Eyestalk Research Articles

The occurrence of luteinizing hormone-like molecule and its receptor in the blue swimming crab, Portunus pelagicus

Knowledge of the neuroendocrine system possibly improves the reproductive performance of captivated crustacean broodstock in aquaculture and it may substitute eyestalk ablation. In this study, we explored the luteinizing hormone (LH)-like molecule and proved the existence of the LH receptor (PpelLHR)-like mRNA in the blue swimming crab, Portunus pelagicus. Using the anti-human LH-β antibody, the immunoreactivities were found in the central nervous system (CNS) and ovary of the crab with the strongest signal in the mature ovary. The full-length PpelLHR-like mRNA sequence contained 4818 bp with deduced protein predicted as seven transmembrane G-protein coupled receptor, made of 1605 amino acids. The phylogenetic tree suggested this protein belonged to the clade of invertebrate LHR/FSHR-like proteins. The PpelLHR-like mRNA expressed in various organs and real-time qPCR revealed significantly higher expression of this mRNA in the brain and lower expression in the ovary of the mature crabs. In situ hybridization of this mRNA was demonstrated in neuronal clusters of the brain, ventral nerve cord, and in the oocyte stage 1–4 of the ovary, respectively. This study was preliminary to prove the existence of LH and its receptor in the blue swimming crab. Functional assay of this receptor should be performed as the next part of experiments to firmly conclude its appearance.

Examination of the antioxidant effects of linalool and Lippia alba essential oil in the Pacific whiteleg shrimp, Litopenaeus vannamei, subjected to eyestalk ablation

Essential oils are considered to have the ability to minimize the effects of stress during handling in aquatic animals. This study evaluated the anesthetic effects of Lippia alba essential oil and its major compound, linalool, on the antioxidant responses of eyestalk-ablated female shrimp Litopenaeus vannamei. Female shrimp were divided into the following treatments: pure seawater (control), ethanol (9000 μL L−1 of ethanol), 750 μL L−1 of Lippia alba essential oil, 1000 μL L−1 of Lippia alba essential oil, 350 μL L−1 of linalool, and 500 μL L−1 of linalool. The shrimp were sedated for 3 min. The eyestalks were then ablated unilaterally by cauterization (cutting the eyestalk with hot scissors). Biochemical variables were analyzed in the gill, muscle, and hepatopancreas. The antioxidant capacity against peroxyl radicals in the group that received 750 μL L−1 of Lippia alba essential oil was significantly higher in the hepatopancreas and muscle than in the control group, whereas the ethanol group had a higher antioxidant capacity in the gills and muscle than the control group. An increase in the total antioxidant capacity in the hepatopancreas and muscle was observed in the 750 μL L−1 Lippia alba essential oil group, but not in the ethanol group, relative to the control group. A significant reduction in lipid peroxidation in the gills was observed in females treated with 500 μL L−1 of linalool compared to the ethanol group. Linalool and Lippia alba essential oil contributed to the improvement in the antioxidant defense systems of shrimp gills. Overall, the obtained results suggest that exposure to Lippia alba essential oil and linalool may activate certain components of the antioxidant system that exist in the gills and scavenge peroxyl radicals; this in turn may help maintain homeostasis and improve the welfare of eyestalk-ablated female shrimp.

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.

Effects of molting on the expression of ecdysteroid responsive genes in the crustacean molting gland (Y-organ)

