scylla rids reported high incidence of maturation and breeding in

78

captivity. Water quality and photoperiod were found to playa major role and

the animals were reared in larger tanks with increased water depth. A major

change from the experiments carried out in the present study was the use of

clam meat as brood stock diet. In the present study, due to limitations in the

natural availability of clams and the abundance of the gastropod Turbo sp.,

the latter was chosen as the standard diet for all experimental purposes.

3.4.4 Gonadosomatic Index

Gonadosomatic Index (GSI) has been used to examine ovarian

development and to obtain information on the spawning season and

reproductive cycle in various crustaceans (Aiken and Wady, 1980; Minagawa,

1997). Although GSI by itself does not provide credible information on ovarian

development, it provides useful information on ovarian cycles which can

supplement detailed information obtained from morphological, anatomical

and histological studies. Studying the reproductive cycle in P. japonicus,

Minagawa (1997) found that ovarian development, particularly, in smaller size

classes, corresponds well with the changes in GSI. This was found to be true

in the present study also. In size classes between 46 and 60 mm CL, in

maturing individuals, the GSI was found to increase. Maximum GSI values

correspond with dominance of mature individuals. Thereafter, in sizes above

70 mm CL, the GSI was found to fluctuate, probably due to the influence of

repetitive breeding cycles and to intermittent growth phases (in female P.

po/yphagus) before the next phase of breeding.

79

3.4.5 Size at Maturity

The size at first maturity is an important indicator of the reproductive

capacity of populations, and thus is an important tool in stock assessment as

alterations in the size at maturity over space and time may be indicative of the

effects of environmental and fishing impacts on the stock. The size at first

maturity is defined as the minimum size at which 50% of the

population has entered into a state of advanced maturation and is

usually estimated by plotting the percentage of mature animals in different

body size classes in a sampled population against size, and directly reading

the size at which 50% are mature. In decapods, the size at first maturity

is usually studied with reference to physiological maturation (the size at

which the gonads attain maturity) and physical maturation (the size at

which the animal is capable of mating and spawning). There is often a

gap in time between the occurrence of physiological and physical

maturity and hence the two phenomenon may not occur at the same

size (Jones, 1988), and both must be known to determine the size at

true sexual maturity (Stewart et aI., 1997). Mac Diarmid and Sainte-Marie

(2006) define three basic indicators of maturity that can each be assessed by

one or more criteria:

• Morphological maturity, detected by development of external body

parts representing secondary sexual characters

• Physiological maturity, reflected in the development of gonads and

accessory glands

80

• Functional maturity, revealed by internal or external features of

behaviour indicating past or current breeding activity

Jones (1988) observed that physiological and physical (or functional) maturity

in scyllarid lobsters may not occur at the same size, and both must be known

to determine the size at true sexual maturity.

Estmates of size at first maturity in lobsters are often limited by several

factors. The probability of the sampled population representing only a small

section of the size groups, or variability in the catchability of mature and

immature lobsters (particularly if the lobsters tend to change habitats with the

onset of maturation) are likely to make the estimates vulnerable to bias

(Farmer, 1974; Tully et al., 2001). The size at first maturity in female lobsters

is usually estimated using functional criteria of egg-bearing (Kensler, 1967;

Aiken and Waddy, 1980), the presence of fresh or spent spermatophores and

resorbing ova, physiological criteria of ovary colour and size, oocyte size and

development of cement glands on the pleopods and morphological criteria of

abdomen and pleopod development (Mac Diarmid and Sainte-Marie, 2006).

Staging based on histological examination of gonads provides more detailed

information than any other indicator (West, 1990). The percentage of

ovigerous (berried) females in the sampled population has been described to

give a reliable estimate of the size at maturity in the spiny lobster J lalandii

(Heydorn, 1969) since he found that after a certain size there was sharp

increase in the percentage of ovigerous condition and the same continued in

higher lengths. However, Berry (1971) found that in P. homarus, the

percentage of ovigerous females showed no marked increase at any

81

particular size and the larger size classes did not show a very high incidence

of ovigerous condition. He attributed this to repetitive breeding in this species.

Kagwade (1988) reported that the percentage frequency of ovigerous P.

po/yphagus in Bombay waters was erratic at various class intervals and at no

length the frequency reached beyond 20.7%. She too attributed this to

repetitive breeding. Observations made in the present study on the

distribution of berried (ovigerous) females in the trawl landings of P.

po/yphagus at Veraval and Mangrol trawl landing centres, indicate a similar

trend. The maximum occurrence (25%) of ovigerous females was observed in

the size range of 81 - 85 mm CL. However, while Kagwade (1988) has

advocated direct observation of the maturity stage of ovaries as a more

reliable method for identifying sexually mature female P. po/yphagus,

decreasing catches and high demand for this commodity has greatly restricted

the availability of sufficient specimens for analysis in the present study

One of the most important external indicators of sexual maturity in

females is the presence of fully developed ovigerous setae on the abdominal

pleopods, to which spawned fertilized eggs remain attached till the larvae are

hatched. Fielder (1964) and Pollock (1982) have recognized the identification

of mature females through the presence of these setae. Paterson (1969)

reported that in some species like J. /a/andii there is a regular cycle of

appearance and disappearance of these setae. Kagwade and Kabli (1996) did

not find any significant relationship between ovigerous setae and maturity in

female T. orienta/is. In the present study it was observed that the appearance

and development of the ovigerous setae for the first time coincides with the

82

onset of sexual maturity in both, P polyphagus and T. orientalis. The number

and size of the setae reach a maximum at the time of egg bearing. After the

first batch of eggs are completely hatched/removed from the pleopods, there

is a reduction in the density of the ovigerous setae, which later increases

during the successive breeding cycle.

