Influence of eyestalk ablation and   5-hydroxytryptamine on the gonadal development of a female crab, Paratelphusa hydrodromous (Herbst)

M. G. Ragunathan* and A. Arivazhagan

Department of Zoology, Post-Graduate Extension Centre, University of Madras, Vellore 632 004, India

The histology of the ovaries shows polymorphic
oocytes in the stage I ovaries and much enlarged
oocytes in the eyestalk-ablated saline-treated
ovaries. The ovarian index has significantly increased in the eyestalk-ablated 5-hydroxytryptamine (5-HT)-treated crabs. The histology of the ovaries of the eye-stalk-ablated 5-HT-treated crabs shows much reduced nucleus and more cytoplasmic contents. The staining intensity is more. Histological study of the spermatheca at stage I reveals fused constituent layers whereas eyestalk-ablated 5-HT-treated crabs shows spermatheca having distinct constituent layers and the cross-section of the spermatheca shows rich secretory substances in the lumen and the staining intensity is more. The spermathecal index is significantly increased in the eyestalk-ablated 5-HT-treated crabs.


AFTER the discovery of gonad inhibiting hormone principles in the eyestalk of Palaemon serratus by Panouse1, various groups of crustaceans have been shown to possess such inhibiting hormones in the X-organ, sinus gland complex2. The surgical removal of the eyestalk in crustaceans in order to promote ovarian maturation has

*For correspondence: No. 3, Vinayagam Street, Velappadi, Vellore 632 001, India

been reported by many workers3–5. Eastman-Reks and Fingerman6 have stated that in general, the removal of both eyestalks in decapod crustaceans induces precocious yolk deposition and vitellogenesis in the ovary. In contrast, injection of eyestalk extracts or partially purified ovary inhibiting hormones results in the inhibition of ovarian maturation.

Richardson et al.7 have reported that the administration of 1.25   10–9 to 1.25   10–7 mole/crab of 5-hydroxytryptamine (5-HT) has showed an increased dose-dependant ovarian development in the fiddler crab, Uca pugilator found in the Florida coast, USA. Sarojini et al.8 have reported that 5-HT exerts an indirect stimulatory effect on the testis by triggering the release of gonad-stimulating hormone in U. pugilator.

Recently, Sharmila9 has reported that the administration of biogenic amines, namely 5-HT, octopamine and dopamine on the stage I female crab, U. pugilator exhibited interesting results. The crabs treated with 5-HT showed significant increase in the gonadal indices, in contrast to those who showed a significant decrease in the gonadal indices when treated with octopamine and dopamine.

From the literature cited above it is clear that studies have been carried out on the eyestalk ablation, treatment of various pharmacological drugs in affecting the reproduction in crustaceans. However, no work has been carried out in studying the combined action of eyestalk ablation and 5-HT treatment especially in a freshwater crab. Hence, the present work has been undertaken to study the concurrent effects of eyestalk ablation and 5-HT on the ovarian and spermathecal development of a female, freshwater crab, Paratelphusa hydrodromous (Herbst).

 

Paratelphusa hydrodromous used in the present study were collected from a lake in Thellur village which is 80 km away from Vellore, by hand picking method. The live animals were brought to the laboratory, stored in cement tanks and were fed with boiled beef mutton ad libitum. The water was changed daily and the crabs acclimatized for a week in the prevailing room temperature.

By examining the pleopods and also by noting the texture of the exoskeleton10, the intermoult stage of the crabs were identified. Stage I ovarian development was determined by selecting only mature crabs by measuring the length and breadth of the carapace and also by using the window method11.

For all the experimental work, only stage I intermoult adult female crabs with carapace length ranging from 18 to 25 mm and breadth ranging from 18 to 34 mm were selected. The physiological saline was prepared following Humason12. 5-HT was obtained in the powdered form from the Sigma Chemical Company, USA. It was prepared in the solution form by using saline at 1 mM concentration.

Forty intermoult, stage I female crabs were selected and were divided into four groups (A, B, C and D) of ten each. Group A was used on the first day of the experiment. The eyestalks of all the crabs of the groups B, C and D were ablated and cauterized with a hot needle. They were left in the tubs for a day to reduce stress. The next day, group B was untreated and considered as the initial control, the group C received saline and was considered as the experimental control and group D received 5-HT and was considered as the experimental group.

