Molt-inhibiting Hormone Research Articles

Role of red pigment concentrating hormone in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain

The eyestalk ganglion is a predominant neuroendocrine organ in crustaceans that synthesize crustacean hyperglycemic hormone (CHH) family participating in physiological activities such as ovarian development, glucose homeostasis, and molting. Red pigment concentrating hormone (RPCH) contributes to a diversity of physiological influences, including ovarian development; however, its role in modulating CHH-family neuropeptides in the eyestalk ganglion remains unclear. In this study, we characterized the putative RPCH receptor (RPCHR) in the mud crab Scylla paramamosain and revealed that it was expressed in a variety of tissues, including the eyestalk ganglion. In vivo administration of mature RPCH peptide significantly restrained vitellogenesis inhibiting hormone (VIH) expression and significantly enhanced RPCHR, crustacean hyperglycemic hormone (CHH) and molt inhibiting hormone (MIH) gene expression, while in contrast, in vivo injection of RPCH-dsRNA for knockdown dramatically enhanced VIH expression and decreased RPCHR, CHH and MIH expression in the eyestalk ganglion. The present study comprehensively reports that RPCH may be responsible for the regulation of ovarian maturation, glucose homeostasis, and molting in S. paramamosain through the modulation of CHH family neuropeptides in the eyestalk ganglion.

Exploring the effects of dietary cholesterol on growth, immunity, and lipid metabolism of juvenile Chinese mitten crabs (Eriocheir sinensis)

This study aims to investigate the nutritional needs and physiological functions of dietary cholesterol in juvenile Chinese mitten crabs (Eriocheir sinensis). In the 8-week feeding experiment, we evaluated the effects of various cholesterol levels (0 control, 0.6, 1.3, 2.6, 5.9, 12.0 g/kg diet) on growth, immune capacity, lipid metabolism, molting, and cholesterol and bile acid metabolism. Crabs (1.06 ± 0.04 g) were divided into six groups with four replicates of 40 crabs per tank. The highest weight gain was found in crabs fed 5.9 g/kg cholesterol (P < 0.05), while the highest molting frequency was observed in the 0.6 g/kg group (P < 0.05). Cholesterol significantly changed fatty acid composition in the hepatopancreas, and 5.9 g/kg cholesterol increased immune enzyme activities (acid phosphatase, alkaline phosphatase, and polyphenol oxidase) (P < 0.05). A 5.9 g/kg cholesterol significantly upregulated lipogenesis- and catabolism-related genes, while higher levels of cholesterol (12.0 g/kg) downregulated lipogenesis-related genes significantly (P < 0.05). Low cholesterol levels upregulated chitinase expression, while ecdysone receptor and molt-inhibiting hormone expressions were upregulated with cholesterol levels (P < 0.05). Cholesterol also upregulated the gene in bile acid metabolism. The total bile acids, the proportion of primary bile acids, and the content of primary bile acids increased significantly compared to the control. Based on quadratic regression model analysis, the optimal dietary cholesterol level for juvenile E. sinensis is 5.74–5.77 g/kg. The study suggests that cholesterol promotes growth and immunity in juvenile E. sinensis by regulating molting frequency, improving lipid and HUFA utilization, and increasing bile acids.

Anatomical and molecular insights into the antennal gland of the giant freshwater prawn Macrobrachium rosenbergii

In this study, the complex organization of the AnG in the giant freshwater prawn Macrobrachium rosenbergii was revealed using various techniques, including conventional histology, histochemistry, scanning electron microscopy, and X-ray tomography. The results showed the diversity of cells in the AnG and the detailed organization of the labyrinth’s tubule into four radiated areas from the central to peripheral zones. The study also demonstrated the expression of some vertebrate kidney-associated homolog genes, aquaporin (AQP), solute carrier family 22 (SLC-22), nephrin, and uromodulin, in the AnG by qPCR. The result of in situ hybridization further showed the localization of SLC-22 and AQP transcript in the bladder and labyrinth’s epithelium, specifically in regions 2, 3, and 4. Additionally, the study revealed neuropeptide expressions in the AnG by qPCR and in situ hybridization, i.e., crustacean hyperglycemic hormone (CHH) and molt inhibiting hormone (MIH), implying that the AnG may have a role in hormone production. Moreover, male and female prawns exhibited different levels of AQP, SLC-22, nephrin, and CHH expressions during the premolt and intermolt stages, suggesting a crucial role relevant to the molting stages. In conclusion, this study clarified the complex structure of the AnG in M. rosenbergii and demonstrated for the first time the expression of vertebrate kidney-associated genes and the possible endocrine role of the AnG. Further investigation is needed to clarify the role of these genes, particularly during ecdysis. The implications of these findings could significantly advance our understanding of the AnG in decapod crustaceans.