Ecdysteroid molting hormones coordinate arthropod growth and development. Binding of 20-hydroxyecdysone (20E) to ecdysteroid receptor EcR/RXR activates a cascade of nuclear receptor transcription factors that mediate tissue responses to hormone. Insect ecdysteroid responsive and Forkhead box class O (FOXO) transcription factor gene sequences were used to extract orthologs from blackback land crab (Gecarcinus lateralis) Y-organ (YO) transcriptome: Gl-Ecdysone Receptor (EcR), Gl-Broad Complex (Br-C), Gl-E74, Gl-Hormone Receptor 3 (HR3), Gl-Hormone Receptor 4 (HR4), Gl-FOXO, and Gl-Fushi tarazu factor-1 (Ftz-f1). Quantitative polymerase chain reaction quantified mRNA levels in tissues from intermolt animals and in YO of animals induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). Gl-EcR, Gl-Retinoid X Receptor (RXR), Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, Gl-Ftz-f1, and Gl-FOXO were expressed in all 10 tissues, with Gl-Br-C, Gl-E74, Gl-E75, and Gl-HR4 mRNA levels in the YO lower than those in most of the other tissues. In MLA animals, molting had no effect on Gl-Br-C, Gl-E74, and Gl-Ftz-f1 mRNA levels and little effect on Gl-EcR, Gl-E75, and Gl-HR4 mRNA levels. Gl-HR3 and Gl-FOXO mRNA levels were increased during premolt stages, while Gl-RXR mRNA level was highest during intermolt and premolt stages and lowest at postmolt stage. In ESA animals, YO mRNA levels were not correlated with hemolymph ecdysteroid titers. ESA had no effect on Gl-EcR, Gl-E74, Gl-HR3, Gl-HR4, Gl-Ftz-f1, and Gl-FOXO mRNA levels, while Gl-RXR, Gl-Br-C, and Gl-E75 mRNA levels were decreased at 3 days post-ESA. These data suggest that transcriptional up-regulation of Gl-FOXO and Gl-HR3 contributes to increased YO ecdysteroidogenesis during premolt. By contrast, transcriptional regulation of ecdysteroid responsive genes and ecdysteroidogenesis were uncoupled in the YO of ESA animals.

Mechanisms of neurocentral-eyestalk-intestinal immunotoxicity in whiteleg shrimp Litopenaeus vannamei under ammonia nitrogen exposure

Ammonia-N, as the most toxic nitrogenous waste, has high toxicity to marine animals. However, the interplay between ammonia-induced neuroendocrine toxicity and intestinal immune homeostasis has been largely overlooked. Here, a significant concordance of metabolome and transcriptome–based “cholinergic synapse” supports that plasma metabolites acetylcholine (ACh) plays an important role during NH4Cl exposure. After blocking the ACh signal transduction, the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) in the cerebral ganglia increased, while the release of NPF in the thoracic ganglia and NE in the abdominal ganglia, and crustacean hyperglycemic hormone (CHH) and neuropeptide F (NPF) in the eyestalk decreased, finally the intestinal immunity was enhanced. After bilateral eyestalk ablation, the neuroendocrine system of shrimp was disturbed, more neuroendocrine factors, such as corticotropin releasing hormone (CRH), adrenocorticotropic-hormone (ACTH), ACh, DA, 5-HT, and norepinephrine (NE) were released into the plasma, and further decreased intestinal immunity. Subsequently, these neuroendocrine factors reach the intestine through endocrine or neural pathways and bind to their receptors to affect downstream signaling pathway factors to regulate intestinal immune homeostasis. Combined with different doses of ammonia-N exposure experiment, these findings suggest that NH4Cl may exert intestinal toxicity on shrimp by disrupting the cerebral ganglion-eyestalk axis and the cerebral ganglion-thoracic ganglion-abdominal ganglion axis, thereby damaging intestinal barrier function and inducing inflammatory response.

Brazilians' perception of shrimp sentience and welfare

With the increase in research aimed at improving the welfare of animals raised in production systems and the recognition of sentience in decapod crustaceans, the welfare of shrimp has become a cause for concern. In shrimp farming, practices contrary to the principles of animal welfare are used and largely unknown to the consuming public. This study aimed to assess the views of Brazilians on the importance of incremental animal welfare improvements in shrimp farming, considering the social and cultural aspects of the respondents. To this end, 300 adult Brazilian participants answered questions on welfare, sentience, and eyestalk ablation of shrimp. Among the respondents, 75.7% expressed concern about the welfare of shrimp, believing that they can experience fear (63.0%), pain (84.0%), and pleasure (47.7%). The latter finding suggests a relative reluctance to recognize positive feelings in shrimp. Regarding eyestalk ablation, 81.7% of respondents were unaware of the procedure, but after a brief explanation, 81.3% considered it unacceptable, with 84.0% believing that shrimps suffer from the practice. Most respondents associated terms such as "painful, cruel, suffering, mercantilist, disrespectful, and mutilating" with the practice of ablation. Furthermore, by using Pearson's chi-square test, it was observed that gender, age, region, education, socio-economic status, profession, and frequency of consumption may be related to the respondents' opinions about shrimp welfare and their perception of shrimp sentience. Therefore, by recognizing that public concern drives changes in production methods, increasing knowledge about shrimp sentience and farming practices can help advance socially acceptable methods.

