Effects of bilateral and unilateral eyestalk ablation on vitellogenin synthesis in immature female kuruma prawns, Marsupenaeus japonicus

In penaeid shrimp species, vitellogenin (VTG) synthesis in the ovary and hepatopancreas is under the inhibitory regulation of a neuroendocrine system, the X-organ/sinus gland complex in the paired eyestalks, and eyestalk ablation (removal of the X-organ/sinus gland complex) is widely used for inducing ovarian development. However, the difference in effects of bilateral and unilateral ablation on VTG gene expression has not been clarified so far. In the present study, VTG synthesis was monitored over a 16-day period after ablation and compared between replicates of immature female kuruma prawns, Marsupenaeus (Penaeus) japonicus, that had been bilaterally or unilaterally ablated and control specimens. After bilateral ablation, ovarian development was induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary increased significantly. Significant VTG mRNA increase was detected 12 h after bilateral ablation. In contrast, after unilateral ablation, ovarian development was not induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary did not change significantly from the control. These results indicate that in immature female prawns, unilateral ablation does not induce VTG gene expression, whereas bilateral ablation induces rapid VTG gene expression (<12 h). The ineffectiveness of unilateral ablation suggests that the remaining X-organ/sinus gland complex in the unilaterally ablated female prawns may secrete sufficient VIH to suppress VTG synthesis.     

Role of eyestalk hormone in the carbohydrate metabolism of a marine penaeid prawn,Parapenaeopsis hardwickii (MIERS) (Crustacea,Decapoda, Penaeidae)

Data furnished here concern with the role of eyestalk hormone in the regulation of carbohydrate metabolism in Parapenaeopsis hardwickii. Bilateral eyestalk ablation has brought about a significant (P < 0.01) fall and rise in the glycogen content in the midgut gland and abdominal muscle respectively. Although eyestalk ablation resulted in a significant (P < 0.01) depletion of fat in midgut gland, n0 significant (P > 0.05) change was observed in the abdominal muscle. Eyestalk extract administration in eyestalk-less prawns has significantly (P < 0.05) restored the glycogen and fat metabolites in the midgut gland. There was an obvious change in the glycogen content of the midgut gland and abdominal muscle of normal prawns when injected with eyestalk extracts from prawns in different molting stages. Eyestalk extract from intermolt prawns caused a significant (P < 0.05) decrease and increase in the glycogen quantity in the midgut gland and abdominal muscle respectively. Eyestalk extract from premolt and postmolt prawns has, although not significantly (P > 0.05), decreased and increased the utilization of glycogen respectively in the midgut gland. The physiological significance of these findings are discussed briefly.Paper forms part IV of the series

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Effects of eyestalk ablation on growth and food conversion efficiency of the freshwater prawn Macrobrachium lanchesteri (de Man)

Bilateral eyestalk ablation in the freshwater prawn Macrobrachium lanchesteri results in high mortality, while unilateral eyestalk ablated prawns exhibited a high survival rate. There was marked increase in the growth of bilateral eyestalk-ablated prawns (47.70 mg/prawn) as compared to those that were unilaterally ablated (19.19 mg/prawn).     

Effects of eyestalk ablation on moulting and growth of penaeid prawn Metapenaeus dobsoni (de Man)

Penaeid prawns M. dobsoni of two different size ranges were ablated both unilaterally (UEA) and bilaterally (BEA) and subjected to individual study. The UEA prawns consumed almost the same amount of food as the control prawns in the size group, 35-40mm, but consumed about 57.8% more food than the control prawns in the larger size group, 48-53mm. Growth was estimated as length and weight changes and significant variation was observed. The moult rate and dry moult weight showed marked differences. Average intermoult period increases with second ablation. Dry weight of moult in every succeeding moult was found to decrease irrespective of the level of ablation. Unilateral eyestalk ablation resulted in increase in dry weight whereas bilateral eyestalk ablation reduced the dry weight of moult and this decrease in the rate increased after each subsequent moult. In the larger size group, values of protein efficiency ratio (PER) were almost same as that of the lower size group. The UEA prawns exhibited highest conversion efficiency. The production rate of UEA prawns was 84% more than that of the control whereas BEA prawns indicated negative production. The same trend followed for net growth efficiency also. The results suggest that unilateral eyestalk ablation can be resorted to accelerate growth and conversion efficiency. Eyestalk ablation has been frequently resorted in matured forms for inducing maturation of gonads. For the first time attempts have been made to compare between the effects of removing one eyestalk versus two and changes taking place in juvenile forms especially in their physiology related to growth.     