The interaction between 20-hydroxyecdysone and AMPK through PI3K activation in Chinese mitten crab, Eriocheir sinensis

In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKβ and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKβ induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.

Effects of astaxanthin in diet on antioxidant capacity and molting-related genes expression of larvae giant freshwater prawn (Macrobrachium rosenbergii)

This experiment was carried out to determine the effects of astaxanthin on reactive oxide species (ROS), antioxidant capacity as well as molting-related genes expression in larvae of giant freshwater prawn, Macrobrachium rosenbergii. A total of 90,000 larvae were assigned to five treatments with six replicates each. Astaxanthin mixed in steamed eggs with graded effective content as 0 (control, A0), 40 (A40), 80 (A80), 160 (A160) and 320 (A320) mg kg−1 were fed to larvae for 14 days. The results showed that ROS of larvae in astaxanthin treatments was significantly lower than the control (P < 0.05). T-AOC, T-SOD and GR activities under astaxanthin treatments generally showed a downward trend and decreased significantly than the control (P < 0.05). No significant difference was observed towards CAT activities among A40 and A80, but the values were markedly greater than A0, A160, A320 (P < 0.05). Larvae in A80 exhibited stronger MDA content than the other (P < 0.05). GSH-Px activity was significantly highest in A320 than the other treatments (P < 0.05). The expression of molt-inhibiting hormone (MIH) in A40 and A80 enhanced markedly than the control (P < 0.05). The elevated expression of Ecdysone receptor (EcR) was recorded only in A160 and A320 (P < 0.05). Chitinase (CHIT) expression among treatments was significantly downregulated than the control except for A40. Heat shock protein (HSP21) and chitooligosaccharidolytic beta-N-acetylglucosaminidase-like expression increased dramatically in A160 and A320 (P < 0.05). For actin 6 gene, higher expression was happened only in A40 compared to control, others presented drop expression in very low level (P < 0.05). This study provides a theoretical basis that 320 mg/kg astaxanthin was excessive level, and might decreased partial antioxidant indexes and influenced molting genes expression of larvae M. rosenbergii.

In silico analysis of crustacean hyperglycemic hormone family G protein-coupled receptor candidates.

Ecdysteroid molting hormone synthesis is directed by a pair of molting glands or Y-organs (YOs), and this synthesis is inhibited by molt-inhibiting hormone (MIH). MIH is a member of the crustacean hyperglycemic hormone (CHH) neuropeptide superfamily, which includes CHH and insect ion transport peptide (ITP). It is hypothesized that the MIH receptor is a Class A (Rhodopsin-like) G protein-coupled receptor (GPCR). The YO of the blackback land crab, Gecarcinus lateralis, expresses 49 Class A GPCRs, three of which (Gl-CHHR-A9, -A10, and -A12) were provisionally assigned as CHH-like receptors. CrusTome, a transcriptome database assembled from 189 crustaceans and 12 ecdysozoan outgroups, was used to deorphanize candidate MIH/CHH GPCRs, relying on sequence homology to three functionally characterized ITP receptors (BNGR-A2, BNGR-A24, and BNGR-A34) in the silk moth, Bombyx mori. Phylogenetic analysis and multiple sequence alignments across major taxonomic groups revealed extensive expansion and diversification of crustacean A2, A24, and A34 receptors, designated CHH Family Receptor Candidates (CFRCs). The A2 clade was divided into three subclades; A24 clade was divided into five subclades; and A34 was divided into six subclades. The subclades were distinguished by conserved motifs in extracellular loop (ECL) 2 and ECL3 in the ligand-binding region. Eleven of the 14 subclades occurred in decapod crustaceans. In G. lateralis, seven CFRC sequences, designated Gl-CFRC-A2α1, -A24α, -A24β1, -A24β2, -A34α2, -A34β1, and -A34β2, were identified; the three A34 sequences corresponded to Gl-GPCR-A12, -A9, and A10, respectively. ECL2 in all the CFRC sequences had a two-stranded β-sheet structure similar to human Class A GPCRs, whereas the ECL2 of decapod CFRC-A34β1/β2 had an additional two-stranded β-sheet. We hypothesize that this second β-sheet on ECL2 plays a role in MIH/CHH binding and activation, which will be investigated further with functional assays.

