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Crabs of the infra-order Brachyura are one of the most diverse groups of crustaceans with approximately 7,000 described species in 98 families, occurring in marine, freshwater, and terrestrial habitats. The relationships among the brachyuran families are poorly understood due to the high morphological complexity of the group. Here, we reconstruct the most comprehensive phylogeny of Brachyura to date using sequence data of six nuclear protein-coding genes and two mitochondrial rRNA genes from more than 140 species belonging to 58 families. The gene tree confirms that the “Podotremata,” are paraphyletic. Within the monophyletic Eubrachyura, the reciprocal monophyly of the two subsections, Heterotremata and Thoracotremata, is supported. Monophyly of many superfamilies, however, is not recovered, indicating the prevalence of morphological convergence and the need for further taxonomic studies. Freshwater crabs were derived early in the evolution of Eubrachyura and are shown to have at least two independent origins. Bayesian relaxed molecular methods estimate that freshwater crabs separated from their closest marine sister taxa ∼135 Ma, that is, after the break up of Pangaea (∼200 Ma) and that a Gondwanan origin of these freshwater representatives is untenable. Most extant families and superfamilies arose during the late Cretaceous and early Tertiary.

Climate-driven warming affects the reproduction of oviparous ectotherms. However, whether oviparous ectotherms possess a protection mechanism against heat stress for oocyte development, which is essential for maintaining the continuity of animal populations, is largely unknown. Under high temperatures, female mud crabs ( Scylla paramamosain ) typically have well-formed ovaries, while a few crabs were found to experience oocyte development failure. To investigate the heat stress protection mechanism of oocyte development in mud crabs, we construct a chromosome-level genome of this species and identify an enhancer of the vitellogenin receptor ( VtgR ) that stimulates its expression under high temperatures. Lacking this enhancer due to an intronic deletion leads to low VtgR expression in abnormal crabs, resulting in abnormal vitellogenic oocyte formation in these individuals when exposed to high temperatures. Furthermore, we identify a similar heat stress protection mechanism in zebrafish. Disruption of Lrp13, a VtgR-like protein in zebrafish, results in impaired vitellogenin absorption and ovarian degeneration in zebrafish exposed to high temperatures. Our results reveal a VtgR-mediated mechanism that protects vitellogenic oocyte formation against heat stress in mud crabs and zebrafish, contributing to their heat adaptability during oocyte development. Climate-driven warming affects the reproduction of oviparous ectotherms. Here, authors identify and characterize a VtgR-mediated heat protection mechanism for oocyte development in both mud crabs and zebrafish.

Background Aeromonas hydrophila , a widespread aquatic pathogen, can infect a range of aquatic organisms, such as fish and crustaceans (including Eriocheir sinensis ). Understanding the host resistance mechanism against A. hydrophila infection is of significant importance. Results In this study, the metabolic and transcriptomic profiles of crabs ( E. sinensis ) at different stages of A. hydrophila infection (early-infection stage: Ah_6h, mid-infection stage: Ah_24h, and late-infection stage: Ah_72h) were investigated. Metabolomic analysis showed that differentially expressed metabolites were predominantly enriched in purine metabolism pathways. Transcriptomic analysis revealed that the infection might activate the crab’s immune response through key signaling pathways, including NF-κB and RIG-I-like receptor pathways, at the early-infection stage, while potentially maintaining immune function throughout infection via apoptosis, phagocytosis, and lysosomal pathways. Notably, the mid-infection stage was the pivotal period in the regulation of the crab’s immune response, with the highest levels of differential metabolites and genes. Integrated transcriptomic and metabolomic analysis further highlighted the potential key roles of the tricarboxylic acid (TCA) cycle and purine and pyrimidine metabolisms in the immune response of E. sinensis against A. hydrophila . Conclusions These findings provide a better understanding of the immunity of E. sinensis in response to bacterial infection.

