Effects Of Eyestalk Ablation Research Articles

Effects of eyestalk ablation and seawater temperature on d-glutamate levels in the reproductive tissues of male kuruma prawn Marsupenaeus japonicus.

D-Glutamate, a novel d-amino acid found in animal tissues, exclusively exists in the male reproductive tissues of the kuruma prawn, Marsupenaeus japonicus. Herein, changes in the d-glutamate content were determined in the male reproductive tissues of M. japonicus during acclimation to breeding seawater temperatures of 18-22°C and unilateral eyestalk ablation. The d-glutamate content in the testis increased with increasing seawater temperature and with unilateral eyestalk ablation. This suggests that both stimulations induced d-glutamate synthesis in the testis. Although the d-alanine content in the testis increased after unilateral eyestalk ablation, it did not change with elevated seawater temperature. Furthermore, we determined the d-glutamate distribution in the M. japonicus spermatophore. This indicates that d-glutamate is crucial in prawn fertilization.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

The Effect of Eyestalk Ablation on Several Immunologic Variables in Litopenaeus vannamei

Eyestalk ablation inflicted upon crustacean is generally performed in captivity to induce ovarian maturation. However, ablation is a hurtful action that may impose physiological stress and even affects survival. The immunologic repercussions of the ablation have not been well understood and thus this study aims to analyze the effect of ablation on immunologic variables through comparing the ablated Litopenaeus vannamei with those of unablated shrimps (control). Unilateral left eyestalk ablation was inflicted on shrimp samples. Shrimp immune responses were observed based on the Total Haemocyte Count (THC), Differential Haemocyte Count (DHC) composing of hyaline cell (HC) counts and granular cell (GC) counts, glucose and uric acid levels, along with the weight gains of shrimps. Observed parametres were taken twice from 10 shrimps for each of the observed and control samples (i.e., on 6 h and 5 d elapsed time). The results show that the ablated shrimps group, after 6 h elapsed time, experienced significant decrease in THC (3.12 ± 0.75 x 106 cells ml-1), but recovered after 5 days (3.89 ± 0.96 x 106 cells ml-1) and were not significantly different from the control (5.16 ± 2.20 x 106 cells ml-1). Meanwhile, glucose level show significant decrease over 6 h and 5 d post ablation (108 and 93 mg dL-1, respectively) compared to the resulting responses from the control (35-36 mg dL-1). Differences in DHC for all cell types as well as uric acid in hemolymph were not significant between the observed and the control samples. Moreover, the ablated treatment did not affect the growth of vannamei after 5 d post ablation. The results revealed that changes occur on the physiological and immune parameters of shrimps due to the eye ablation treatment as shown by the decrease in the THC and glucose levels, especially within the early moments post treatment. However, at the end of the observation (5 d post ablation), shrimps exhibit signs of recovery from stress indicated by an increase in THC close to normal.

Evaluation of different induced molting methods in Callinectes ornatus (Crustacea, Decapoda, Portunidae) as a tool for the commercial production of soft-shell crabs.

We investigated the effects of eyestalk ablation and cheliped autotomy in inducing molting in Callinectes ornatus. The specimens in intermolt stage were divided into two size classes: 1 (30-50 mm) and 2 (51-70 mm) and were further divided into four experimental groups. In the CA group (Cheliped Autotomy, n=76), crabs were submitted to cheliped autotomy; in the UA group (Unilateral Ablation, n=66) and BA group (Bilateral Ablation, n=66) to unilateral and bilateral eyestalk ablation, respectively. The C group (Control, n=70) was used as control. The animals were individually kept in tanks interconnected to a recirculation system for 30 days. The highest frequency of premolt was recorded in the BA group (32%), followed by the CA group (16%). The premolt frequency of class 1 (28%) was significantly higher (p<0.05) than of class 2 (8%). The mean time until molt in the BA group was significantly lower (p <0.05) than other groups tested. The highest mortality rates were 55% and 25% in crabs from the BA and CA groups, respectively. The results indicate bilateral eyestalk ablation and cheliped autotomy are potentially capable of inducing molt in C. ornatus, but these techniques have limited efficiency for the commercial application of large-scale soft-shell crab.