Antidepressants – The new endocrine disruptors? The case of crustaceans

Antidepressants are high-volume pharmaceuticals that accumulate to concentrations in the μg·L−1 range in surface waters. The release of peptide hormones via neurosecretory cells appears as a natural target for antidepressants. Here I review research that suggests that antidepressants indeed disrupt endocrine signalling in crustaceans, by acting on the synthesis and release of neurohormones, such as crustacean hyperglycaemic hormone, moult inhibiting hormone and pigment dispersing hormone in decapods, as well as methyl farnesoate in Daphnids. Hence, antidepressants can affect hormonal regulation of physiological functions: increase in energy metabolism and activity, lowered ecdysteroid levels, potentially disrupting moult and somatic growth, reducing colour change capacity and compromising camouflage, as well as induction of male sex determination. Several studies further suggest effects of antidepressants on crustacean reproduction, but the hormonal regulation of these effects remains elusive. All things considered, a body of evidence strongly suggests that antidepressants are endocrine disrupting compounds in crustaceans.

Activation and characterization of G protein-coupled receptors for CHHs in the mud crab, Scylla paramamosain

Crustacean hyperglycemic hormone (CHH) superfamily peptides constitute a group of neurohormones, including the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), which reportedly play an essential role in regulating various biological activities by binding to their receptors in crustaceans. Although bioinformatics analyses have identified G protein-coupled receptors (GPCRs) as potential CHH receptors, no validation through binding experiments has been carried out. This study employed a eukaryotic expression system, HEK293T cell transient transfection, and ligand-receptor interaction tests to identify the GPCRs of CHHs in the mud crab Scylla paramamosain. We found that four GPCRs (Sp-GPCR-A34-A37) were activated by their corresponding CHHs (Sp-CHH1-v1, Sp-MIH, Sp-VIH) in a dose-dependent manner. Of these, Sp-GPCR-A34 was exclusively activated by Sp-VIH; Sp-GPCR-A35 was activated by Sp-CHH1-v1 and Sp-VIH, respectively; Sp-GPCR-A36 was activated by Sp-CHH1-v1 and Sp-MIH; Sp-GPCR-A37 was exclusively activated by Sp-MIH. The half-maximal effective concentration (EC50) values for all CHHs/GPCRs pairs (both Ca2+ and cAMP signaling) were in the nanomolar range. Overall, our study provided hitherto undocumented evidence of the presence of G protein-coupled receptors of CHH in crustaceans, providing the foothold for further studies on the signaling pathways of CHHs and their corresponding GPCRs.

The effect of dietary vitamin A supplementation on growth, gonadal development, molting, immunity, and ammonia nitrogen resistance in sub-adult male Chinese mitten crab, Eriocheir sinensis