George (2005) mentions the role of decalcified windows on the female

sternum in the spiny lobster genus Panulirus, which can be used to determine

maturity (e.g. Lindberg, 1955; Velazquez, 2003). However, while George

(2005) has described the presence of three pairs of mating windows in female

p, polyphagus, the present study clearly shows the presence of four pairs of

mating windows in this species - one pair each at the base of the third and

fourth pairs of pereiopods and two pairs at the base of the fifth pair of

pereiopods. The process of decalcification, beginning from the fifth pair of

pereiopods and progressing up to the third, is found to be a good indicator of

the functional maturity of the female, indicating that it is ready for

impregnation.

In animals with highly complicated and specialized reproductive

development and behaviour, it would always be advantageous to assess the

sexual maturity on the basis of more than two external or internal indicators of

maturity. The size at which physical maturity occurs in lobsters has been

identified by examining discontinuities (changes in slope) in the linear

relationships between body size and certain externally visible features.

Female maturity has been associated with allometric changes in the length of

83

pleopods, the length of the pleopodal setae, telson length or the width of the

abdomen relative to carapace length, which all change in relation to

preparation for first spawning (Street, 1969: Hossain, 1978b: Aiken and

Waddy, 1980: Pollock and Augustyn, 1982: Jones, 1988: Stewart et al., 1997;

Lizarraga-Cubedo et aI., 2003; Kulmiye, 2004; DeMartini et ai, 2005).

MacDiarmid and Sainte-Marie (2006) recommend that easily measured

appendage length to body size relations should be routinely applied to provide

estimates of female SOM (Size at Onset of Maturity) in lobsters, but only after

this approach has been validated by undertaking histological studies of

gonadal maturation. In the present study, the size at maturity in females and

males of P polyphagus and T. orientalis was estimated from different indices

of maturity and corroborated by histological observations on the progress of

gonadal development.

Assessment of size at maturity in males is much more difficult than in

females. Male physiological maturity has been determined by the presence of

mature spermatozoa in vasa deferentia of several spiny lobsters (Heydorn,

1969; Berry, 1970; MacDiarmid, 1989; Turner et ai, 2002) and in the clawed

lobsters H. american us and Nephrops norvegicus (Farmer, 1974). However,

physiological maturity need not necessarily indicate functional maturity. Male

functional maturity has often been associated with a change in the dimensions

of the first cheliped in clawed lobsters or of the second or third pereiopods in

spiny lobsters, relative to CL (MacDiarmid and Sainte-Marie, 2007). Berry

(1970), Lipcius et al., 1983, Bertelsen and Horn (2002) have associated the

extreme development of the second and third pereiopods of some male spiny

84

lobsters with their ability to extract females from their dens. The change in the

size of the pereiopods normally occurs after physiological maturity has been

attained. George and Morgan (1979) described a method of determining this

by plotting the leg size against the CL, for immature and mature lobsters. The

size at maturity is then indicated by the point of upward deflection or the point

of intersection of the regression lines of immature and mature lobsters. In the

present study comparative analyses were done from regression lines of

different somatic length against the carapace length in two size groups within

each sex of either species and from regression lines generated for males and

females without size differentiation. From the inferences drawn on size at

maturity for the two species, it is evident that in both species, the males

mature earlier than the females. While smaller males mate with larger

females, possibly of the same clutch, in T. orientalis, larger males mate with

smaller females, possibly of the same clutch, in P. polyphagus. This is due to

the differential growth exhibited by the sexes. Between the two species, T.

orienta/is exhibits more potential for faster maturation and completion of

breeding cycles. The larval phase is much shorter and larval rearing is also

relatively more feasible in T. orienta/is (Kizhakudan et ai, 2004). These

observations suggest the candidature of T. orientalis for aquaculture while the

development of husbandry practices for P po/yphagus will depend on

improved captive breeding and larval rearing to settlement.

3.4.6 Fecundity

Fecundity is a measure of fertility of an animal. It is usually

expressed in terms of the number of eggs capable of being produced

85

by an individual during a breeding cycle. Fecundity in lobsters is greatly

influenced by external factors like water temperature, pH and nutritional

factors. The number of eggs produced is also directly related to the

size of the lobster. In semi-enclosed areas and indoor experiments,

density and competition for food is also an important factor determining

the reproductive status and fecundity of lobsters. The total number of

eggs produced by an animal during its life time is the net result of the

interplay between two variable factors - the breeding frequency and the

number of eggs produced in each breeding cycle. Since both these

factors are independently subject to alterations in the environment, a

measure of the total observed fecundity deviates greatly from the actual

fecundity capacity of the animal. A better picture of the animal's

fecundity can be obtained by taking into account the fecundity per

breeding cycle. However, such observations require data collected over a few

years, from a large number of animals, which is beyond the scope of the

present study since lobsters are a high-demand, high-value commodity facing

the crisis of dwindling catches.