Insights on Molecular Mechanisms of Ovarian Development in Decapod Crustacea: Focus on Vitellogenesis-Stimulating Factors and Pathways.

Vitellogenesis in crustaceans is an energy-consuming process. Though the underlying mechanisms of ovarian maturation in decapod Crustacea are still unclear, evidence indicates the process to be regulated by antagonistically-acting inhibitory and stimulating factors specifically originating from X-organ/sinus gland (XO/SG) complex. Among the reported neuromediators, neuropeptides belonging to the crustacean hyperglycemic hormone (CHH)-family have been studied extensively. The structure and dynamics of inhibitory action of vitellogenesis-inhibiting hormone (VIH) on vitellogenesis have been demonstrated in several species. Similarly, the stimulatory effects of other neuropeptides of the CHH-family on crustacean vitellogenesis have also been validated. Advancement in transcriptomic sequencing and comparative genome analysis has led to the discovery of a large number of neuromediators, peptides, and putative peptide receptors having pleiotropic and novel functions in decapod reproduction. Furthermore, differing research strategies have indicated that neurotransmitters and steroid hormones play an integrative role by stimulating neuropeptide secretion, thus demonstrating the complex intertwining of regulatory factors in reproduction. However, the molecular mechanisms by which the combinatorial effect of eyestalk hormones, neuromediators and other factors coordinate to regulate ovarian maturation remain elusive. These multifunctional substances are speculated to control ovarian maturation possibly via the autocrine/paracrine pathway by acting directly on the gonads or by indirectly exerting their stimulatory effects by triggering the release of a putative gonad stimulating factor from the thoracic ganglion. Acting through receptors, they possibly affect levels of cyclic nucleotides (cAMP and cGMP) and Ca2+ in target tissues leading to the regulation of vitellogenesis. The “stimulatory paradox” effect of eyestalk ablation on ovarian maturation continues to be exploited in commercial aquaculture operations, and is outweighed by the detrimental physiological effects of this procedure. In this regard, the development of efficient alternatives to eyestalk ablation based on scientific knowledge is a necessity. In this article, we focus principally on the signaling pathways of positive neuromediators and other factors regulating crustacean reproduction, providing an overview of their proposed receptor-mediated stimulatory mechanisms, intracellular signaling, and probable interaction with other hormonal signals. Finally, we provide insight into future research directions on crustacean reproduction as well as potential applications of such research to aquaculture technology development.

Effect of Eyestalk Ablation on the Ovarian Maturation Stages of Blue Swimming Crab, Portunus pelagicus

Effect of Eyestalk Ablation on the Ovarian Maturation Stages of Blue Swimming Crab, Portunus pelagicus

Effects of Eyestalk Ablation on Feeding, Gonad Development, and Energy Conversion of Penaeus monodon Broodstock

Effects of Eyestalk Ablation on Feeding, Gonad Development, and Energy Conversion of Penaeus monodon Broodstock

Gonadal maturation, fecundity and hatching performance of wild caught tiger shrimp Penaeus monodon using unilateral eyestalk ablation in captivity