Blood glucose in marine penaeid prawns: I. Neuroendocrine regulation in Parapenaeopsis hardwickii (Miers). (Crustacea,Decapoda, Penaeidae)

In the present investigation on femaleP. hardwickii, the blood glucose level in bilateral eyestalk extirpated, reinjected with ES extract as well as normal prawns, administered with ES, different concentrations of BR and ThG extracts, was estimated quantitatively after fifteen minutes and two hours. During all treatments there was an immediate hike in blood glucose level after fifteen minutes but after 24 hr. of eyestalk ablation the level does not differ significantly (P > 0.05) from controls. There was a significant (P < 0.01) enhancement in blood glucose level in the cauterized eyestalkless and normal prawns injected with ES extract after 2 hr. There was a considerable (93% level of confidence) decrease in blood glucose level of normal prawns after injecting higher concentrations of BR and ThG extracts. There was an obvious change in blood glucose level due to the endocrine hormones injected from other prawns of the related family. ES extracts ofP. stylifera produced a significant (P < 0.01) increase, whereas higher concentration of BR and ThG extracts caused considerable (92% level of confidence) decrease in blood glucose level after injecting in cauterized eyestalkless prawns. The extracts of the same endocrine centres fromMetapenaeus monocerus did not induce any significant (P > 0.05) alteration in blood glucose quantity ofP. hardwickii. Hence from the above findings it may be inferred that, the hormones produced by eyestalks (X organ-Sinus gland Complex) posses a hyperglycemic factor, whereas hormones released from BR and ThG might be having a property to cause hypoglycemia (?).     

Hormonal regulatory role of eyestalk factors on growth of heart in mud crab,Scylla serrata

The present study was attempted to know the growth regulation of eyestalk factors on the growth of heart in Scylla serrata using eyestalk extractions and bilateral eyestalk ablations. The bilateral eyestalk ablation led to the maximum growth indices of the heart ((H) indices) to 0.162 and 0.158 in ablated male and female, respectively, in comparison to 0.153 and 0.167 in the control male and female and 0.147 and 0.157 in injected male and female, respectively. The data have shown that the heart of male crabs grows faster than female crabs. The study has also shown that bilateral eyestalk ablation resulted in a significant increase in the heart indices in males and has least effect on the growth of the female heart. The results presented strongly support a potential role of the eyestalk factors and molting hormone regulating the growth of the heart in S. serrata.     

Hyperglycaemic effects of 5-hydroxytryptamine and dopamine in the freshwater prawn, Macrobrachium malcolmsonii

The effects of 5-hydroxytryptamine (5-HT; serotonin) and dopamine (DA) on tissue carbohydrate metabolism and haemolymph glucose levels in the freshwater prawn, Macrobrachium malcolmsonii, were investigated. Injection of 5-HT and DA produced hyperglycaemia in a dose-dependent and time-dependent manner, with DA being more effective than 5-HT. Interestingly, 5-HT and DA induced hyperglycaemia only in intact prawns but not in bilaterally eyestalk-ablated individuals. Total carbohydrate (TCHO) and glycogen levels decreased and phosphorylase activity increased in the hepatopancreas and muscle of intact prawns after being injected with 5-HT or DA. However, bilateral eyestalk ablation decreased haemolymph glucose and tissue phosphorylase activity and increased TCHO and glycogen levels of the hepatopancreas and muscle. Injection of 5-HT or DA did not cause significant changes in these variables in eyestalk-ablated prawns. It is hypothesized that 5-HT and DA induce hyperglycaemia in prawns by stimulating the release of crustacean hyperglycaemic hormone (CHH) from the X-organ sinus gland (XO-SG) complex located in the eyestalk.     

Methyl farnesoate: Its site of synthesis and regulation of secretion in a juvenile crustacean

Analogy to Insecta suggests that JH(s) may play important roles in the development and reproduction of Crustacea. It has recently been shown by Laufer et al. (1987), using LC and GC/MS that the mandibular organ of adult crabs synthesized methyl farnesoate, the unepoxidated from of JH III, and that synthesis varied according to the sex, as a function of eyestalk ablation, and stage of vitellogenesis. In experiments reported here, we found that eyestalk removal from juvenile spider crabs, Libinia emarginata, resulted in a two-fold increase in the rate of MF synthesis by MOs in vitro. Furthermore, 2 h incubations of MOs from eyestalk ablated animals with eyestalk extracts inhibited the rate of synthesis of MF by about 60%. These results suggest that MF may be a gonad stimulating hormone (GSH) reported by other workers. Secretion by the MO is inhibited by an eyestalk factor (MO-IH), and MO-IH may be similar or identical to the gonad inhibiting hormone (GIH) reported by others.     