Fig.4a

p .... ~.'- .... :c, _~ .. _' _f

V

<1.

b.

Walking legs of P. polyphagus a. female (i) III WL (ii) IV WL (iii) V WL

(iv) propodus and dactylus of V WL in adult female forming a claw like structure

b. male (i) III WL (ii) IV WL (iii) V WL

Fig.4b Abdominal pi eo pods of adult female P. polyphagus showing exopods (ex ), endopods (en) and ovigerous setae (as) (i) I (ii) 1\ (iii) 1\1 (iv) IV

Fig.4c Development of penile process in male P. po/yphagus a. immature b. early maturing c. maturing d. developed e. active & ripe s. setae p. pigmentation m. membranous cuticle

cl. b.

c.

e· r.

Fig. 5 Formation of decalcified mating windows in female P. po/yphagus

a. clear ridges; no decalcification b. decalcification begins from posterior tip of sternum c. one pair of windows (w ) on V thoracic sternite d & e. 3 windows (2 on V and 1 on IV) f. 4 windows (2 on V, 1 on IV, 1 very small on III)

Fig. Sa

~.-:-.~ I

Iv

Walking legs and gonopore (g) of T. orientalis a. female (i) I WL (ii) II WL (iii) III WL (iv) IV WL (v) V WL (vi) coxa of III WL with gonopore

b. male (iv) IV WL

(i) I WL (v) V WL

(ii) II WL (iii) III WL (vi) coxa of V WL with gonopore

Fig.6b Abdominal pleopods of adult female T. orientalis showing exopods (ex), en do pods (en) and ovigerous setae (as) (i) I (ii) II (iii) III (iv) IV

Fig. 7

c.

Development of male reproductive system in P. polyphagus a. early maturing b. mature c. active adult t - testis, at - anterior testis, mt - middle testis, pt -posterior testis, p - proximal vas deferens, d - distal vas deferens, pp - penile process, ej - ejaculatory duct, tb -transverse bridge

Fig. 8

o

a·

c.

Development of female reproductive system in P. polyphagus a. immature b. maturing c. mature d. ripe o - ovary, ao - anterior ovary, mo - middle ovary, po _ posterior ovary, ov - oViduct, tb - transverse bridge

Fig. 9

~.

d..

e·

Developmental stages of oocytes in P. polyphagus, as observed under a Light Microscope (scale: 125 11m) a. immature non-vitellogenic stage b. stage 1 of primary vitellogenesis c. stage 2 of primary vitellogenesis d. stage 3 of primary vitellogenesis e. stage 1 of secondary vitellogenesis f. stage 2 of secondary vitellogenesis em - egg membrane, vo - vacuole, yg - yolk granule

Fig. 10

t

ct.

Development of male reproductive system in T. orientalis a. immature b. mature c. active adult t - testis, at - anterior testis, mt - middle testis, pt -posterior testis, p - proximal vas deferens, d - distal vas deferens, g - gonopore, ej - ejaculatory duct

Fig. 11

:1:.

Co.)

Gi--r:rl-~ SF~~

''-J---f--~'n=<8 ~

Schematic illustration of the developmental stages of male spermatophore in P. polyphagus and T. orientalis a. testis (te) with proximal and distal vas deferens (p &d), b. c.s. of anterior testis showing spermatocytes packed in acini (a), c. c.s. of proximal vas deferens showing spermatophore and gelatinous matrix, d. C.s. of distal vas deferens showing typhlosole (t ) and spermatophores (s ), e. c.s. of distal ejaculatory duct, f. loop-like convoluted arrangement of spermatophore tubules (sf) in distal vas deferens, g. c.s. of distal vas deferens in T. orientalis showing typhlosole (t ) and spermatophores (s ) embedded in gelatinous matrix, h. loop-like convoluted arrangement of spermatophore tubules (sf) in distal vas deferens in T. orientalis

Fig. 12

-, o o

c.

e.

Development of ovary in female T. orientalis

o o

t.

a. immature b.& c. early maturing d. late maturing f. ripe g. spent recovery

.. ; . • • • ~ . •

o - ovary, ao - anterior ovary, mo - middle ovary, po _ posterior ovary, ov - oviduct, tb - transverse bridge

Fig. 13

4..

b.

Diagrammatic representation of courting and mating behaviour a. P. polyphagus b. T. orientalis

iii Cl

iii Cl

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0 II) ...

Fig. 14 a

10

9

8

7

6

5

4

3

2

1 ... 0

'" ... ~ ...

Fig. 14 b

• 1m

.... -- • Ern

• Lm/M

o II)

CD ... II) II)

o CD

CD II)

'" CD o .... CD CD

CL (mm)

'" .... o co

CD .... '" co

o

'" CD co

Gonadosomatic Index in male P. po/yphagus

1m

.. --- -Em

• Lm/M

)( Sp

.!! . -I •

Jf.

0 '" 0 II) 0 II) 0 '" 0 '" '" '" CD CD .... .... co co '" '" CD ~ CD ~ CD ~ CD ~ CD ~ ... '" II) CD CD .... .... co co '"

CL (mm)

Gonadosomatic Index in female P. po/yphagus

II)

'"

0 0 ~

CD

'"

4

• 1m 3.5 - -- ·Em

3 • LmlM r

r 2.5 ~~ T

en 2 1

CJ ·r 1.5 ,

• I . - ~.