Molt is a critical developmental process in crustaceans. Recent studies have shown that the hepatopancreas is an important source of innate immune molecules, yet hepatopancreatic patterns of gene expression during the molt cycle which may underlie changes in immune mechanism are unknown. In this study, we performed Illumina sequencing for the hepatopancreas of the mud crab, Scylla paramamosain during molt cycle (pre-molt stage, post-molt stage, and inter-molt stage). A total of 44.55 Gb high-quality reads were obtained from the normalized cDNA of hepatopancreas. A total of 70,591 transcripts were assembled; 55,167 unigenes were identified. Transcriptomic comparison revealed 948 differentially expressed genes (DEGs) in the hepatopancreas from the three molt stages. We found that genes associated with immune response patterns changed in expression during the molt cycle. Antimicrobial peptide genes, inflammatory response genes, Toll signaling pathway factors, the phenoloxidase system, antioxidant enzymes, metal-binding proteins and other immune related genes are significantly up-regulated at the post-molt stage and inter-molt stage compared with the pre-molt stage, respectively. These genes are either not expressed or are expressed at low levels at the pre-molt stage. To our knowledge, this is the first systematic transcriptome analysis of genes capable of mobilizing a hepatopancreas immune response during the molt cycle in crustaceans, and this study will contribute to a better understanding of the hepatopancreas immune system and mud crab prophylactic immune mechanisms at the post-molt stage.

Background Ecdysis is an innate behaviour programme by which all arthropods moult their exoskeletons. The complex suite of interacting neuropeptides that orchestrate ecdysis is well studied in insects, but details of the crustacean ecdysis cassette are fragmented and our understanding of this process is comparatively crude, preventing a meaningful evolutionary comparison. To begin to address this issue we identified transcripts coding for neuropeptides and their putative receptors in the central nervous system (CNS) and Y-organs (YO) within the crab, Carcinus maenas, and mapped their expression profiles across accurately defined stages of the moult cycle using RNA-sequencing. We also studied gene expression within the epidermally-derived YO, the only defined role for which is the synthesis of ecdysteroid moulting hormones, to elucidate peptides and G protein-coupled receptors (GPCRs) that might have a function in ecdysis. Results Transcriptome mining of the CNS transcriptome yielded neuropeptide transcripts representing 47 neuropeptide families and 66 putative GPCRs. Neuropeptide transcripts that were differentially expressed across the moult cycle included carcikinin, crustacean hyperglycemic hormone-2, and crustacean cardioactive peptide, whilst a single putative neuropeptide receptor, proctolin R1, was differentially expressed. Carcikinin mRNA in particular exhibited dramatic increases in expression pre-moult, suggesting a role in ecdysis regulation. Crustacean hyperglycemic hormone-2 mRNA expression was elevated post- and pre-moult whilst that for crustacean cardioactive peptide, which regulates insect ecdysis and plays a role in stereotyped motor activity during crustacean ecdysis, was elevated in pre-moult. In the YO, several putative neuropeptide receptor transcripts were differentially expressed across the moult cycle, as was the mRNA for the neuropeptide, neuroparsin-1. Whilst differential gene expression of putative neuropeptide receptors was expected, the discovery and differential expression of neuropeptide transcripts was surprising. Analysis of GPCR transcript expression between YO and epidermis revealed 11 to be upregulated in the YO and thus are now candidates for peptide control of ecdysis. Conclusions The data presented represent a comprehensive survey of the deduced C. maenas neuropeptidome and putative GPCRs. Importantly, we have described the differential expression profiles of these transcripts across accurately staged moult cycles in tissues key to the ecdysis programme. This study provides important avenues for the future exploration of functionality of receptor-ligand pairs in crustaceans.

