Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: behavioral evaluation

Eyestalk ablation allows the removal of neurohormones that inhibit early reproduction and decreases the time to complete the molting cycle. The present study evaluated the impact of unilateral eyestalk ablation on the behavior of blue crab breeders Callinectes arcuatus in the short term under laboratory conditions. Immature male and female crabs and individuals in the intermolt stage were collected in Ohuira Bay, Ahome, Sinaloa, Mexico. Three bioassays were performed: males, females, and male-females each with three treatments; control + xylocaine (50 μL, 5%), squash ablation + xylocaine (50 μL, 5%), ablation by string ligation + xylocaine (50 μL, 5%), each with three replicas and three periods of evaluation after the ablation procedure (10 min, 2 h, and 24 h). Kruskal-Wallis and Chi-square (χ2) tests were used to evaluate significances (P < 0.05) in the behavior of C. arcuatus. The sizes of blue crab C. arcuatus were 9.72 ± 1.0 cm of carapace width. The feeding behavior of the male-females bioassay was significant at 2 h (P < 0.05). The results obtained were satisfactory using two techniques, unilateral eyestalk ablation by squash and ligation in which pain and aggressiveness indicators were mitigated. Disorientation and aggressiveness in males were significant at 2 h (P < 0.05), and in females after 10 min, and 2 h after ablation (P < 0.05). In the male-females bioassay, aggressiveness was significant at 2 h (P < 0.05). Courtship and copulation were recorded in those crabs subjected to unilateral eyestalk squash ablation (13 and 8, respectively).

Transcriptome Analysis Reveals the Endocrine Regulation on the Expression of IAG in Litopenaeus vannamei

The insulin-like androgenic gland hormone (IAG) plays a key role in male sexual differentiation and spermatogenesis in crustaceans. The expression of IAG is usually negatively regulated by neuropeptide hormones through the “eyestalk-AG” endocrine axis. However, the underlying mechanism is still largely unknown. In the present study, we performed a comparative transcriptome analysis on the androgenic gland (AG), the main secretory organ of IAG, of L. vannamei before and after unilateral eyestalk ablation. A total of 67 differentially expressed genes (DEGs) were identified, including some putative genes involved in sexual development. Interestingly, several genes related to molting and endocrine processes were found differentially expressed between the two treatments. Further investigation on the expression profiles of these genes and Lv-IAG showed that their expression patterns were closely correlated throughout the molting cycle. In addition, injection of 20-hydroxyecdysone (20E) could inhibit the expression of Lv-IAG at different molting stages in a dosage-dependent manner. The data indicated that ecdysteroids played important roles in regulating the Lv-IAG expression and might bridge the endocrine axis between eyestalk and AG. The present study provided new insights into understanding the regulation of sexual development in male crustacean.

Distinct gene expression dynamics in developing and regenerating limbs

Regenerating animals have the ability to reproduce organs that were originally generated in the embryo and subsequently lost due to injury. Understanding whether the process of regeneration mirrors development is an open question in most regenerative species. Here we take a transcriptomics approach to examine to what extent leg regeneration shows the same temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, global patterns of gene expression during leg regeneration show a high degree of variation, related to the physiology of individual animals. A major driver of this variation is the molting cycle. After dissecting the transcriptional signals of individual physiology from regeneration, we obtain temporal signals that mark distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although both processes use largely the same genes, the temporal patterns in which these gene sets are deployed are different and cannot be systematically aligned.

The FTZ-F1 gene encodes two functionally distinct nuclear receptor isoforms in the ectoparasitic copepod salmon louse (Lepeophtheirus salmonis)

The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic crustacean that annually inflicts substantial losses to the aquaculture industry in the northern hemisphere and poses a threat to the wild populations of salmonids. The salmon louse life cycle consists of eight developmental stages each separated by a molt. Fushi Tarazu Factor-1 (FTZ-F1) is an ecdysteroid-regulated gene that encodes a member of the NR5A family of nuclear receptors that is shown to play a crucial regulatory role in molting in insects and nematodes. Characterization of an FTZ-F1 orthologue in the salmon louse gave two isoforms named αFTZ-F1 and βFTZ-F1, which are identical except for the presence of a unique N-terminal domain (A/B domain). A comparison suggest conservation of the FTZ-F1 gene structure among ecdysozoans, with the exception of nematodes, to produce isoforms with unique N-terminal domains through alternative transcription start and splicing. The two isoforms of the salmon louse FTZ-F1 were expressed in different amounts in the same tissues and showed a distinct cyclical expression pattern through the molting cycle with βFTZ-F1 being the highest expressed isoform. While RNA interference knockdown of βFTZ-F1 in nauplius larvae and in pre-adult males lead to molting arrest, knockdown of βFTZ-F1 in pre-adult II female lice caused disruption of oocyte maturation at the vitellogenic stage. No apparent phenotype could be observed in αFTZ-F1 knockdown larvae, or in their development to adults, and no genes were found to be differentially expressed in the nauplii larvae following αFTZ-F1 knockdown. βFTZ-F1 knockdown in nauplii larvae caused both down and upregulation of genes associated with proteolysis and chitin binding and affected a large number of genes which are in normal salmon louse development expressed in a cyclical pattern. This is the first description of FTZ-F1 gene function in copepod crustaceans and provides a foundation to expand the understanding of the molecular mechanisms of molting in the salmon louse and other copepods.