1

0.5 ~ 0

I() 0 I() 0 I() 0 I() 0 I() .... I() I() '" '" .... .... "" "" , , , , , , , , , ~ '" ~ '" ~ '" ~ '" ~ .... .... I() I() '" '" .... .... "" CL (mm)

Fig. 15 a Gonadosomatic Index in male T. orientalis

7 1m

6 - .-- -Em

• LmlM I 5

~ I .. I )( Sp

4 en CJ

3

. ' I· -• of 2

, • -'" 1 ! ~ ""

• 0

I() 0 I() 0 I() 0 I() 0 I() '" '" .... .... ex> ex> Ol

~ '" ~ '" ~ '" ~ '" I() lD to to .... .... en IX)

CL (mm)

Fig. 15 b Gonadosomatic Index in female T. orientalis

100

90

Ii: 80 ::J 70 >-..: ::;; 60 >-...J 50 ...J ..:

40 ::J >< w 30 '" ~ • 20

10

0 0 ~

'" M

Fig. 16 a

100

90

Ii: 80 ::J

70 >-..: ::;; 60 >-...J 50 ...J ..: ::J 40 x w

30 '" ;;'1 20

10

0 0 ~

'" M

Fig. 16 b

-.- penile process

-o.-mature testes

n =261

'" 0 '" 0 '" 0 '" 0 '" 0 '" ~ '" '" '" '" .... .... co co (7) (7) , :;;: '" ~ '" ~ '" ;:: '" 0; '" ~

~ '" '" '" '" .... co (7)

CL (mm)

Size at sexual maturity in male P. po/yphagus (wild) derived from physiological and functional maturity indices

----.- penile process

n =38

'" 0 '" 0 '" 0 '" 0 '" 0 ~ '" '" '" '" .... .... co co (7) , , , , , , :;;: '" ~ "' U; "' ~ "' 0; "' ~ '" '" "' .... .... co

CL(mm)

Size at sexual maturity in male P. po/yphagus (captive) derived from functional maturity index

'" (7) , ~ (7)

" R' = 0.9893

" a<50mmCL

• ::0&0111111 Cl

" '55 g u ~" "

35

~. ,.

,. 35 " " " " " ~ ~ '" el!mm)

a. CL- MWC

" R'.O."" 20

.<60mmCL

o::o60mmCL

-" E g ~ • • 10

.~--~--~----~--~--~--~ •

"" 200

E 150 g ~ ~

> 100

.. •

20 .. .. ellmm)

c. CL - PPL

,,<50mlll CL

·>50mmCL

20 60

CLlmm)

.. 10 •

R" -0.9923

80 100

e. CL-IV LL

120

120

"

" .<60mmCL R2"O.ni

• >60mmCL

" • ~46 ~

~

>

" 26 /..~. " 26 " .. .. " " .. .. 10 • 11.

ellmm)

b. CL - VSL

'80

.<50mmCL R''' 0.9921

'" o>50mmCL

180

,,,

80

"~~--~~--~--r--r--~~--' 30 40 50 60 70 80 90 100 110 120

CLlmm)

d. CL -III LL

, .. HO • <liOm",CL

R"" 0."2

'" o>50mmCL

I"

I 110 ::I > ..

"

30 40 50 60 70 ISO 90 100 110 120

ellmm)

f. CL - V LL

Fig. 17 Relationship between carapace length and somatic lengths

as indices of sexual maturity in male P. po/yphagus

100

90

80

70

Ii: ::J 60 .... « 50 ::;; >-...J 40 ...J « ::J 30 t;j I/)

20 '" • 10

0 0

'" .;, "

Fig.18a

Fig.18 b

--* ----..- ovigerous setae ~Mature ovary -x- mating window -egg brush ~berried females

n=289

'" 0 '" 0 '" 0 '" 0 '" 0 '" 0 "! '" '" ... '7 eo '" 0> '<' 0 0 ~

.;, .;, .;, .;, ~ ";" ~

;;; ;;; ~ ~ ;;; .;, .;, '" '" r- r- eo eo ~

0> 0 0 CL(mmj ~ ~

Size at sexual maturity in female P. po/yphagus (wild) derived from physiological and functional maturity indices

Size at sexual maturity in female P. po/yphagus (captive) derived from physiological and functional maturity indices

80 " 70 R2 .. 0.993

46

.CSOmmCL 40 80 o>60mmCl

" " 30 E E

~ 40 ~ " U " ~ 00

~ > 20 R' = 0.'361 30

16 20

R' = 0.9637 10

10

30

200

180

180

140

E 120

~ 10. " " 80

80

" 2.

J.

Fig. 19

" " " 70 BO

CL(mm)

a. CL- MWC

R' = 0.99

.<GOmmeL

o>SOmmCL

R'= 0.9693

,. 5. •• 7.