The dose was given on the 1st, 5th and 10th days of the experiment. The dose given to each crab was 50 m l/crab. All the crabs of the three groups (B, C and D) were placed separately in round plastic tubs and the water was poured in all the tubs so that the crabs were half immersed. Small pieces of stones were kept in the centre of the tubs for the crabs to rest comfortably. The water was changed daily and the crabs were fed with boiled beef mutton ad libitum. The experiment lasted for 15 days.

On the 15th day, the crabs were weighed and then used in the experiment. The ovary and spermatheca were removed, blotted dry and were then weighed. The indices were calculated by using the following formula as suggested by Giese13.

 

Wet weight of the tissue

 


Gonadal index =  100

Wet weight of the crab

 

The data were statistically analysed.

 

Histological sections of group A (stage I) ovaries reveal that the oocytes are polygonal in shape. The nucleus and nucleolus are distinctly seen. The cytoplasm of the oocytes stained moderately (Figure1 a). The ovarian lobes of the group B (eyestalk ablated, untreated) are intact. The cytoplasm is moderately filled with ooplasmic substances when compared with the group A (Figure 1 b). The ovaries of the group C (eyestalk ablated saline treated) show smaller nucleus and the oocytes are richly filled with ooplasmic substances. The rich ooplasmic substance in some areas are homogenous so that the connective tissues are not clearly visible. The staining intensity of the oocytes are high with haematoxylin-eosin (Figure 1 c). The ovaries of the group D (eyestalk ablated 5-HT treated) shows the oocytes with full of ooplasmic substance. In some oocytes nucleus and nucleolus are absent. Most of the oocytes show vacuolization. The oocytes are mostly darkly stained with haematoxylin-eosin (Figure 1 d).

The histological sections of the spermatheca of the group A (stage I) exhibit three layers, an outer cuticle, middle muscular and inner epithelial layers. All the three layers are slightly fused together one above the other encircling the central lumen with refractile fluid content. The three layers stain slight pink colour (Figure 2 a). The section of the spermatheca of the group B shows that the three constituent layers are slightly enlarged and the central lumen is filled with granular substances (Figure 2 b). The section of the spermatheca of the group C shows much more darkly stained constituent layers than the group B and the lumen is darkly stained due to its secretory function (Figure 2 c). The section of the spermatheca of the group D reveals that the three constituent layers are clearly visible. The epithelial layer is very much enlarged with tall columnar cells and are darkly stained (Figure 2 d).

The ovarian index of the group A is 0.3706; group B is 0.7156; group C is 0.7382 and the group D is 0.7574. When the results are statistically analysed, it shows a significant increase (P < 0.001) in the ovarian index of the group B when compared with group A and a significant increase (P < 0.001) in the ovarian index of the group C when compared with the group B. Likewise, there is a significant increase (P < 0.001) in the ovarian index in the group D when compared with the group C (Table 1).

The spermathecal index of the group A is 0.4517; group B is 0.6132; group C is 0.6462 and group D is 0.6928. When the results are statistically analysed, it shows a significant increase (P < 0.001) in the spermathecal index of the group B when compared with group A, and a significant increase (P < 0.001) in the spermathecal index of the group C when compared with the group B. Similarly, there is a significant increase (P < 0.001) in the spermathecal index in the group D when compared with the group C (Table 1).

Reproductive strategy is mostly based on the objective of successfully producing large number of individuals for which the animal totally adapts itself to the immedi-

585.gif (114759 bytes)

 

Figure 1.  Ovary of a, stage I normal; b, eyestalk ablated untreated; c, eyestalk ablated saline treated and; d, eyestalk ablated 5-HT treated crab (  120); Nu, Nucleus; Cy, Cytoplasm; Nl, Nucleolus; Oo, Oocytes; Op, Ooplasmic substances; V, Vacuoles.