In this study, the effects of eyestalk ablation on maturation, moulting, spawning and hatching rate of wild caught Penaeus monodon were investigated. Twenty males (M) and 30 eyestalk–ablated and 30 non–ablated females (F), all individually marked (M: F=1:3) were stocked into two 15–ton capacity (each) maturation tank. All the ablated females moulted in 7.92±0.24 (mean±standard error) days, and first spawning occurred 3.2±0.20 days following eyestalk ablation. While twenty-five ablated females spawned, producing an average of 296,160±26,589 eggs, only eight non–ablated females spawned, producing an average of 195,462±20,565 eggs during the investigation period of 120 days. A significant positive correlation between fecundity and female body weight (P&lt;0.05; P&lt;0.001) was observed. Throughout the experiment, the average number of spawning per female was 1.8±0.14 for ablated and one for non–ablated females. Multiple spawning (up to three times) occurred within the same moulting cycle in ablated females. Among the ablated P. monodon females, 36% spawn once, 48% second time, and 16% third time. The average fertility rate of the eggs was high, ranging between 80 and 90% in non-ablated females, while in ablated females; the range was between 72 and 88% and differ significantly (p&lt;0.05) ablated and non ablated females. Average hatching rate ranges between 70 and 80% for ablated and 75 and 82% for non-ablated females (p&lt;0.05). From eggs to nauplii production per female was 167,838 for ablated and 127,500for non–ablated females. This study indicates that eyestalk ablation and environmental condition were important inducing tools for re-maturation of spent P. monodon that continued supply of seed stock for effective commercial shrimp farming.J. Bangladesh Agril. Univ. 13(2): 315-322, December 2015

Role of Halloween genes in ecdysteroids biosynthesis of the swimming crab (Portunus trituberculatus): Implications from RNA interference and eyestalk ablation

Molting, including metamorphosis molting in arthropods are controlled by the ecdysteroids that are synthesized and secreted by the crustacean Y-organ (YO) or the insect prothoracic gland (PG). The Halloween genes encoding the enzymes mainly involved in the biosynthesis of ecdysteroids are well studied in insects but not in crustaceans. Given the importance of Halloween genes in ecdysteroids biosynthesis, we have previously reported the cDNA cloning of disembodied (Dib) in P. trituberculatus. Here, cDNA sequences of another two Halloween genes, Spook (Spo) and Shadow (Sad), were further identified and characterized. The predicted amino acid sequences for these two Halloween genes of Portunus trituberculatus were compared to those of several other arthropods, and several typical domains of the cytochrome P450 mono-oxygenase (CYP) were identified. Similar to the tissue distribution of Dib, the Spo and Sad also showed high specificity to the YO. RNA interference (RNAi) of these 3 genes indicated they all play essential role in ecdysteroids biosynthesis. To investigate the relationships of the Halloween genes to the eyestalk neuropeptides such as molt-inhibiting hormone (MIH), effects of eyestalk ablation (ESA) on the expression of Dib, Spo and Sad were detected. Expression of Dib and Sad, but not Spo, was significantly induced by ESA. The result indicated that the inhibition of MIH in ecdysteroids biosynthesis may be partly through the transcriptional regulation of certain Halloween genes, such as Dib and Sad, while the Spo might not be the target for MIH signal.

Differential expression of X-box binding protein 1 during ovarian development and association between its SNP and growth-related parameters of the giant tiger shrimp Penaeus monodon