CL(mrn)

c. CL -III LL

". ". 12<l

, .. E ~ •• " " > ••

,. 2 •

•

80

30

" 100 30 " 50 80 70 80 " 100

CL(mm)

b. CL - VSL

180

160 R' ... 0.9928

• <50mm CL 140 o:;.50mmCL

120

E 100 ~

" " 80 ~

60

" 20

90 100 J. ,. •• 7 • so .. 100

CL(mm}

d. CL-IV LL

R';; 0.9717

RZ '" 0.9204

.. " .. 7. •• 90 100

CL(mm)

e. CL-V LL

Relationship between carapace length and somatic lengths

as indices of sexual maturity in female P. pofyphagus

'" 80

70

60

E 50 .s ~ '" ..

30

20

10

0

0

250

200

E 150

.s ~ ~

- 100

.0

0 0

Fig. 20

70

60 -M

50 -F

E 40

.s ~ ~ >

30

20

10

0 10 20 30 40 '0 .0 70 80 90 100 0 10 20 30 40 .0 .0 70 80 90 100

CL(mm) CL(mm}

3. CL-MWC b. CL - VSL

250

-M 200 -M

-F -F

E 150

E :r ~

~ 100

50

0

10 20 30 40 .0 60 70 80 gO 100 0 10 20 30 40 '0 60 70 80 90 100

CL(mm) CL{mm)

c. CL -III LL d. CL-IV LL

180

160

-M 140

-F

120

E 100 .s ~

80 ~

> 60

40

20

0

0 10 20 30 40 .0 .0 70 80 90 100

CL(mm)

e.CL-VLL

Comparison of regression lines between carapace length and somatic lengths in male and female P. po/yphagus

-c: Q)

E a. 0 Qi > Q)

'0

'" .,

Q) '0 .. ., If) S8-~8 c: Q) 0 .. Q) Cl ~ c: :::l 08-9L 0 .. ., '0

::!!; Q) If)

~ <1\ 0 SL-~L .c

t "C

!. OL-99 c VI Il'l - '-M -II E .l!l c: S9-~9 E c: - Q)

..J '\: () 0

09-9S ....: ..!!:! ., E

SS-~S c: >--.;:

OS-9v ::J -., E

Sv-~v iii ::J >< Q) VI

0 0 0 0 0 0 0 0 0 0 0 -0 '" CO r- <D Il'l '<t <'"> N .... <1\ .... Q)

.!::! nml'lfW A llVnX3S % en

.... N

Cl u..

100

90 I" <50mm CL

" E 10 g

~ " <

" "

'>50mmCL

R'.0.9836

R2·0.986 •

JO+---__ ---+----~--~--~--~ JO 40 50 50 7D " ..

CL[mm)

a. CL-AWC

45

" • <50mmCL R'·0.9868

·"50mmCL

"

"

'" R''' 0.982

15+---~--__ ~--__ --~----~--_,

100

" ao

7D

E " g " :I ~ "

30

20

10

30 50 .. 7D " .. CL(mm)

c. CL- VSL

R".0.9158

.. <SOmmeL

·>50mmCL ..

R''' 0.8636

o+---~--~--__ --__ --~--~---. 20 30 .. 60 7D .. "

CLlmm)

e. CL-IV LL

" 7D .. <SOmmel R"·0.98 •

• >50 mm CL

" E .s 50

~

R"·0.9634 2O+---__ ----.l--____ --~--~--_,

JO

100

..

.. 7D

.. "

.. GO

CLlmm)

7D

b. CL·MWC

.. <50mm CL R'·0.902 •

• >50mm CL

:

R".0.871

ao to

,o~--__ --~----~--~----~--~ JO

so

7D

GO

JO

JO

.. 50 " CL[mm)

d. CL -III LL

.. <50mmCL

·,.60mmCL

" GO

CL(mm)

f. CL - V LL

7D " to

7D .. ..

Fig. 22 Relationship between carapace length and somatic lengths

as indices of sexual maturity in male T. orientalis

100

90

80

i 70

60

; 50

40

~ 30 0

20

10

0

Fig. 23 a

100

90

80

i 70

60

i 50

40

30 ~ 0

20

10

0

Fig. 23 b

-X-X-X -o--mature ovary

-x- ovigerous setae

--f.3-berried females

n =221

~ ~ ~ Hi Sl III 8 , , \'" 1:: i!O !B Iii

, II!

CL (mm)

Size at sexual maturity in female T. orienta/is (wild) derived from physiological and functional maturity indices

-x- ovigerous setae

--f.3-berried females

Y--"'v--x-x-x-x

n =64

CL (mm)

8 \'" , !R

Size at sexual maturity in female T. orientalis (captive) derived from morphological Land functional maturity indices

100 '" 90 .<50mmCL RI- 0.9661 70 .. <Sammet. R''' 0.7966

'>SammeL '>50mmCL 80

60

70 , , !'. .. e -" ~

~ • < ., " 40

R' = 0.9206 JO

JO

20 JO 40 " 70 " 20

60 20 JO 40 " 60 70 80

CL(mm) CL{mm)

a. CL-AWC b. CL-MWC

" 90

R'=O.7126

40 .. <60mmeL. R2 =0.8036 ... <5Omm CL 80

o>60mmCL • >50mmCL

" , E 10

.§. 30 !'. ~

~ ~

~ :: 60 >

" If = 0.9883

20 50 R' -0.9'18

" 40

JO 40 " " 70 80 JO ., 50 SO 70 80

CL(mm) CL(mm)

c. CL- VSL d. CL -III LL

90 70

1f·0.78S9

80 ... <SOmmeL R'=O.772 ... <!iOmm CL.