Vitamin A (VA) is closely related to male gonadal development; however, whether it can promote the speed of gonadal maturity in male Chinese mitten crab, Eriocheir sinensis, is unknown. This study investigated the effects of dietary VA on growth, gonadal development, molting, immunity, and ammonia nitrogen resistance in sub-adult male E. sinensis to determine the appropriate dietary VA supplementation level. A total of 280 crabs (61.22 ± 0.45 g) were divided into seven experimental groups with four replicates (n = 10 per treatment replicate). Crabs in the experimental treatments were fed a basal diet (1846 IU/kg VA; 38.11% crude protein, 7.65% crude lipid) supplemented with 0, 10,000, 20,000, 40,000, 80,000, 160,000, or 320,000 IU/kg VA for 8 weeks. Subsequently, a 48-h ammonia nitrogen stress test was conducted on a subset of crabs (total 105 crabs, seven treatments with three replicates, n = 5 per treatment replicate). Orthogonal polynomial contrasts were used to determine the associations between VA levels and outcome measures. In the feeding trial, dietary VA supplementation was significantly negatively associated with final body weight and significantly positively associated with feed conversion ratio (both P < 0.01 for linear model). Final total weight of crabs in a rearing unit, hepatopancreas index, and survival rate (SR) was significantly linearly and quadratically associated with VA supplementation, with the highest levels in crabs fed 10,000–20,000 IU/kg VA-supplemented diets (both P < 0.05). VA supplementation was significantly associated with gonadosomatic index (whole gonad weight / body weight) and testis index (testis weight / body weight; all P < 0.01 for linear and quadratic models), with crabs fed a 20,000 IU/kg dietary VA-supplemented diet having the highest levels that were 1.19-fold and 1.27-fold higher than those supplemented with 0 IU/kg VA, respectively; mRNA expression of genes related to testis development [DNA meiotic recombinase 1, nanos, and cullin 4] were in alignment with this pattern. Molting frequency was also highest in crabs receiving 20,000 IU/kg dietary VA (2.00 ± 0.08) among all experimental diets, and lowest in those receiving the 320,000 IU/kg treatment (1.75 ± 0.13; linear and quadratic models, both P < 0.01); mRNA expression of molting-related genes [crustacean hyperglycemic hormone, molt-inhibiting hormone, ecdysone receptor, and retinoid X receptor] exhibited analogous patterns. Evaluation of immunity-related biochemical indicators [lysozyme, acid phosphatase, and alkaline phosphatase] and gene expressions [toll-like receptor 2, relish, myeloid differentiation factor 88, crustin, anti-lipopolysaccharide factor, and prophenoloxidase] indicated that the 20,000 IU/kg VA treatment conferred the highest nonspecific immunity (quadratic associations, all P < 0.05). In the acute ammonia nitrogen stress test, crabs fed a 20,000 IU/kg VA-supplemented diet for 8 weeks had the highest SR of 60%, while no crab survived in VA320000 treatment (linear and quadratic models, both P < 0.001). Antioxidant-related biochemical indicators [superoxide dismutase (sod), glutathione peroxidase (gpx), malondialdehyde, and total antioxidant capacity] and gene expressions [sod, gpx, thioredoxin 1, and peroxiredoxin 6] were also significantly quadratically associated with VA levels (all P < 0.01). In conclusion, 20,000 IU/kg dietary VA supplementation proved advantageous for growth, gonadal development, molting, immunity, and antioxidant capacity in sub-adult male E. sinensis; however, dietary VA supplementation levels over 80,000 IU/kg appeared to be detrimental to crabs.

Effects of Different Sources of Culture Substrate on the Growth and Immune Performance of the Red Swamp Crayfish (Procambarus clarkii).

The substrate in the aquatic environment plays a crucial role in nutrient deposition and recovery for the growth of aquatic organisms. In order to optimize the culture medium of Procambarus Clarkii, culture media from different sources were selected in this study to explore their effects on the growth and immune performance of red swamp crayfish. The results showed that the weight gain rate (WGR), body length growth rate (BLGR) and specific growth rate (SGR) in group I2 were the highest, followed by group I1 and group I3. The WGR and SGR of crayfish in the I1 and I2 groups were significantly higher than those in the I3 group (p < 0.05). The activities of acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) were the highest in group I2, followed by group I3, and the lowest in group I1. The expression trends in growth-related genes, nuclear hormone receptor (E75), molt-inhibiting hormone (MIH) and chitinase genes were similar, and the expression levels in the I2 group were higher than those in the I1 and I3 groups. It was noted that the expression levels of E75 and MIH genes in the I2 group were significantly higher than those in the I3 group (p < 0.05). α diversity analysis of 16S rRNA data showed that there was no statistically significant difference in the abundance of intestinal flora among the three culture substrate groups. The β diversity in the Xitangni group, crayfish Tangni group and Shuitangni group was significantly different. These changes in microbiota suggest that using different substrates to culture crayfish leads to differences in gut microbiota diversity. To sum up, the growth in crayfish and immune performance influenced by the culture substrate condition and aquatic breeding sediment substrates, rather than crab pool and paddy field pond sediment substrates, showed a better effect.