Abstract The full-length cDNA of X-box binding protein 1 ( PmXbp1 ) in the giant tiger shrimp, Penaeus monodon was identified. It was 1762 bp in length containing an ORF of 855 bp corresponding to 284 amino acids. The expression level of PmXbp1 in different ovarian stages of wild intact broodstock was comparable but greater than that of cultured juveniles ( P PmXbp1 in stages III (late vitellogenic) and IV (mature) ovaries of wild P. monodon broodstock ( P PmXbp1 in ovaries of domesticated 18-month-old broodstock at 24 hpi ( P PmXbp1 (185 bp) were examined in domesticated 3-month-old juveniles (average body weight and total length = 12.32 ± 5.13 g and 11.30 ± 1.58 cm, N = 162) by single-strand conformational polymorphism (SSCP) analysis. Association between SSCP patterns of PmXbp1 and growth-related parameters was found where the average body weight (15.07 ± 5.04, N = 44) of juveniles carrying pattern A (corresponding to a T/T 349 SNP) was significantly greater than that (11.37 ± 4.80, N = 116) of juveniles carrying pattern B (corresponding to a T/C 349 SNP). The relative expression levels of PmXbp1 in the hepatopancreas of juveniles carrying different SSCP/SNP genotypes were significantly different (A P PmXbp1 in shrimp exhibiting a greater growth performance (22.08 ± 2.97 g and 13.60 ± 0.56 cm; N = 11) was significantly lower than that with a lesser growth performance (5.00 ± 0.89 g and 8.60 ± 0.98 cm; N = 8) ( P PmXbp1 seems to play an important role in reproduction and growth of P. monodon . Statement of relevance In aquaculture, the primary goal of genetic selection is the growth improvement of economically important species. Molecular markers that allow for selection of juveniles and broodstock with a high breeding value for reproductive maturation and/or growth rates are useful for the shrimp industry. Information on correlation between genotypic and phenotypic variations in penaeid shrimp is rather limited. Identification of molecular markers linked with growth/reproduction or those regulating the growth/reproduction mechanisms can be applied to improve artificial selection processes of P. monodon . To assess the possible biological roles of X-box binding protein 1 in reproduction and growth of P. monodon , the PmXbp1 cDNA sequence was characterized. Expression patterns of PmXbp1 during ovarian development in wild P. monodon broodstock were examined. Effects of eyestalk ablation (wild broodstock) and serotonin induction (domesticated broodstock) on expression levels of ovarian PmXbp1 were examined. In addition, analysis of relationships between SNP detected by SSCP and DNA sequencing and expression levels of PmXbp1 , and growth traits in juvenile shrimp was examined.

Abnormal morphology in megalopae of the swimming crab,Portunus trituberculatusduring seed production: causes and prevention

The occurrence of morphologically immature megalopae, which retain zoeal features such as dorsal spines and furcae of telson, is closely correlated with larval mass mortality during seed production of the swimming crab Portunus trituberculatus in Japanese hatcheries. To determine the cause of immature megalopal morphology, zoeae were reared with various supplementary schedules and density of diets (rotifer, Artemia and phytoplanktons including Chlorella vulgaris and Nannochloropsis oculata). In addition, to assess the relationship between immature morphology and endocrine control, the effect of causative dietary factor was compared with the effect of eyestalk ablation at various timing during zoeal development. Megalopal immature morphology was more distinct in Chlorella-supplemented groups than in Nannochloropsis-supplemented groups. High density Chlorella supplementation was associated with the highest incidence of immaturity and resulted in larval mass mortality. The premoult of the third zoeal stage was identified as a critical period at which Chlorella supplementation led to the highest incidence of immaturity. This critical period coincided with the critical period at which larval metamorphosis was regulated by the eyestalk neurosecretory system. Our results suggested that the occurrence of immature megalopal morphology under culture conditions is most likely caused by phytoplankton (especially, Chlorella) supplementation, which disrupts the endocrine regulation. On the basis of our results, we successfully prevented the occurrence of immature megalopal morphology in 500 L tanks by excluding the influence of phytoplankton before the critical period (i.e. discontinuing phytoplankton supplementation and supplying rotifer cultured with non-phytoplankton materials).

Ecdysone receptor gene from the freshwater prawn Macrobrachium nipponense: Identification of different splice variants and sexually dimorphic expression, fluctuation of expression in the molt cycle and effect of eyestalk ablation

The full-length cDNA of an ecdysone receptor gene (MnEcR) from Macrobrachium nipponense was cloned and the expression of the gene was investigated. MnEcR maintained a relatively low expression level in the early stages of embryos, but from nauplius stage, a steady increase in MnEcR expression was detected, it had the highest expression level in zoea stage. MnEcR was highly expressed in the hepatopancreas and gills among ten different tissues examined. MnEcR was rapidly upregulated in the premolt stage and rapidly downregulated in the postmolt stage. The expression of MnEcR was remarkably downregulated after eyestalk ablation in M. nipponense. An 18-amino-acid insertion/deletion and a 49-amino-acid substitution were found in the coding region of MnEcR, resulting in four splice variants: MnEcR-L1, -L2, -S1 and-S2. The expression of four splice variants of MnEcR in gonads was investigated using RT-PCR. Interestingly, the expression patterns of these splice variants differed between males and females. The dominant splice variants in testis were MnEcR-S1 and -S2, while in ovary they were MnEcR-L1 and -S2, indicating specific roles for these splice variants in male and female individuals.