o;>ofiOmmCL. 60 ->fiOmmCL

e 70 , !'. !'. " .-~ ~ ~ ., ~ 60 >

40

" Rl .. 0.9665

40 JO

JO 40 " 60 70 80 JO 40 60 80 70 80

CI..\mm) Cl..lmm)

e. CL-IV LL f. CL - V LL

Fig. 24 Relationship between carapace length and somatic lengths

as indices of sexual maturity in female T. orientalis

140 100

90 12<>

-M 80 -M

100 -F 70

E 80 E " ~ ~ " ~ " ~ « ~ 40

40 30

20 20

10

0 0 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

CL(mm) CL(mm)

3. CL- AWC b. CL -MWC

60 140

50 120

-M

-F 100

40

E E 80

~ 30 ~ ~ ~

!l ~ " 20

40

10 20

0

0 10 20 30 40 " 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

CL(mm) CL(mm)

c. CL- VSL d. CL-III LL

120 100

90

100 -M 80 -F

80 70

E E 60

E 60 ~ 50 :; ~

~ ~

~ > 40

40 30

20 20

10

0 0

0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 5060 70 80 90 100

CL(mm) CL(mm)

e. CL-IV LL f. CL - V LL

Fig. 25 Comparison of regression lines between carapace length and somatic lengths in male and female T. orientalis

1000 • 900 •

800 Y = 12.531 x - 636.73 R' = 0.9685

0- 700 0 ? 600 -,..

500 .'" "0 c: ::l u 400

" n =47 u. 300

200

100

0 50 60 70 80 90 100 110 120

CL (mm)

Fig. 26 a Relationship between fecundity and carapace Length in P. po/yphagus

70

60

50 0-0 0 :.... 40 ,.. "" "0 c: 30 ::l U

" u.. 20

10

0 50

y = 0.7285x - 19.153 R' = 0.9424

60 70

•

•

n =53

80 90 100 110

CL (mm)

Fig. 26 b Relationship between fecundity and carapace Length in T. orienta/is

Plate VI

l' ! ,~ i

i

" ,

f

f f

,

~/' ,/ ~ ___ ...i .... __ ..... _:~ .... __ ..J

L. eft j

Thoracic and abdominal appendages of P. polyphagus

a. Walking legs - III, IV & V : sub-adult female b. Walking legs - III, IV & V : sub-adult male c. Walking legs - III, Iv & V : adult female d. Setae on dactylus: adult female e. Setae on dactylus: adult male f. Abdominal pi eo pods : sub-adult female g. Abdominal pleopods with ovigerous setae: adult female

Plate VII

04

I

l

d~ ______ "'.~ ...... '.~ r

I

\ \

I J .. ~ . c.t:::!: __

Penile process in male P. po/yphagus

a. Sub-adult male with penile process on coxa of fifth pair

of walking legs

b. Magnified view of penile process (sub-adult male)

c. Mature adult male: penile process with pigmentation

Plate VIII

,

r --.v - r-

4-

. .---" b·

Maturation related changes in ventral sternum in

P. po/yphagus

a. Ventral sternum in sub-adult male

b. Ventral sternum in sub-adult female

c-f. Formation of mating window (w ) in adult female

Plate IX

d,

~.

.... "

:;.... "

• " ~.~-';O::::~ >,

. ~" ~ -. e,

@

,.,;. , ';'/ ... /' " ,

/'

t' 1 ".

F.

Thoracic and abdominal appendages of T. orienta/is

a. Pereiopods -III, IV & V : adult female b. Pereiopod - V : adult male & adult male

~-;~:-: 2 ._z:. :; ---.

.2 t,.

. {'" -......,'

c, Dactylus of V pereiopod : adult male (m ) & adult female (f) d. Abdominal pleopods : adult male e. Abdominal pleopods : sub-adult female f. Abdominal pleopods with ovigerous setae: adult female g. Ovigerous setae on abdominal pleopods

Plate X

~- . .~ ,

" , 1

-' . --- ... ..,...

... ...... b.

,.

Gonopores in T. orientalis a. Male - ventral sternum (s ) with gonopore (g ) on coxa

(c) of V pereiopod b. Enlarged view of male gonopore c. Female - ventral sternum (s ) with gonopore (g) on

coxa (c ) of III pereiopod d. Enlarged view of female gonopore

,c

,It'

.-

,~

~ . ..1';;:...:;;' '--'

"-

. .....:, (

.~~, .. -

) ./

..

3

·,C ,

\ ,. . ~"1~ .. , > •

~ 3 " 2

Plate XI Abdominal pleopods of adult female T. orienta lis with eggs

attached to ovigerous setae

[ Exopod : (ex) Endopod : (en) 1

Plate XII

-~

I j) , .

II J • L 4,

-. j --1

-@

.@ '-. !

, ®,...J

,

l. •

• -c. d, e,

Reproductive system in male p, po/yphagus

a, in situ view of vas deferens showing convoluted proximal part (p) and enlarged distal part (eI)

b, Testis (I) with vas deferens c, Median vas deferens (m) d. Distal vas deferens (eI) e, Ejaculatory duct (e) terminating in extensible penile

process (pp)

1

• .. j

I

c·

r L. ....