Effects of eyestalk ablation on growth, feeding behavior, movement and circadian gene expression in Haliotis discus hannai

The present study aimed to investigate the effects of eyestalk ablation on growth, feeding behavior, circadian movement activity, and circadian gene expression in the abalone Haliotis discus hannai. Abalones were divided into two groups: eyestalk-ablated (EA) and control (C), and were cultured for 60 days. Results showed that the EA group exhibited significantly higher growth rates in body weight compared to the C group. However, the EA group also had a higher mortality rate, indicating the negative consequences of eyestalk ablation. The feeding rate was significantly higher in the EA group, especially during nocturnal feeding, suggesting that eyestalk ablation may specifically affect nocturnal feeding behavior in abalone. Both EA and C groups displayed rhythmic oscillations in movement activity, with no significant differences in mesor and amplitude. Nevertheless, the peak of populational movement proportion in the EA group occurred approximately 2 h earlier than that in the C group. Transcriptome analysis identified 417 circadian genes, with the most significant enrichment found in the growth-related thyroid hormone signaling pathway. The expression of five core clock genes (clock, cry, rho, tipin, per) exhibited significant circadian rhythms in H. discus hannai, while their rhythmic oscillation significance was weaker in the EA group, except for the tipin transcript. These findings reveal that eyestalk ablation affects growth, feeding and locomotor behavior, and clock gene expression in abalone, providing valuable insights for understanding the circadian regulatory mechanisms and physiological ecological adaptations of abalone. However, due to its harmful effects on animal welfare and the limited promotion of growth, this technology is not advocated for use in aquaculture practices.

Trilobite hypostome as a fusion of anterior sclerite and labrum

The trilobite hypostome is a biomineralized ventral plate that covers the mouth, but its evolutionary origin remains controversial. The labrum is a lobe-like structure that can take on variety of shapes in front of the mouth in arthropods, while the anterior sclerite refers to a cuticular plate articulated to the anterior margin of the head in some Cambrian arthropods. Here I present a perspective that views the trilobite hypostome as a fusion of the anterior sclerite and the labrum based on anatomical, topological, and developmental evidence. According to this perspective, the anterior lobe of the hypostome originated from the anterior sclerite, while the posterior lobe reflects a remnant of the sclerotized cover of the labrum. The convex anterior lobe housed the root of the eye stalks, represented by the palpebral ridges and the hypostomal wing, and the posterior lobe occasionally developed a pair of posterolateral extensions, as do the labra. The position of the antennal insertion was located in front of the posterior lobe, displaying a similar topology to the Cambrian arthropods with the labrum. The hypostome was present in many artiopodans except for the Conciliterga, in which the anterior sclerite was separate from the labrum.

The immunotoxicity mechanism of NH4Cl exposure to Litopenaeus vannamei based on the cerebral ganglion-eyestalk-haemocytes axis

Ammonia nitrogen, as a water environmental toxin, poses a potential threat to aquatic animals. Although NH4Cl stress is known to cause immunotoxicity, mechanistic pathways linking stress networks in the neuroendocrine system to immunotoxicity remain poorly understood. In this study, firstly, using transcriptome analysis of cerebral ganglion and eyestalk in shrimp, we identified significant changes in genes related to biogenic amines, acetylcholine, crustacean hyperglycemic hormones, and neuropeptide F. Additionally, expression patterns of neuroendocrine factors in different tissues of shrimp were evaluated to explore the sources of these factors. Here, we showed that NH4Cl exposure activates acetylcholine (ACh) neurons in cerebral ganglion of shrimp and dramatically upregulates high affinity choline transporter 1 (ChT1) gene expression. The knockdown of ChT1 gene enhanced the immunity of haemocytes in shrimp compared with saline and GFP dsRNA groups. And after eyestalk ablation, the levels of neuroendocrine factors in the cerebral ganglion and thoracic ganglion were disturbed, and haemocytes parameters induced by NH4Cl were significantly decreased. Combined with different doses of NH4Cl exposure experiments, we demonstrated that: (1) In a short period of NH4Cl exposure, the neuroendocrine factors CRH–ACTH–cortisol and 5-HT–DA in the cerebral ganglion–eyestalk axis of shrimp play a major role in regulating haemocytes immunity; (2) With the prolongation of exposure, the immunotoxicity induced by NH4Cl was mainly due to the release of more ACh in the cerebral ganglion, which promoted the release of NPF in the thoracic ganglion, and CHH and NPF in the eyestalk, as well as weakened the effect of biogenic amines. Subsequently, these neuroendocrine factors regulate immunity through intracellular signaling pathways. Collectively, these results established a new mechanism that NH4Cl might directly regulate haemocytes immunotoxicity through the cerebral ganglion and thoracic ganglion; or through the cerebral ganglion-eyestalk axis or cerebral ganglion-thoracic ganglion axis cause haemocytes immunotoxicity.