Cottonseed Meal Protein Hydrolysate Improves the Growth Performance of Chinese Mitten Crab (Eriocheir sinensis) by Promoting the Muscle Growth and Molting Performance.

Growth retardation and prolonged marketing cycle have been noticed in the practical aquaculture of Chinese mitten crab (Eriocheir sinensis) fed with artificial feed. Plant protein hydrolysates contain a large number of small peptides and free amino acids, which can improve the growth performance of aquatic animals. However, the potential mechanisms are still not well elucidated. In this research, the influences of cottonseed meal protein hydrolysate (CPH) on the growth, feed utilization, muscle growth, and molting performance were investigated in E. sinensis. A total of 240 crabs (mean body weight 37.32 ± 0.38 g) were individually randomly distributed to six diets supplemented with 0%, 0.2%, 0.4%, 0.8%, 1.6%, and 3.2% of CPH for 12 weeks. These findings indicated that the addition of CPH at 0.4% significantly increased the survival rate, body protein gain, apparent protein utilization, trypsin and pepsin activities, and the methyl farnesoate content. When the dose reached 0.8%, the weight growth ratio, meat yield, ecdysone concentration, and the transcription of the ecdysteroid receptor all significantly increased, while the transcriptions of both myostatin and molt-inhibiting hormone significantly decreased. When CPH was added at 1.6%-3.2%, the feed conversion ratio, body crude protein content, Na+/K+-ATPase activity, and the molting ratio were all significantly improved, while the opposite was true for the transcription of the transforming growth factor-β type I receptor. The investigation results indicated that when added above 0.4%, CPH could stimulate the growth performance of E. sinensis and promote the muscle growth and molting performance.

FOXO-like Gene Is Involved in the Regulation of 20E Pathway through mTOR in Eriocheir sinensis

The Forkhead Box O (FOXO) gene plays a key role in various biological processes, such as growth, metabolism, development, immunity and longevity. Molting is an essential process for crustacean growth, which is mainly regulated by 20-hydroxyecdysone (20E) and molt-inhibiting hormone (MIH). Although the role of FOXO in regulating the immune response of crustaceans is well documented, its involvement in controlling crustacean molting remains unclear. In this study, a FOXO-like gene (designed as EsFOXO-like) was identified in Eriocheir sinensis, and the regulation of the 20E pathway by EsFOXO-like was also investigated. The coding sequence of EsFOXO-like was 852 bp, which consisted of 283 amino acids including a conserved Forkhead (FH) domain. EsFOXO-like shared high similarity with FOXO genes from other crustaceans, and the mRNA expression levels of the EsFOXO-like gene were highest in the hepatopancreas and lowest in the hemocytes. However, transcription and protein expression of the EsFOXO-like gene were found to be up-regulated only during the pre-molt stage in the hepatopancreas, with lower expression levels observed at the post-molt stage. To explore the role of EsFOXO-like in the 20E pathway, EsFOXO-like was firstly inhibited by a specific FOXO inhibitor (AS1842856) and then through an EsFOXO-like dsRNA injection, respectively, and the results showed that the relative expression levels of EsFOXO-like were notably decreased in the hepatopancreas after both the inhibitor and dsRNA treatments. The 20E concentration, the mRNA expression levels of the 20E receptors including the ecdysone receptor (EcR) and the retinoid-X receptor (RXR) and EsmTOR transcription in the AS1842856 group or the EsFOXO-RNAi group were all significantly higher than that in the control group, while the mRNA expression level of EsMIH was significantly decreased after EsFOXO-like inhibition. To further investigate whether the EsFOXO-like acts through mTOR or not, Rapamycin was administered to inhibit mTOR activity in EsFOXO-like inhibited crabs. The results revealed a significant reduction in the concentration of 20E and the expression level of EsMIH in the AS1842856 + Rapamycin group compared to the AS1842856 + DMSO group, accompanied by an increase in EsEcR and EsRXR expression. These findings collectively suggest that EsFOXO-like regulates the 20E pathway through mTOR, which offered valuable insights into the understanding of the molting process in crustaceans.