Variants in the histochemical patterns of lipids in the hepatopancreas of Scylla serrata (Forskal) (Brachyura, Decapoda).

The highest concentrations of phospholipid, neutral lipid and fatty acids were observed in the R cells and connective tissue of the hepatopancreas of Scylla serrata. The basal parts of the B cells and apical parts of the cells lining the main duct also showed moderate presence of these substances. The E cells however, except at their cell membranes were found to be devoid of lipids. F cells on the other hand exhibited lipoid complexes. Considerable reduction in the staining intensity of fatty acids were noticed 4 h after the bilateral ablation of eyestalks, neutral lipid undergo depletion 24 h after the operation whereas phospholipid reserves increase 48 h after the eyestalk removal. A fall in the quantity of neutral lipid and phospholipid was conspicuous when eyestalk extract was injected into normal or destalked crabs. From the present data it appears that R cells and connective tissue form major sites of lipid storage and in an intermolt animal eyestalk factor(s) may have an important role in the control of lipid metabolism.     

Identification of steroid hormones and fatty acids during gonadal maturation of spiny lobster Panulirus homarus

Information on steroid hormones and fatty acids that play roles in lobster reproduction is still very limited although the data are indispensable to seed production in hatchery. The study was designed to identify steroid hormones and fatty acids during gonadal maturation of spiny lobster (Panulirus homarus). Male spiny lobsters were treated with and without thyroxine injection. Female spiny lobsters were treated with and without eyestalk ablations during mature and immature gonad developments. Androst-5-en-17-one,3β (androst) and estran-3-one,17β (estran), two steroid hormones were identified at different levels of gonadal maturity of spiny lobsters. High concentrations of androst and estran were detected in the male spiny lobsters treated with thyroxine injections. Estran showed high concentrations in female brood stock of spiny lobsters during oogenesis stages both without eyestalk ablation and with ablation of one or two eyestalks, except in the immature female gonads. It was found that stearic acid was the highest and dominant fatty acid in mature male spiny lobster. Stearic acid, oleic acid, palmitic acid and caprylic acid were fatty acids with high concentrations in immature and mature female spiny lobsters. After 30 days in captivity, only stearic acid and oleic acid were found dominantly in eyestalk ablated mature female spiny lobsters.     

Effect of x-organ sinus gland extract on [(35)S] methionine incorporation to the ovary of the red swamp crawfish Procambarus clarkii

The presence of gonad-inhibiting hormone in the x-organ sinus gland complex was evaluated in female Procambarus clarkii. Elimination of gonad-inhibiting hormone by way of eyestalk removal resulted in a large acceleration of ovarian development. Daily injection of four sinus gland equivalents reduced ovarian growth of eyestalk-ablated females by about 50% on day 6. Use of the radiotracer [(35)S] methionine showed that gonad-inhibiting activity reached its peak effect between 12 and 24 h following sinus gland injection. Dose-response showed that at least two sinus gland equivalents were needed to significantly counter the accelerated growth induced by eyestalk ablation. The high dose of extract needed to cause significant inhibition was attributed to this delayed response, which subsequently may have required a relatively prolonged exposure to the hormone.     

Metabolic effects of eyestalk removal in the crab Varuna litterata (Fabricius)

Metabolic effects of eyestalk removal in the crab V. litterata is studied. Bilateral eyestalk ablation results in hypoglycaemia and a fall and rise in the glycogen content of hepatopancreas and muscle respectively. Injection of unboiled eyestalk extract evokes hyperglycaemia, whereas boiled eyestalk extract does not produce any significant change in the glycaemia level. The other effect of eyestalk removal is a fall in the fat content of the hepatopancreas and these results are discussed.     