Effect of eyestalk ablation in &amp;lt;i&amp;gt;Eriocheir sinensis&amp;lt;/i&amp;gt; on physiological and biochemical metabolism

Eyestalk ablation is the most common procedure to induce gonadic maturation of Eriocheir sinensis. In addition to reproduction, other physiological and metabolic processes are also affected by the removal of X-organ sinus complex located in the eyestalk. In this study, we studied physiological and biochemical effects of eyestalk ablation of E. sinensis, and the results were as follows. The amylase activity of male crabs with unilateral or bilateral eyestalk ablaion was increased with the days increasing, and the maximal value was on the 20th day (P P

The Effects of Eyestalk Ablation on the Reproductive and Immune Function of Female Macrobrachium americanum

Eyestalk ablation is the most common procedure used to induce crustacean reproduction in commercial hatcheries. However, other physiological and metabolic processes are affected by the removal of the X-organ sinus gland complex located in the eyestalk. In this study, the effect of unilateral eyestalk ablation on the concentration of several hemolymph metabolites, reproduction and immune function in female Macrobrachium americanum was investigated. Molting cycle time was not significantly (P=0.17) shortened in eyestalk-ablated females. However, an increased number of molts was observed in the eyestalk-ablated group because survival was significantly higher due to increased passivity and lowered aggression. As M. americanum has closed thelyca, reproduction was not accelerated after eyestalk ablation compared to control prawns. Food intake (P=0.007) and oxygen consumption (P=0.047) were both higher in eyestalk-ablated females. Concentrations of protein and glucose in hemolymph were not affected (P=0.54 and P=0.19, respectively), indicating that the demand for these metabolites was met. Glucose was metabolized by aerobic metabolism as shown by the lower lactate concentration (P=0.02) and higher respiration rate after ablation. Protein was metabolized for growth as revealed by the 100% higher growth rate in eyestalkablated females (P=0.017). Triglycerides were more highly concentrated (P=0.02) in eyestalk-ablated females. This difference was most likely due to being transported from the digestive gland to the gonad.By analyzing the immune system it was observed that only the total hemocyte count was higher (P=0.002) in eyestalk-ablated females. The protein content in hemocytes, the prophenol oxidase system and the coagulation time were not affected. In conclusion, the eyestalk ablation procedure did not cause female M. americanum to have more reproductive events over time. Despite this failure to accelerate reproduction, eyestalk ablation reduced aggression, and thus increased survival, and promoted superior growth. This result suggests that eyestalk ablation is promising for the cultivation of M. americanum.

Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): Cloning and effects of eyestalk ablation on gene expression