Identification and profiling of long non-coding RNAs during molt cycle: An involvement of lnc1182 in the molt of white shrimp, Litopenaeus vannamei

Molt is an essential physiological process for crustacean growth. It is known to be controlled by factors such as hormones and steroids, but the role of long non-coding RNA (lncRNA), a potential regulatory molecule, has not been studied. In this study, we investigated the role of lncRNA in the molt process of white shrimp, Litopenaeus vannamei. The 9437 of 58,142 unigenes of de novo assembled transcriptome were identified as putative lncRNAs, and 768 lncRNAs were differentially expressed during molt cycles. Five differentially expressed lncRNAs were selected to determine their expressions in different tissues and molt stages. Most were expressed more in gills and epidermis than other tissues. Expressions of lnc1182 and lnc336 significantly decreased during premolt stages and increased at postmolt in gills and epidermis, while lnc179 expression increased during premolt stages. RNA interference was used to investigate the role of lnc1182 in the molt process. The molt of lnc1182 knockdown shrimp progressed to early premolt faster than shrimp injected with dsGFP, and also shortened molt duration. In addition, total sugar level in hemolymph was higher in the lnc1182 knockdown shrimp than the control shrimp, whereas gill Na+/K+ ATPase activity, total hemocyte count, and expression of molt-inhibiting hormone (MIH) were not changed. These suggested that lnc1182 is likely involved in the control of sugar metabolism during molting but not in ion exchange activity and MIH expression. Furthermore, lnc1182 and 20-HE probably regulated each other. RNA sequencing was used to investigate the set of genes associated with lnc1182. Fifty-five transcripts were differentially expressed in gills of lnc1182 knockdown shrimp. Expressions of candidate metabolism and immune-related genes were validated for their differential expressions in lnc1182 knockdown shrimp. Our work produced lncRNA profiles and indicated possible functions of lnc1182 in metabolism and immunity during the molt cycle in shrimp.

Sex-specific effects of propiconazole on the molting of the Chinese mitten crab (Eriocheir sinensis)

Given the inevitable exposure of Eriocheir sinensis (E. sinensis) to fungicides in rice-crab co-culture systems, understanding the potential effect of fungisides is important for practical application. Molting is a crucial development process of E. sinensis, which is regulated by endocrine system and genetic factors, and is susceptible to exogenous chemicals. However, the impact of fungicides application on the molting of E. sinensis have been rarely reported. In the present study, propiconazole, a widely used fungicide for rice disease management, was found to exert potential effects on the molting of E. sinensis at residual-related level in the rice-crab co-culture fields. After 14 days of short-term exposure to propiconazole, female crabs exhibited remarkably higher levels of hemolymph ecdysone than males. When the exposure was extended to 28 days, propiconazole markedly accelerated molt-inhibiting hormone expression by 3.3-fold, ecdysone receptor expression by 7.8-fold, and crustacean retinoid X receptor expression by 9.6-fold in male crabs, while it showed the opposite effect in females with suppressed gene expression. Propiconazole also induced the activity of N-acetylglucosaminidase in male crabs rather than females during the experiments. Our study suggests that propiconazole exerts sex-specific effects on the molting of E. sinensis. The impact of propiconazole application in the rice-crab co-culture systems remains more assessment to avoid affecting the growth of cultured E. sinensis.