Plate XIII T.S. of testis (light micrograph, x400) in early maturing

male P. polyphagus showing spermatogonioa (sg)

and primary spermatocytes (ps) packed in seminiferous

lobules (acini) (a) and seminiferous duct (ei)

a & b anterior testis c. middle testis

d. posterior testis

• - • .J '~~-

, t I

Plate XIII e. T.S. of testis in conjunction with proximal vas deferens

(light micrograph, x400) in early maturing male P.

po/yphagus showing lumen (f) with columnar nucleated

epithelial cells (e ) and connective tissue ( ct)

f. T.S. of testis (light micrograph, x400) in maturing male P.

po/yphagus showing peripheral spermatogonia (sg) and

centrally placed spermatocytes (ps)

Plate XIII T.S. of anterior portion of proximal vas deferens (light

micrograph) in male P. po/yphagus showing lumen (r) with

columnar epithelial lining (e)

g. early maturing stage (x100) h. maturing stage (x100)

i. enlarged view of lumen (x400)

j. wall of posterior vas deferens showing epithelial layer,

(e), muscular layer (m) and connective tissue (c) (x400)

Plate XIII T.S. of anterior portion of distal vas deferens (light

micrograph) in early maturing male P. po/yphagus showing

developing epithelial villi (v) on central typhlosole (t) with

connective tissue (c ). muscular layer (m) and epithelial

layer (e).

k. entire view ofT.S. (x 100)

I. typhlosole with villi and connective tissue (x400)

m. three-layered outer wall (x400)

h.

Plate XIII T.S. of distal portion of distal vas deferens (light

micrograph) in maturing male P. po/yphagus showing

inner glandular epithelial lining (g), typhlosole (t ) and

enlarged lumen (r).

n. entire view of T.S. (x 100)

o. three-layered outer wall (x400)

"p. villi (v) of typhlosole (x400)

Plate XIII

"I r ,.~.

T.S. of distal vas deferens (light micrograph) in adult male

P. po/yphagus showing muscle and connective tissue of

thinner outer wall, glandular epithelium (g), typhlosole (I),

spermatophores (s ), spermatophore wall (matrix secreted

by proximal vas deferens) (m1), matrix secreted by the

typhlosole (m2) and matrix secreted by peripheral

epithelium (m3)

q. entire view of T.S. (x 100)

r. villi (v) of typhlosole (x400)

s & t. spermatophore with spermatophore wall

Plate XIV Spent-recovery stage of adult female P. po/yphagus

ovary, with thick ovarian wall and oocytes in secondary

vitellogenesis stage

Plate XV a. T.S. of ovary (light micrograph. x400) in immature female P. po/yphagus showing the proliferative previtellogenesis phase with germinative zone (gz), primordial gonias (g ) and oogonial eells( 00 )

b. T.S. of anterior portion of previtellogenie ovary (light micrograph, x400) in early maturing female P. po(yphagus showing oogonial eel/s(oo ) and primary ooeytes (oc)

/

• •. __ .~ «-.f

.~..:;- " . .. "" ." .. " I' \ ........ I, ,

t I

e.

Plate XV c. T.S. of previtellogenic ovary (light micrograph, x1000) in early maturing female P. po/yphagus showing the peripheral chromatin nucleoli (n ) and follicle cells (f)

d. T.S. of developing ovary (light micrograph, x400) in primary vitellogenesis phase in maturing female P. po/yphagus showing central germinal zone (gz) and developing oocytes (oc) towards the peripheral region

e. T.S. of developing ovary (light micrograph, x400) in primary vitellogenesis phase in maturing female P. po/yphagus showing packed oocytes (oc) in which yolk deposition has begun

I.

Plate XV T.S. of oviduct (light micrograph) in female P. po/yphagus

showing three-layered wall with outer epithelial layer (e ),

central connective tissue layer (c ) and inner high

columnar epithelium (ce), lumen (r), and villi (v).

j. maturing stage: entire view of T.S. (x100)

k. maturing stage: view of a portion (x400)

I. mature stage : enlarged view (x400 & x6, digital loom)

showing columnar epithelial cells and villi, forming

semi-closed channels (ch )

I.

Plate XV f. I.S. of mature ovary (light micrograph, x100) of female P. po/yphagus in stage I of secondary vitellogenesis phase, showing mature oocytes (oc) in the ovarian cavity

g. I.S. of developing ovary (light micrograph, x400 & x6, digital zoom) in stage II of primary vitellogenesis phase in maturing female P. po/yphagus showing packed oocytes (oc) in yolk platelet stage

h. I.S. of developing ovary (light micrograph, x400) in early maturing female P. po/yphagus showing ovarian wall (ow)

i. I.S. of mature ovary (light micrograph, x400) of female P. po/yphagus in stage I of secondary vitellogenesis phase, showing mature oocytes (oc)

,

,

. j

b· c.

Plate XVI Reproductive system in male T. orientalis

a. in situ view of mature testis (t ) with vas deferens b. Maturing testis with vas deferens c. Ripe testis with vas deferens of sexually active male [ Proximal vas deferens (p ) Distal vas deferens (d) 1

c. d.