Six cDNAs encoding chitinase proteins in Pandalopsis japonica were isolated by using polymerase chain reaction (PCR) cloning methods and bioinformatic analysis of expressed sequence tags (ESTs). The cDNAs, designated Pj-Cht1, 2, 3A, 3B, 3C, and 4, encoded proteins ranging from 388 to 607 amino acid residues in length (43.61-67.62kDa) and displayed a common structural organization: an N-terminal catalytic domain, a Thr/Pro-rich linker region, and either 0 (Pj-Cht2, 3A), 1 (Pj-Cht1, 3B, and 3C), or 2 (Pj-Cht4) C-terminal chitin-binding domain(s) (CBD). Pj-Cht1 and 2 lacked the 5' end of the open reading frame (ORF); the other Pj-Chts contained the complete ORF. All known decapod crustacean chitinases were segregated into at least four groups based on phylogenetic analysis and domain organization. Group 1 chitinases, represented by Pj-Cht1, were most closely related to insect group I chitinases and may function in the digestion of the peritrophic membrane. Group 2 chitinases including Pj-Cht2 show different domain organizations and pI value from other chitinases and appear to function in degradation of the old exoskeleton during the premolt period. Group 3 chitinases, represented by Pj-Cht3A, 3B, and 3C, may function in digestion of chitin-containing food and defense against pathogens. Group 4 chitinases, represented by Pj-Cht4, have two CBDs and their functions are unknown. Five Pj-Chts (Pj-Cht1, 3A, 3B, 3C, and 4) are expressed in the hepatopancreas and intestine, whereas Pj-Cht2 is expressed in epidermis and SG/XO complex suggesting crustacean chitinases can be classified into two groups (hepatopancreatic and epidermal) based on the expression profile. Eyestalk ablation (ESA) down-regulated the hepatopancreatic chitinase expression (Pj-Cht1, 3A, and 3C); Pj-Cht3B expression was not significantly affected by ESA. By contrast, mRNA levels of Pj-Cht2 were significantly upregulated in 7days post-ESA. Pj-Cht4 mRNA levels were too low for measurement with quantitative polymerase chain reaction. ESA had no significant effect on chitinase expression in the intestine. These data indicate that Pj-Cht1, 3A, 3B, 3C, and 4 are hepatopancreatic chitinases that may function in the digestion of ingested chitin and the modification of peritrophic membrane in the intestine. By contrast, epidermal chitinase, Pj-Cht2 may play a role in chitin metabolism during molt cycle as shown in other crustacean group 2 chitinases.

Two juvenile hormone esterase-like carboxylesterase cDNAs from a Pandalus shrimp (Pandalopsis japonica): Cloning, tissue expression, and effects of eyestalk ablation

Methyl farnesoate (MF), a crustacean juvenile hormone (JH) analog, plays important roles in the regulation of a number of physiological processes such as molting, metamorphosis, and reproduction. Understanding its metabolic pathway is a key for various potential applications in crustacean aquaculture, including artificial seed production and enhancement of growth. Although the synthetic pathway of MF is well established, little is known about its degradation and recycling in crustaceans. In insects, juvenile hormone esterase (JHE), a carboxylesterase, is responsible for JH inactivation. Two cDNAs, encoding JHE-like carboxylesterases (CXEs) from the hepatopancreas and ovary of Pandalopsis japonica, were isolated by using a combination of in-silico data mining from an expressed sequence tag (EST) database and traditional PCR-based cloning. The full length Pj-CXE1 (2084bp) and Pj-CXE2 (1985bp) cDNAs encoded proteins composed of 584 and 581 amino acids, respectively. The active site sequence and domain organization of the Pj-CXEs were highly conserved, including the catalytic triad and other motifs, which suggested that both Pj-CXEs are biologically active carboxylesterases. Phylogenetic analysis of the deduced sequences of Pj-CXEs showed that both were most closely related to the JHEs from non-lepidopteran insects. End-point RT-PCR showed that Pj-CXE1 was expressed primarily in the gonad, whereas Pj-CXE2 was expressed in both the hepatopancreas and hindgut. Quantitative PCR showed that Pj-CXE1 was upregulated in the gonads by eyestalk ablation (ESA). In contrast, ESA had no significant effect on Pj-CXE2 expression in hepatopancreas or gonad. This is the first report of the cloning of two JHE-like CXE cDNAs in decapods and the upregulation of Pj-CXE1 by acute withdrawal of eyestalk neuropeptides. Further study is needed to understand the function of CXEs in MF metabolism and its regulation by eyestalk neuropeptides.