Thermal Tolerance and Physiological Changes in Mud Crab, Scylla paramamosain Crablet at Different Water Temperatures

This study was carried out to determine the physiological changes (survival, growth, molting cycle, sex differentiation, and gill condition) of mud crab, Scylla paramamosain crablet at different water temperatures of 24, 28 and 32 °C, and ambient temperature of 27 to 30 °C. Thermoregulatory behavior, represented by preferred temperature (29.83 ± SD 2.47 °C), critical thermal minimum (17.33 ± SD 0.58 °C), critical thermal maximum (40 ± SD 0.00 °C), and thermal tolerance interval (22.67 ± SD 0.58 °C), were checked for Crablet 1 stage only (with ambient temperature as acclimation temperature).Both low (24 °C) and high (32 °C) temperatures were associated with lower growth performance, and survival rate (p < 0.05), in comparison with both 28 °C and ambient temperature treatments.Male ratio at low temperaturetreatment (24 °C) was higher (80.09 ± SD 18.86%) than for other treatments (p < 0.05), observed as 44.81 ± D 10.50%, 41.94 ± SD 19.44%, and 76.30 ± SD 5.13% for 28 °C, 32 °C and ambient temperature treatments, respectively. However, there was no significant difference observed between 24 °C, 28 °C, and ambient temperature treatments. Anatomical alterations of gill lamellae of S. paramamosain crablet for both 32 °C, and 24 °C treatments, appeared thinner and paler than at both 28 °C, and ambient temperature treatments. Based on this study, temperature of 28 to 30 °C was recommended as the optimal temperature for the long-term nursery phase of S. paramamosain.

Transcriptome Analysis of The Whole Body Provides Novel Insights Into The Molecular Mechanisms of Growth, Immunity, and Metabolism During The Molting Cycle of The Juvenile Mud Crab Scylla Paramamosain

Background: Molting is an inherent cyclic process that occurs in all crustaceans and is essential for growth, metamorphosis, and reproduction. Recent studies have shown that many organs and systems are involved in molting regulation, including the hepatopancreas, the immune system, and production of ecdysteroids by Y-organs. However, the molecular mechanisms underlying the dynamic process of crab molting remain poorly understood at the whole-individual level. Therefore, we investigated global expression changes in the transcriptomes of the mud crab Scylla paramamosain, the most cultured crab species worldwide. We revealed a cascade of sequential expression events of molting-related genes involved in various aspects of the molting process by using whole-body sequencing of juvenile crabs. Results: RNA-sequencing (RNA-seq) produced 139.49 Gb of clean reads and 20,436 differentially expressed genes (DEGs) among different molting stages. The expression patterns for genes involved in several molecular events critical for molting, such as cuticle reconstruction, cytoskeletal structure remodeling, hormone regulation, immune responses, and metabolism, were characterized and considered as mechanisms underlying molting in S. paramamosain. Among these genes, we identified 10,695 DEGs in adjacent molting stages. In further GO and KEGG analyses, the significantly enriched pathways included structural constituents of cuticle, binding and chitin metabolic processes, steroid hormone biosynthesis, insulin resistance, and amino sugar metabolic processes. The expression profiles of 12 functional genes detected via RNA-Seq were corroborated via real-time RT-PCR assays. The results indicated gene expression profiles across the molting cycle and identified possible activation pathways for future investigation of the underlying molecular mechanisms. Conclusions: This study establishes a comprehensive transcriptomic repertoire for the molting process in S. paramamosain. The data generated in this study will serve as an important transcriptomic resource for the crab research community to facilitate gene and genome annotation and provide fundamental support for future research on molecular mechanisms in this species. Furthermore, the comprehensive analysis results will improve our understanding of morphological variation in the molting cycle and serve as a potential blueprint for future research on molting in crustaceans and other animals.

Identification of Pathways and Key Genes in Venous Remodeling After Arteriovenous Fistula by Bioinformatics Analysis

The arteriovenous fistula (AVF) is the first choice for vascular access for hemodialysis of renal failure patients. Venous remodeling after exposure to high fistula flow is important for AVF to mature but the mechanism underlying remodeling is still unknown. The objective of this study is to identify the molecular mechanisms that contribute to venous remodeling after AVF. To screen and identify the differentially expressed genes (DEGs) that may involve venous remodeling after AVF, we used bioinformatics to download the public microarray data (GSE39488) from the Gene Expression Omnibus (GEO) and screen for DEGs. We then performed gene ontology (GO) function analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA) for the functional annotation of DEGs. The protein-protein interaction (PPI) network was constructed and the hub genes were carried out. Finally, we harvested 12 normal vein samples and 12 AVF vein samples which were used to confirm the expressions of the hub genes by immunohistochemistry. A total of 45 DEGs were detected, including 32 upregulated and 13 downregulated DEGs. The biological process (BP) of the GO analysis were enriched in the extrinsic apoptotic signaling pathway, cGMP-mediated pathway signaling, and molting cycle. The KEGG pathway analysis showed that the upregulated DEGs were enriched in glycosaminoglycan biosynthesis and purine metabolism, while the downregulated DEGs were mainly enriched in pathways of glycosaminoglycan biosynthesis, antifolate resistance, and ABC transporters. The GSEA analysis result showed that the top three involved pathways were oxidative phosphorylation, TNFA signaling via NF-K B, and the inflammatory response. The PPI was constructed and the hub genes found through the method of DMNC showed that INHBA and NR4A2 might play an important role in venous remodeling after AVF. The integrated optical density (DOI) examined by immunohistochemistry staining showed that the expression of both INHBA and NR4A2 increased in AVF compared to the control group. Our research contributes to the understanding of the molecular mechanism of venous remodeling after exposure to high fistula flow, which may be useful in treating AVF failure.