Regulation of CHH family in eyestalk by A-type allatostatin in the mud crab Scylla paramamosain

The eyestalk ganglion is a principal neuroendocrine organ in crustaceans for the synthesis, storage and release of the crustacean hyperglycemic hormone neuropeptide (CHH) family. A-type allatostatin (AST-A) is involved in a variety of physiological functions, although its function in regulating the CHH family in the eyestalk ganglion has not been reported. The present study identified AST-A in the mud crab Scylla paramamosain. AST-A was highly expressed in the ganglia as shown by reverse transcription PCR, while AST-A receptor (AST-AR) was expressed in multiple tissues, such as the eyestalk ganglion. Both in vitro and in vivo experiments revealed that AST-A mature peptide significantly increased mRNA expression of AST-AR, vitellogenesis-inhibiting hormone (VIH), molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH). In contrast, RNA interference by injection of AST-AR-dsRNA into crabs significantly reduced the expressions of VIH, MIH, and CHH in the eyestalk ganglion. This study reports for the first time that AST-A might play roles in ovarian development, molting and glucose metabolism in S. paramamosain via regulating CHH family neuropeptides in the eyestalk ganglion.

A first glimpse into the m6A modification machinery of shrimp: Genomic features, expression patterns and potential roles in molting regulation

N6-methyladenosine (m6A) is the most abundant type of RNA modification in eukaryotes, yet information on the m6A methylation enzymatic machinery of decapod crustaceans is quite scarce. Here, three essential genes for m6A modification, including two m6A methyltransferases (LvMETTL3 and LvWTAP) and a m6A-binding protein (LvYTHDF2), were identified from the Pacific whiteleg shrimp Litopenaeus vannamei. Their genomic structures were dissected in an evolutionary context. Common transcription factor binding sites and conserved functional motifs were identified, but the three m6A modification genes also possessed some unique features, illustrating lineage-specific diversification of regulatory elements and functions of these genes. The three genes displayed ubiquitous tissue expression profiles, and showed dynamic expression changes at different molting stages. Following suppression of LvMETTL3, LvWTAP or LvYTHDF2 by RNA interference, the molting rate of shrimp was significantly accelerated, and expression of the molt inhibiting hormone MIH plummeted, while major genes in the ecdysone signaling pathway including EcR, RXR and E75 all showed significant induction. The results indicated that m6A modification may regulate shrimp molting process via MIH and the ecdysone signaling pathway. The current study provided the first molecular characterization of several key components of the m6A enzymatic machinery in penaeid shrimp, and established a foundation for further exploration of the roles of m6A modification in shrimp development and growth.

The Influence of Photodynamic Antimicrobial Chemotherapy on the Microbiome, Neuroendocrine and Immune System of Crustacean Post Larvae

Photodynamic antimicrobial chemotherapy (PACT), employing a combination of light and natural photosensitizer molecules such as curcumin, has been accepted as a safe modality for removing aquatic pathogens which cause diseases such as cholera in humans and vibriosis in aquatic animals. Curcumin and its photodegradation products are generally considered as safe to animals, but the impact of reactive oxygen species (ROS) generated by these products on the growth and survival of organisms at a cellular level has not been studied in detail. The ROS generated by curcumin on photoexcitation using blue light (λmax 405 nm, 10 mW cm-2) disinfects more than 80% of free-living Vibrio spp. in the rearing water of Penaeus monodon. However, it is less effective against Vibrio spp. colonized inside P. monodon because the carapace of the animal prevents the transmission of more than 70% of light at the 400-450 nm range and thus reduces the formation of ROS. The influence of curcumin and photoexcited curcumin on the microbiome of P. monodon were revealed by nanopore sequencing. The photoexcited curcumin induced irregular expression of genes coding the moult-inhibiting hormone (MIH), Crustacean hyperglycaemic hormone (CHH)), prophenoloxidase (ProPO), and crustin, which indicates toxic effects of ROS generated by photoexcited curcumin on the neuroendocrine and immune systems of crustaceans, which could alter their growth and survival in aquaculture settings. The study proposed the cautious use of photodynamic therapy in aquaculture systems, and care must be taken to avoid photoexcitation when animals are experiencing moulting or environmental stress.