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

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

Predatory behavior under monocular and binocular conditions in the semiterrestrial crab Neohelice granulata.

Neohelice granulata crabs live in mudflats where they prey upon smaller crabs. Predatory behavior can be elicited in the laboratory by a dummy moving at ground level in an artificial arena. Previous research found that crabs do not use apparent dummy size nor its retinal speed as a criterion to initiate attacks, relying instead on actual size and distance to the target. To estimate the distance to an object on the ground, Neohelice could rely on angular declination below the horizon or, since they are broad-fronted with eye stalks far apart, on stereopsis. Unlike other animals, binocular vision does not widen the visual field of crabs since they already cover 360° monocularly. There exist nonetheless areas of the eye with increased resolution. We tested how predatory responses towards the dummy changed when animals' vision was monocular (one eye occluded by opaque black paint) compared to binocular. Even though monocular crabs could still perform predatory behaviors, we found a steep reduction in the number of attacks. Predatory performance defined by the probability of completing the attacks and the success rate (the probability of making contact with the dummy once the attack was initiated) was impaired too. Monocular crabs tended to use frontal, ballistic jumps (lunge behavior) less, and the accuracy of those attacks was reduced. Monocular crabs used prey interception (moving toward the dummy while it approached the crab) more frequently, favoring attacks when the dummy was ipsilateral to the viewing eye. Instead, binocular crabs' responses were balanced in the right and left hemifields. Both groups mainly approached the dummy using the lateral field of view, securing speed of response. Although two eyes are not strictly necessary for eliciting predatory responses, binocularity is associated with more frequent and precise attacks.

Eyestalk ablation and acclimation temperatures affect juveniles Penaeus vannamei thermo-tolerance: molecular biomarkers of cell protection oxidative stress, and compensatory mechanisms

This study investigated the effect of acclimation temperatures and eyestalk ablation (EA) on the thermal tolerance of juvenile Penaeus vannamei. In each case, the relative expression of genes involved in cell protection (hsp70 and hsp90), oxidative stress (cMnSOD and GPx), and anaerobic metabolism (hif1a) was assessed. For this purpose, shrimp were acclimated to 20, 26, and 32°C for 21 days. After acclimation, the critical thermal maxima (CTmax) was determined in non-eyestalk ablated, unilaterally, and bilaterally eyestalk ablated organisms. An effect of acclimation temperatures on CTmax values was observed, with shrimp acclimated at 32°C having the highest rates. Likewise, EA resulted in lower thermal tolerance to CTmax in organisms acclimated at 20 and 26°C. The shrimp's protective and cellular repair responses were evidenced by increased hsp70 and hsp90 gene expression after CTmax and were intensified by the EA. In contrast, the results showed that cMnSOD was very sensitive to CTmax, and its expression was intensified with EA, while for GPx, there was an increase in the relative gene expression, mainly in shrimp acclimated at 20°C. In the case of hif1a, overexpression was observed at the acclimation temperature of 26°C, showing the activation of compensatory mechanisms such as anaerobic metabolism. EA caused a significant molecular response during CTmax of molecular biomarkers involved in heat stress response, oxidative stress, and compensatory mechanisms.