Effects of Eyestalk Ablation on Maturation, Spawning, Hatching, Molting and Growth of Precocious Pond Reared Penaeus monodon

Effects of Eyestalk Ablation on Maturation, Spawning, Hatching, Molting and Growth of Precocious Pond Reared Penaeus monodon

Effect of eyestalk ablation on the metabolic activities of two penaeid prawns: Metapenaeous monoceros and M. dobsoni (De Man)

Metabolic activities in two penaeid prawns Metapenaeus monoceros and M. dobsoni in two size ranges of 35–40 mm and 65–70 mm for the former, and 35–40 mm and 48–53 mm for the latter, were observed for 16 days to study the effect of unilateral (UEA) and bilateral eyestalk ablation (BEA). In both species, BEA prawns of the two size groups consumed more oxygen than the UEA prawns, leading to higher metabolic activity. Small size groups revealed more metabolic activity than the large size ones, In both species, trend for the oxygen consumption (OC) was a steadily decreasing function of time in the small size group, but a steady state condition was observed in the large size group. The small size prawns showed higher amount of ammonia excretion (AE) than the large ones. BEA prawns of both size groups excreted more ammonia than the UEA with higher ammonia quotient (AQ) values, leading to higher metabolic activity, suggesting that measures should be taken for keeping the level of ammonia in the culture to the minimum for the safety of the culture. Changes in AQ are highly age‐sensitive, this being explained by higher order polynomials, unlike that observed in the OC and AE trend, with respect to time. These changes induced in the metabolic activities are discussed.

Effects of eyestalk ablation on carbonic anhydrase activity in the euryhaline blue crabCallinectes sapidus: neuroendocrine control of enzyme expression

Carbonic anhydrase (CA) activity in the gills of the euryhaline blue crab, Callinectes sapidus, was measured in response to acute low-salinity transfer and treatment with eyestalk ablation (ESA) in an attempt to elucidate potential regulatory mechanisms of salinity-mediated CA induction. ESA alone resulted in an approximate doubling of CA activity in the posterior, ion-transporting gills of crabs acclimated to 35 ppt. Transfer of intact crabs to 28 ppt, a salinity at which the blue crab is still an osmotic and ionic conformer, had no effect on CA activity, but treatment with ESA prior to transfer resulted in a 5-fold increase. Hemolymph osmolality was unaffected by ESA. There was a 7-fold induction of CA activity in posterior gills of intact crabs transferred from 35 to 15 ppt, and this was potentiated by about 100% by ESA. Hemolymph osmolality was slightly elevated in the ESA-treated crabs. CA activity in anterior gills did not increase in response to any treatment. Hemolymph concentrations of methyl farnesoate (MF) were measured for all experimental animals. MF concentrations were undetectable in all intact crabs, regardless of salinity. Treatment with ESA resulted in elevated levels of hemolymph MF, but these levels were still relatively low and unrelated to salinity. These results suggest that CA induction is under the control of a regulatory substance located in the eyestalk. This substance appears to be a CA repressor, keeping CA expression at low levels in the gills of crabs acclimated to high salinity. Exposure to low salinity, or treatment with ESA, removes the effects of this putative repressor and allows CA induction to occur.

Circadian Locomotor Activity Rhythm in the Freshwater Crab Pseudothelphusa americana (De Saussure, 1857): Effect of Eyestalk Ablation

The circadian rhythm of locomotor activity in the freshwater crab, Pseudothelphusa americana, was studied in aquaria using infrared crossing sensors. Individuals with ablated eyestalks were compared with intact individuals in constant darkness (DD) and in light-dark cycles (LD). Our results showed that intact animals in DD displayed bimodal rhythms. In LD conditions the two peaks were associated with lights on and lights off, respectively. A significant difference in the free running periods before and after LD was observed in all intact animals. After eyestalk ablation (ES-X), the circadian rhythm of locomotor activity disappeared immediately, but reappeared several days later. Diurnal activity was seen in some ES-X animals when exposed to LD. Our results indicate that locomotor activity rhythm in P. americana is driven primarily by oscillators located outside the eyestalks, and that extraretinal photoreceptors mediate either entrainment or masking effects.