Plate XVII T.S. of anterior portion of maturing testis (light micrograph)

in male T. orientalis showing spermatogonioa (sg)

and primary spermatocytes (ps) packed in seminiferous

lobules (acini) (a) and seminiferous duct (d)

a.x100 b. enlarged view of wall (x400)

c. & d. enlarged view of acini with spermatogonia (x400)

, .' t

~.

9· h.

Plate XVII T.S. of distal vas deferens (light micrograph) in adult male·

T. orientalis

e & f. showing typhlosole (t ) and spermatophores (s)

embedded in gelatinous matrix (x 100)

g. enlarged view of typhlosole (x400)

h. gelatinous matrix (x400) ® i. spermatophore with spermatophore wall embedded

in the matrix (x400)

•••

... . , ,

b.

i./.· .. '" 1~ ;: f

~~.

\ I l I

.:1; !

, • I

Plate XVIII Reproductive system in female T. orienta/is

a. in situ view of early maturing ovary (0 ) b. in situ view of late maturing ovary (0)

do.

Plate XIX

t,:,.::::-~;

~1; .) ,.l 1'. ,

" "- \ f , I.':.; ,

:( ·L

rl j", ~ :-- "'-

I i i / I : 1

> ,

I , I

- \ I '!-~ \ ) :% ,

. , I , !

I I

~f .. I a. ,

Development of ovary in female T. orientalis

a. Immature b.& c. Early maturing d.& e. Late maturing f. Ripe g. Spawning [ Oviduct (CJv ) 1

Plate XX a, b. T.S. of anterior portion of ovary (light micrograph, x400) of maturing female T. orientalis in late secondary vitellogenesis stage showing polyhedral shaped mature oocytes (0 ), germinal zone (gz ) and thick ovarian wall (ow)

Plate XX c. T.S. of anterior portion of ovary (light micrograph, x400) of maturing female T. orienta/is in late primary vitellogenesis stage showing maturing oocyte (oc ), with nucleus

d. T.S. of posterior portion of ovary (light micrograph, x400) of mature female T. orientalis showing non-nucleated mature oocytes (oc) and germinal epithelium (ge)

e. T.S. of posterior portion of spent ovary (light micrograph, x400) of female T. orientalis showing resorbed oocytes (oc) and infiltration of connective tissue (ct)

Plate XX f.

g.

h.

i.

T.S. of oviduct (light micrograph, x50) of maturing female T. orientalis showing lumen (() with columnar epithelial cells (ce )

enlarged view of oviduct (x100)

enlarged view of oviduct (x 1 00 & x6, digital zoom), showing columnar epithelial cells

enlarged view (x400), showing columnar epithelium connective tissue (ct ) and oviduct wall (w ) with peripheral oocyte (o )

, 0/ \;i

),~ "

~;-

• c.

1 , ' i

N .. • I I •

N Q

•

Plate XXI a - c Butterfly-shaped spermatophore attachment (s ) on

ventral sternum in female P. polyphagus

[ Dorsal view (d) Ventral view (v) 1

Plate XXI

\

e·

) .-I.J

(

~" ':-

, , " ,

"

.. • •

-~ \

"

" 0 ,

,

\ .itA\ ~ d - e Freshly extruded spermatophoric mass of P.

po/yphagus showing loop-like convolution of the

spermatophore tube

"!F-_1I"

r -" ., ___ ..J

r.\. l If".. • \~

<

, ,

c.

Plate XXII a - c Gelatinous spermatophore mass (s ) on 1st and 2nd

abdominal sternites in female T. orientalis

e.

-".,::- .. ~<t. -, . ~. .

. '''-',.,.. '~.'" " , .#'.

t : r

~.' .•

":'-."'"

.-

,f.

h.

Plate XXII d - e Freshly extruded spermatophoric mass of T. orienta/is, embedded in gelatinous matrix

f. Ruptured spermatophore wall after release of spermatozoa

g. Live spermatozoa inside freshly extruded spermatophore (100 X)

h. Spermatids inside spermatophore in the distal vas deferens (light microscope, x400)

Plate XXIII Ovigerous female P. po/yphagus with fertilized eggs on

pleopods and spermatophore scar on ventral sternum

r. ,

(

. ,

• c. -

, ~-...

. .

;

..." 9' >!

"../, ... {--

Plate XXIV a. Freshly spawned eggs of P. po/yphagus

b. Fertilised eggs attached to ovigerous setae

c. Final stage of embryonic development - Eyed stage

(folded naupliosoma visible inside the eggs)

•

• -,'

;I - -b.

Plate XXV a. Ovigerous female T. orientalis with fertilized eggs on

pleopods

b. Freshly spawned eggs of T. orientalis

I. I • ~ q,. ,

, r

~ .. -c.

r 4 * "

I - '-

Plate XXVI a. Freshly spawned eggs of T. orientalis b. Final stage ova from ripe ovary, with yolk granules c. Early stage of embryonic development d. Eyed stage e. Chromatophore development in embryo f. Final stage of embryonic development

(folded naupliosoma visible inside the eggs)

~.

•

h.

, ~ "' '~ ~ .... '

• ~ , I j

I

\ t ~ ,

, --\_.~

Plate XXVI g. Hatching 9 capsule h. Emptyeg