Effects of molting on the expression of ecdysteroid biosynthesis genes in the Y-organ of the blackback land crab, Gecarcinus lateralis

A pair of Y-organs (YOs) synthesize ecdysteroids that initiate and coordinate molting processes in decapod crustaceans. The YO converts cholesterol to secreted products through a biosynthetic pathway involving a Rieske oxygenase encoded by Neverland (Nvd) and cytochrome P450 monooxygenases encoded by Halloween genes Spook (Spo; Cyp307a1), Phantom (Phm; Cyp306a1), Disembodied (Dib; Cyp302a1), and Shadow (Sad; Cyp315a1). NAD kinase (NADK) and 5-aminolevulinic acid synthase (ALAS) support ecdysteroid synthesis in insects. A 20-hydroxylase, encoded by Shed in decapods and Shade in insects, converts ecdysone to the active hormone 20-hydroxyecdysone (20E). 20E is inactivated by cytochrome P450 26-hydroxylase (Cyp18a1). Contigs encoding these eight proteins were extracted from a Gecarcinus lateralis YO transcriptome and their expression was quantified by quantitative polymerase chain reaction. mRNA levels of Gl-Spo and Gl-Phm were four orders of magnitude higher in YO than those in nine other tissues, while mRNA levels of Gl-NADK and Gl-ALAS were similar in all ten tissues. In G. lateralis induced to molt by multiple leg autotomy, YO mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-NADK, and Gl-ALAS were highest in intermolt and premolt stages and lower in postmolt. Gl-Dib mRNA level was not affected by molt stage. mRNA level of Gl-Sad, which converts 2-deoxyecdysone to ecdysone, was higher in mid- and late premolt stages, when YO ecdysteroidogenic capacity is greatest. Gl-Cyp18a1 mRNA level was highest in intermolt, decreased in premolt stages, and was lowest in postmolt. In animals induced to molt by eyestalk ablation, YO mRNA levels of all eight genes were not correlated with increased hemolymph 20E titers. These results suggest that YO ecdysteroidogenic genes are differentially regulated at transcriptional and translational levels.

Molecular Characterization Related to Ovary Early Development Mechanisms after Eyestalk Ablation in Exopalaemon carinicauda.

Eyestalk ablation is an effective method to promote ovarian development in crustaceans. Herein, we performed transcriptome sequencing of ovary and hepatopancreas tissues after eyestalk ablation in Exopalaemon carinicauda to identify genes related to ovarian development. Our analyses led to the identification of 97,383 unigenes and 190,757 transcripts, with an average N50 length of 1757 bp. In the ovary, four pathways related to oogenesis and three related to oocyte rapid growth were enriched. In the hepatopancreas, two vitellogenesis-associated transcripts were identified. Furthermore, short time-series expression miner (STEM) and gene ontology (GO) enrichment analyses revealed five terms related to gamete generation. In addition, two-color fluorescent in situ hybridization results suggested that dmrt1 might play a vital role in oogenesis during the early stage of ovarian development. Overall, our insights should support future studies focusing on investigating oogenesis and ovarian development in E. carinicauda.

Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

Mud crabs (Scylla spp.) are commercially important crustacean species that can be found throughout the Indo-West Pacific region. During culture, the induction of ovarian maturation is important to meet the consumer demand for mature mud crabs and hasten seed production. Eyestalk ablation is an effective tool to enhance ovarian maturation in mud crabs. However, there is no standard protocol for the eyestalk ablation of mud crabs. In this study, two eyestalk ablation techniques are described: cauterization (the use of hot metal to ablate the eyestalk of an anesthetized crab) and surgery (the removal of the eyestalk using surgical scissors). Before eyestalk ablation, sexually mature females (CW > 86 mm) were anesthetized using an ice bag (-20 °C) with seawater. When the water temperature reached 4 °C, the ice bag was removed from the water. Flowing seawater (ambient temperature: 28 °C) was used for recovery from the anesthesia immediately after eyestalk ablation. Mortality did not occur during or after the process of eyestalk ablation. The eyestalk ablation protocol presented here accelerated the ovarian maturation of the mud crabs.

Structure and function analyses of the Mmd2 gene in pacific white shrimp Litopenaeus vannamei.

Monocyte to macrophage differentiation factor 2 gene (Mmd2) encodes a member of the progestin and adipoQ receptor (PAQR) family, and plays a key role in growth and development. Our previous studies had found Mmd2 (Monocyte to macrophage differentiation factor 2) is a new candidate gene for growth traits in Pacific white shrimp (Litopenaeus vannamei). For the purpose of understanding the underlying mechanism of LvMmd2 affecting the growth of shrimp, we analyzed the gene structure, phylogeny, expression profiles and RNA interference of this gene in L. vannamei. We found the LvMmd2 gene sequence was highly conserved in transmembrane regions, it was widely expressed in different tissues, with the highest expression level in the eye stalk. Knockdown LvMmd2 could significantly promote body length and body weight gain, suggesting it is a growth suppressor. Through transcriptome analysis we identified 422 differentially expressed genes (DEGs) between the dsMmd2 group and control group, among which 337 genes were upregulated in the dsMmd2 group, including numerous muscle-related genes and protein synthesis genes. Further bioinformatics analysis showed that growth, metabolism, and immune-related signal pathway had changed significantly. The above results greatly increase our understanding on the conservative structure and function of LvMmd2 gene, and provide potential application prospects in genetics and breeding.