Effects of triazole plant growth regulators on molting mechanism in Chinese mitten crab (Eriocheir sinensis)

Rice crab co-culture is a new integrated farming model in China. The application of triazole plant growth regulators (PRGs) is often used as an advantageous option to combat rice lodging. However, there is still a gap regarding the toxicity of these PRGs on the growth and development of the Chinese mitten crab (Eriocheir sinensis, E. sinensis). Here the effect of triazoles (paclobutrazol and uniconazole) on the molting mechanism of E. sinensis was investigated. Monitoring of regulatory genes associated with molting showed that the two PRGs were found to inhibit the expression of ecdysteroid hormone (EH), ecdysteroid receptors gene (EcR), and retinoid X receptors gene (RXR) and induce secretion of molt-inhibiting hormone (MIH) gene. In addition, the activities of chitinase (CHIA) and N-acetyl-β-d-aminoglucosidase (β-NAGase) were also inhibited by exposure to PRGs. Exposure to PRGs also elevated the mRNA expression of the growth-related myostatin gene (MSTN). These results revealed that there is a long-term risk of exposure to triazoles PRGs that may inhibit molting and affect normal development and immune system of E. sinensis.

CasEcR and CasMIH Genes in the Blue Crab, Callinectes sapidus: A Temporal Evaluation and Melatonin Effects

Environmental cues synchronize endogenous rhythms of many physiological processes such as hormone synthesis and secretion. Little is known about the diurnal pattern of hormones and gene expression of the Callinectes sapidus molt cycle. We aimed to investigate in the eyestalk and hepatopancreas of premolt and intermolt C. sapidus the following parameters: 1) the diurnal expression of the ecdysteroid receptor CasEcR isoforms, and the molt inhibiting hormone CasMIH; 2) the diurnal hemolymph ecdysteroid and melatonin levels; and 3) melatonin effects on the transcripts of the above-mentioned genes in intermolt C. sapidus. Ecdysteroid levels were higher in the premolt than the intermolt animals at all time points evaluated (ZTs). Premolt crabs displayed a variation of ecdysteroid concentration between time points, with a reduction at ZT17. No difference in the melatonin level was seen in either molt stage or between stages. In the eyestalk of intermolt animals, CasEcR expression oscillated, with a peak at ZT9, and premolt crabs have a reduction at ZT9; CasMIH transcripts did not vary along 24 h in either molt stage. Moreover, the evaluated eyestalk genes were more expressed at ZT9 in the intermolt than the premolt crabs. In the hepatopancreas, CasEcR expression showed a peak at ZT9 in premolt crabs. Exogenous melatonin (10−7 mol/animal) reduced the expression of both genes in the eyestalk at ZT17. In the hepatopancreas, melatonin markedly increased the expression of the CasEcR gene at ZT9. Taken altogether, our results are pioneer in demonstrating the daily oscillation of gene expression associated to molt cycle stages, as well as the daily ecdysteroid and melatonin levels and the remarkable influence of melatonin on the molt cycle of C. sapidus.

Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain

The eyestalk ganglion serves as an essential endocrine organ in Crustacea. Although the short neuropeptide F (sNPF) is involved in a broad range of physiological functions via its counterpart receptor (sNPFR), its roles in regulating the crustacean hyperglycemic hormone (CHH) family in the eyestalk ganglion is still unclear. In this study, reverse transcription PCR indicates that the sNPF was most highly expressed in the eyestalk ganglion, whereas sNPFR was expressed in 11 tissues of the mud crab Scylla paramamosain, including eyestalk ganglion. Immunohistochemistry analyses further display that sNPF immunoreactive neurons were localized in the X-organ of eyestalk ganglion. Moreover, the stimulatory effect of sNPF on the gene expression of vitellogenesis-inhibiting hormone (VIH), molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH) in the eyestalk ganglion was confirmed by in vivo experiment. In contrast, RNA interference by injection of sNPF dsRNA or sNPFR dsRNA into crabs significantly reduced VIH, MIH, and CHH expressions in the eyestalk ganglion, respectively. This study first reported that sNPF might play roles in the reproduction, molting and glucose metabolism in S. paramamosain via regulating CHH family neuropeptides in the eyestalk ganglion.