Table 1.  Ovarian and spermathecal indices of a eyestalk ablated,
5-HT treated Paratelphusa hydrodromous

ate environmental conditions, thus sometimes leading to an entirely different type of reproductive cycle of other species of the same genus14. Reproductive cycles of invertebrates are generally determined by the gonadal indices, potentiality of sperms, appearance of mature gametes in the gonads and the breeding of eggs15.

The gonadal index as an indicator of reproductive activity of marine invertebrates was first described by Bennett and Giese16. The gonadal index is an important tool in reproductive biology, which clearly forecasts the development of the gonads. In the present investigation both the ovarian and spermathecal indices have significantly increased in the 5-HT-treated crabs when compared with their experimental controls. The spermathecal index shows a direct correlation with the ovarian index suggesting the possibility of a single hormone theory, viz. gonad stimulating hormone, controlling the maturation of ovary and spermatheca. A similar synchrony was also postulated by Stephens17 in the development and control of cement glands and ovary by a single hormone, suggesting a close relationship between them, using eyestalk ablation and injection studies of brain and thoracic ganglion extracts.

Deecaraman and Subramoniam4 in Squilla holoschista have postulated a controlling mechanism of ovary and cement glands by a single hormone produced by neurosecretory cells of thoracic ganglion and brain. Viswanathan18, Kulasekharan19, Ragunathan20 and Sharmila9 have further demonstrated the similar correla-

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Figure 2.  C.S. of spermatheca of a, stage I normal; b, eyestalk ablated untreated, c, eyestalk ablated saline treated and d, eyestalk ablated 5-HT treated crab (  130). Ep, Epithelial layer; C, Cuticular layer; Mu, Muscular layer; Lc, Luminal contents; Sb, Secretory substances.

 

tion existing between the ovary and spermatheca of different species of crabs.

The ovary and spermatheca of the stage I female crabs, after eyestalk ablation and subsequent administration of saline and 5-HT produced significant changes in their histology when compared to their respective controls.

In the eyestalk intact, untreated crab the oocytes of the ovary shows clear nucleus occupying a major portion of the oocyte. All the oocytes are clear and are enclosed in a connective tissue. The ovarian lobe is intact. The eyestalk ablated untreated crabs show a slight increase in the ovarian contents. A further increase in the ooplasmic contents of the ovaries is noticed in the eyestalk-ablated saline-treated crabs. In the eyestalk-ablated 5-HT treated crabs the ovaries show much reduced nucleus in the oocytes and are rich with yolk granules.

Histological sections of the spermatheca of eyestalk intact untreated female crabs show the fusion of three layers and staining reaction of the luminal contents are not intense. Whereas, the cross section of the spermatheca of the crabs of eyestalk-ablated 5-HT-treated crabs shows the presence of three distinct layers and also intense staining reaction of the luminal contents.

The development of gonads after eyestalk ablation has also been reported by earlier workers in different species of crustaceans. A gonad-inhibiting hormone principle in the eyestalk of Palaemon serratus was first reported by Panouse1. Various groups of crustaceans have been shown to possess such inhibiting hormones in the X-organ–sinus gland complex2. The surgical removal of the eyestalk in crustaceans in order to promote ovarian maturation has been reported by many workers3,5.

The administration of 5-HT and their effects on the gonadal development have been reported by several workers. Richardson et al.7 found that 5-HT has a stimulatory effect on the development of ovaries in U. pugilator, while Kulkarni et al.21 and Fingerman22 have also given supporting evidence showing the stimulatory role of 5-HT in the reproduction of female crustaceans.

Sarojini et al.23 have reported the in vitro stimulatory action of 5-HT in P. clarkii. There are also reports demonstrating the effects of 5-HT in the testis of male fiddler crab, U. pugilator8. Sarojini et al.23 have also reported the testicular maturation after administration of 5-HT in P. clarkii. Recently Sharmila9 has reported a significant increase in the ovarian and spermathecal indices in U. (celuca) lactea annulipes after administration of 5-HT.

From the present investigation it is evident that both the eyestalk ablation and 5-HT have played an important role either acting directly or indirectly on the target organs and could have helped in the gonadal development.

 


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Received 14 August 1998; accepted 5 December 1998.