Sesquiterpenoid pathway in the mandibular organ of Penaeus monodon: Cloning, expression, characterization of PmJHAMT and its alteration response to eyestalk ablation

Methyl farnesoate (MF), a crustacean equivalent of juvenile hormone (JH) of insects, is known to be produced from the mandibular organ (MO). This study reports transcriptome analysis of Penaeus monodon MO and identifies putative genes encoding enzymes in the sesquiterpenoid pathway. A total of 44,490,420 clean reads were obtained and utilized for subsequent analysis. De novo assembly created 31,201 transcripts and 31,167 unigenes. To archive the functional annotation, all unigenes were annotated with KOG, KEGG, and GO. Putative genes encoding enzymes and regulatory proteins involved in the sesquiterpenoid pathway were obtained from the MO transcriptome data based on the conserved domains and sequence homology. They included S-adenosylmethionine synthetase, farnesyl pyrophosphate synthase, short chain dependent dehydrogenase/reductase (SDR), NAD(P) + -dependent aldehyde dehydrogenase, S-adenosylmethionine-dependent methyltransferases or juvenile hormone acid-O-methyl transferase (JHAMT), farnesoic acid O-methyl transferase (FAMeT), juvenile hormone binding protein, cytochrome C/P-450 family 15 (CRYP15A1)/methylfarnesoate epoxidase (MFE), juvenile hormone epoxide hydrolase (JHEH), and juvenile hormone esterase (JHE). We first identified and characterized JHAMT orthologs inP. monodon(PmJHAMT). The complete cDNA sequence ofPmJHAMTconsisted of 1,221 nt encoded 271 amino acids with a conserved S-adenosyl methionine (SAM) binding domain. Phylogenetic analysis clusteredPmJHAMTinto the group JHAMT with the same clade of the crabPortunus trituberculausJHAMT. Moreover, the predicted three-dimensional structure of PmJHAMT showed remarkable similarity with the recent crystal structure ofthe Bombyx moriJHAMT homodimer. RT–PCR analysis revealed that PmJHAMT was exclusively expressed in MO and initially expressed at stage 3 postlarvae. In situ hybridization with a specific probe to PmJHAMT validated the specific expression of this gene in MO cells. Finally, we evaluated the regulation of MO by eyestalk inhibitory peptides. Diminishing MO inhibitory hormone through unilateral eyestalk ablation resulted in a significantly higher expression ofPmJHAMTin MO by quantitative PCR. This result indicated that the eyestalk inhibitory hormone inhibited MF synthesis byPmJHAMTgene suppression in the MO. This finding provides insight into the crustacean sesquiterpenoid pathway and improves our understanding of crustacean endocrinology.

RNA interference analysis of potential functions of cyclin A in the reproductive development of male oriental river prawns (Macrobrachium nipponense).

Cyclin A (CycA) plays essential roles in regulating multiple steps of the cell cycle, and it affects gonad development in mammals and invertebrates. Previous RNA interference (RNAi) analysis revealed that knocking-down the expression of CycA in female oriental river prawns (Macrobrachium nipponense) inhibited ovarian development. CycA was also predicted to have regulatory roles in reproductive development of male M. nipponense based on significant changes of Mn-CycA expression after eyestalk ablation. The goal of this study was to investigate the potential functions of CycA in the reproductive development of male M. nipponense using RNAi and histological observations. Quantitative real-time PCR analysis revealed that both single-side and double-side eyestalk ablation stimulated the expressions of Mn-CycA, and the expression was higher in prawns with double-side eyestalk ablation (p < 0.05). Mn-CycA expression was significantly higher in the testis and androgenic gland during the reproductive season than during the non-reproductive season (p < 0.05). In the RNAi analysis, Mn-CycA expression significantly decreased after prawns were injected with dsCycA, and the expression of insulin-like androgenic gland hormone (Mn-IAG) also decreased as Mn-CycA expression decreased. This result indicated that CycA positively regulated the expression of IAG in M. nipponense. Histological observations revealed that the number of sperm decreased dramatically to <5% of the total cells in the testis of the dsCycA-treated group compared to that of control group on day 14, indicating that knockdown of Mn-CycA expression inhibited testis development by affecting the expression of Mn-IAG in M. nipponense. These results highlighted the functions of CycA in male reproductive development of M. nipponense, which can be applied to future studies of male reproduction in other crustacean species.