Methyl farnesoate induced ovarian maturation in the spider crab,Libinia emarginata

Summary

To overcome the problem of getting crustaceans to reproduce in captivity, eyestalk ablation or X-organ sinus gland removal is commonly utilized in commercially important species such as shrimp. We have investigated the effect of unilateral and bilateral eyestalk ablation on methyl farnesoate (MF) production by mandibular organs (MOs) and on ovarian maturation in female spider crabs Libinia emarginata, a useful model since these animals are in a terminal molt and are devoid of a functional Y-organ. Non-reproductive, over-wintering female L. emarginata were induced to be reproductive by feeding and increasing the holding temperature to stimulate the endocrine system. In addition, we removed X-organ sinus glands by eyestalk ablation either unilaterally (UEA) or bilaterally (BEA) to further stimulate MF synthesis by MOs. Endogenous MF in the hemolymph was extracted and quantified by means of HPLC and in some cases by GC/MS. Oocyte growth and egg quality were studied simultaneously to determine how they were related to MF levels found during vitellogenesis. The initial MF concentration in unablated controls was low, 0.31 ng/ml of hemolymph, and this increased (p<0.05) to about 1 ng/ml by 2 weeks, remaining at about that level for the remainder of the experiment. Eyestalk ablation significantly stimulated MF concentrations by week 1 to nearly 2 and 3.5ng/ml in the UEA (p <0.01) and BEA (p <0.001) animals, respectively. Oocytes appeared to respond to increased MF levels, as ovarian maturation was initiated from the point at which MF increased (p <0.05). Thereafter, the rate of oocyte growth was directly correlated with the extent of elevation of MF. The gonado-somatic index [(GSI) = gonad weight/body weight × 100] of controls at the start was about 1.5 and increased to 6.5 by week 4. Mature oocytes were reached at a GSI around 7. Oocyte maturation was accomplished at week 2 in BEA, week 3 in UEA, and later than week 4 in controls. After maturation, oocytes started to degrade in some ablated animals, particularly in the bilaterally ablated ones where the highest MF concentrations were observed. These data indicate that MF elevations are required for stimulating ovarian maturation in Crustacea. MF appears to accelerate gonad development during the vitellogenic process, but may be deleterious at high concentrations. These results have a significant and important application and implications for aquaculture.     

Calcium–calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting

Calcium–calmodulin dependent protein kinase I is a component of a calmodulin-dependent protein kinase cascade and involved in many physiological processes. The full-length cDNA of calcium–calmodulin dependent protein kinase I (MnCaMKI) was cloned from the freshwater prawn Macrobrachium nipponense and its expression pattern during the molt cycle and after eyestalk ablation is described. The full-length cDNA of MnCaMKI is 3262 bp in length and has an open reading frame (ORF) of 1038 bp, encoding a 345 amino acid protein. The expression of MnCaMKI in three examined tissues was upregulated in the premolt stage of the molt cycle. Its expression was induced after eyestalk ablation (ESA): the highest expression level was reached 1 day after ESA in hepatopancreas, and 3 days after ESA in muscle. By dsRNA-mediated RNA interference assay, expression of MnCaMKI and ecydone receptor gene (MnEcR) was significantly decreased in prawns treated by injection of dsMnCaMKI, while expression of these two genes was also significantly decreased in prawns treated by injection of dsMnEcR, demonstrating a close correlation between the expression of these two genes. These results suggest that CaMKI in M. nipponense is involved in molting.     

Histochemical localization of oxidative enzymes in the hepatopancreas of scylla serrata (forskål) (brachyura: decapoda)

A histochemical study on the distribution of mitochondrial and oxidative enzymes viz., succinic, NAD and NADP-linked isocitrate and malic dehydrogenases and cytochrome oxidase in the hepatopancreas of Scylla serrata (Forskål) shows that the R cells possess the highest concentration of them at the apical parts; F and B cells showed poor staining reactions whereas E cells and connective tissue exhibited trace staining reactions. A moderate staining for these was obtained in the cells lining the main ducts.

The bilateral removal of eyestalks resulted in the stimulation of succinic and NAD-linked malic dehydrogenases and cytochrome oxidase, observed 4 h after the operation; after 24 h, however, these enzymes showed a slight reduction in the activity. A significant increase in the activity of NADP-linked isocitrate and malic dehydrogenases was noted 24 h after eyestalk ablation. The major alterations were noticed in the R cells.

After injecting eyestalk extract into destalked or intact crabs, a fall in the staining intensity of succinic, NAD-linked malic and isocitrate dehydrogenases in the R cells was apparent 2–4 h after the treatment but was subsequently re-established after 24 h.

It seems from the present results that there may be a factor(s) in the eyestalk of S. serrata which regulates the oxidative metabolism in the hepatopancreas. The physiological significance of oxidative enzymes in various cytologic elements is discussed.     