Cloning and function of the cyclin‐dependent kinase 9 gene in ovarian development of the ridgetail white prawn, Exopalaemon carinicauda

To investigate the key genes affecting ovarian development in the ridgetail white prawn, Exopalaemon carinicauda, we cloned the full-length cDNA of the CDK9 gene and discussed its role in ovarian development. Our results show that the gene has an open reading frame of 1155 bp, encoded 383 amino acids, and it has a conserved cyclin binding motif S_TKc. The open reading frame of CDK9 was expressed as a 43.96 Kda molecular weight protein. The phylogenetic tree analysis showed that the CDK9 gene has the highest homology with the CDK9 genes of Penaeus vannamei and Penaeus japonicus. The results of qRT-PCR show that the CDK9 gene was also expressed in the eye stalk, stomach, heart, gill, intestine, muscle, ovary, hepatopancreas and ventral cord. The expression level was highest in the ovary, which was significantly different from the other tissues (p < 0.05). In the five stages of ovarian development, the expression level of the CDK9 gene was highest in stage I, which was significantly different from the other stages (p < 0.05). In situ hybridization tests showed strong expression signals in the membrane, cytoplasm and nucleus of oocytes and previtellogenic oocytes. Nevertheless, following the proliferation of oocytes, the expression signals became much stronger in the oocyte membrane and in the whole follicular cells located in the interstitial space. The RNA interference results show that the expression of the CDK9 gene significantly affects the expression of the Vg and VgR genes. Our findings suggest that CDK9 plays an important role in the synthesis and transportation of vitellin.

Discovery of a hidden form of neuropeptide F and its presence throughout the CNS–gut axis in the mud crab, Scylla olivacea

The mud crab Scylla olivacea (Scyol) is an economically crucial crustacean species in Thailand, due to its high market demand and nutritional value. The neuropeptide F (NPF) has been implicated in the coordinated regulation of feeding and metabolism in invertebrates. While various isoforms of neuropeptide F (NPF) have been previously explored in the mud crab, some knowledge gaps in relation to the NPF family, and ambiguities in the nomenclature from previous reports, remain. In this study, NPF was firstly localized in the central nervous system and gastrointestinal tract of the mud crab, S. olivacea, using a polyclonal antibody against Macrobrachium rosenbergii, Macro-NPF. The NPF immunoreactivity (ir) was detected dominantly in the X-organ/sinus gland complex of the eyestalk (ES) and the various neuronal clusters (cluster 6, 9/11, and 14/15) and neuropils (anteromedial and posteromedial protocerebral neuropils, olfactory and accessory olfactory neuropils, and medial antennule neuropil, columnar neuropil) of the brain (BR), commissural ganglia, and suboesophageal ganglion of the ventral nerve cord (VNC). Interestingly, this study also presented the NPF immunoreactivity (NPF-ir) in the acinar gland-like cell and spindle-shaped epithelial cells of S. olivacea intestine. The full-length Scyol-NPFII was characterized by molecular cloning and revealed 414 nucleotides with 375 nucleotides of an open reading frame which encoded 124 deduced amino acids. A 124-amino acid precursor protein of Scyol-NPFII included a 26-residue signal peptide and a 69-amino acid mature peptide. The Scyol-NPFII showed the highest percentage of hit similarity to S. paramamosain-NPFII and clustered in the NPFII family, separated from the other forms of NPFs in this species. The spatial gene expression in various tissues revealed that Scyol-NPFII was found dominantly in the ES and BR, VNC, heart, intestine, and muscle. This study provided a novel form of NPF in the female mud crab, S. olivacea, which could open the possibility of its functioning in the brain-to-gut controlling axis. This study could provide essential information for further application in the cultured system of S. olivacea in the near future.