Neuroendocrine control of reproduction in the male penaeid prawn,Parapenaeopsis hardwickii (Miers) (Crustacea,Decapoda, Penaeidae)

Effects of bilateral eyestalks ablation and injections of eyestalks, (Es) brain (Br) and thoracic ganglia (ThG) extract separately on the androgenic gland and testis development of Es-ablated (Experiment 1) and normal (Experiment 2) P. hardwickii, were investigated. The androgenic gland activity increased significantly in prawns having Es-ablated (Group II), and eyestalkless (Groups VI & VII) and normal (Groups D & E) prawns received Br and ThG extracts, separately, as compared to their respective controls (Groups I & A). Injection of unboiled Es extract in eyestalkless (Group V) and normal (Group C) prawns arrested the androgenic gland activity. Simultaneously, there was a significant (p<0.05) increase in the diameter of testicular follicles, testis weight, testis index and number of mature spermatocytes per follicle in the prawns of groups II, VI & VII of experiment 1 and of groups D & E of experiment 2 when compared to their respective controls. Injection of Es extract into the eyestalkless (Group V) and normal (Group C) prawns ceased testicular development. A significant (<0.05) decrease in testicular protein and midgut gland glycogen and lipid was found in groups II, VI & VII of experiment 1 and in groups D & E of experiment 2, but the glycogen and lipid content of the testis increased. These results indicate that the hormones released from the neuroendocrine centres regulate androgenic gland activity and spermatogenesis.     

Influence of eyestalk removal on the histochemical distribution of oxidative enzymes in the cheliped muscle of Scylla serrata (Forskål)

Histochemical studies on the oxidative enzymes, NAD- and NADP-dependent isocitrate (IDH) and malate (MDH) dehydrogenases, succinic dehydrogenase (SDH), and cytochrome oxidase of the cheliped muscle of Scylla serrata (Forskål) indicated that their concentrations are relatively lower than those of vertebrate muscle. The site of action of various oxidative enzymes is found to be common in the component fibres varying in diameter. The sarcolemma generally exhibited stronger positive reactions for the enzymes than the sarcoplasm.The bilateral removal of eyestalks had a stimulatory effect on the activity of oxidative enzymes. Initially increased activity of SDH, IDH and MDH (NAD-linked) and cytochrome oxidase 2–4 h after eyestalk removal was found to be maintained after 24 h; a noticeable increase in the NADP-linked MDH was also apparent by this time.The eyestalk extract when injected into de-stalked animals, caused a decrease in the levels of SDH, NAD-linked IDH and MDH, and cytochrome oxidase. Biochemical estimations of SDH clearly indicate that bilateral eyestalk extirpation results in remarkably enhanced enzyme activity; conversely, the administration of eyestalk extract brings about a sharp decline in the enzyme concentration. Thus, it seems that the eyestalks may contain a factor regulating oxidative metabolism.     

Current progress in shrimp endocrinology-a review

One problem in aquaculture is obtaining brood because many commercially important species are incapable of spontaneous maturation under artificial conditions. Commercial shrimp hatcheries commonly use eyestalk ablation to stimulate gonadal maturation in shrimps. Research has been conducted on the inhibition of reproductive maturation by hormones originating in the eyestalk glands and on other endocrine sources (e.g.,brain, thoracic ganglion, ovary, mandibular organ, androgenic gland and Y-organs) to determine their roles. Alternate techniques for acceleration of gonad maturation through the use of synthetic hormones or neurotransmitters may benefit aquaculture. Neurohormones and neuroregulators have been shown to accelerate gonadal maturation but an effective delivery technique must be developed for use in a large-scale aquaculture operation.     

Methionine-enkephalin induces hyperglycemia through eyestalk hormones in the estuarine crab Scylla serrata

The hypothesis is tested that methionine-enkephalin, a hormone produced in and released from eyestalk of crustaceans, produces hyperglycemia indirectly by stimulating the release of hyperglycemic hormone from the eyestalks. Injection of methionine-enkephalin leads to hyperglycemia and hyperglucosemia in the estuarine crab Scylla serrata in a dose-dependent manner. Decreases in total carbohydrate (TCHO) and glycogen levels of hepatopancreas and muscle with an increase in phosphorylase activity were also observed in intact crabs after methionine-enkephalin injection. Eyestalk ablation depressed hemolymph glucose (19%) and TCHO levels (22%), with an elevation of levels of TCHO and glycogen of hepatopancreas and muscle. Tissue phosphorylase activity decreased significantly during bilateral eyestalk ablation. Administration of methionine-enkephalin into eyestalkless crabs caused no significant alterations in these parameters when compared to eyestalk ablated crabs. These results support the hypothesis that methionine-enkephalin produces hyperglycemia in crustaceans by triggering release of hyperglycemic hormone from the eyestalks.