The crustacean hepatopancreas has different functions including absorption, storage of nutrients and vitellogenesis during growth, and ovarian development. However, genetic information on the biological functions of the crustacean hepatopancreas during such processes is limited. The swimming crab, Portunus trituberculatus, is a commercially important species for both aquaculture and fisheries in the Asia-Pacific region. This study compared the transcriptome in the hepatopancreas of female P. trituberculatus during the growth and ovarian maturation stages by 454 high-throughput pyrosequencing and bioinformatics. The goal was to discover genes in the hepatopancreas involved in food digestion, nutrition metabolism and ovarian development, and to identify patterns of gene expression during growth and ovarian maturation. Our transcriptome produced 303,450 reads with an average length of 351 bp, and the high quality reads were assembled into 21,635 contigs and 31,844 singlets. Based on BLASTP searches of the deduced protein sequences, there were 7,762 contigs and 4,098 singlets with functional annotation. Further analysis revealed 33,427 unigenes with ORFs, including 17,388 contigs and 16,039 singlets in the hepatopancreas, while only 7,954 unigenes (5,691 contigs and 2,263 singlets) with the predicted protein sequences were annotated with biological functions. The deduced protein sequences were assigned to 3,734 GO terms, 25 COG categories and 294 specific pathways. Furthermore, there were 14, 534, and 22 identified unigenes involved in food digestion, nutrition metabolism and ovarian development, respectively. 212 differentially expressed genes (DEGs) were found between the growth and endogenous stage of the hepatopancreas, while there were 382 DEGs between the endogenous and exogenous stage hepatopancreas. Our results not only enhance the understanding of crustacean hepatopancreatic functions during growth and ovarian development, but also represent a basis for further research on new genes and functional genomics of P. trituberculatus or closely related species.

Background The Sox (SRY-related HMG-box) transcription factor family plays central roles in cell fate determination, neurogenesis, and reproductive development, yet its evolutionary history and molecular diversification in crustaceans remain poorly understood. Here we present a genome-wide comparative study of Sox genes in three representative crabs— Portunus trituberculatus , Scylla paramamosain , and Eriocheir sinensis —based on their high-quality reference genomes. Results We identified 16, 16, and 15 Sox genes, respectively, and established a robust HMG-box–based phylogeny and nomenclature, assigning all members to the canonical B–F subgroups and an ungrouped Sox-like clade. Comparative analyses uncovered striking lineage-specific events, including the complete loss of SoxB1 in E. sinensis and an expansion of the SoxC subgroup, accompanied by a mosaic of deeply conserved and newly derived motifs and domains. Integrative transcriptomic analyses revealed that many Sox genes are strongly expressed in neural tissues and testes, and that certain SoxB and SoxC members exhibit stage-specific activation during embryonic, larval, or testicular development, highlighting key roles in neurogenesis, pluripotency maintenance, and spermatogenesis. Conclusions Together, these findings provide the first comprehensive evolutionary framework for the crab Sox family, uncover unexpected gene loss and subgroup expansion, and offer new insights into how conserved transcriptional regulators diversify to shape neural and reproductive programs in arthropods.

Osmoregulation and osmoconformation are two mechanisms through which aquatic animals adapt to salinity fluctuations. The euryhaline crab Scylla paramamosain, being both an osmoconformer and osmoregulator, is an excellent model organism to investigate salinity adaptation mechanisms in brachyurans. In the present study, we used transcriptomic and proteomic approaches to investigate the response of S. paramamosain to salinity stress. Crabs were transferred from a salinity of 25 ppt to salinities of 5 ppt or 33 ppt for 6 h and 10 days. Data from both approaches revealed that exposure to 5 ppt resulted in upregulation of ion transport and energy metabolism associated genes. Notably, acclimation to low salinity was associated with early changes in gene expression for signal transduction and stress response. In contrast, exposure to 33 ppt resulted in upregulation of genes related to amino acid metabolism, and amino acid transport genes were upregulated only at the early stage of acclimation to this salinity. Our study reveals contrasting mechanisms underlying osmoregulation and osmoconformation within the salinity range of 5–33 ppt in the mud crab, and provides novel candidate genes for osmotic signal transduction, thereby providing insights on understanding the salinity adaptation mechanisms of brachyuran crabs.

The Chinese mitten crab (Eriocheir sinensis), an economically important crustacean that is endemic to China, has recently experienced high-temperature stress. The high thermal tolerance of E. sinensis points to its promise in being highly productive in an aquacultural context. However, the mechanisms underlying its high thermal tolerance remain unknown. In this study, female E. sinensis that were heat exposed for 24 h at 38.5 °C and 33 °C were identified as high-temperature-stressed (HS) and normal-temperature-stressed (NS) groups, respectively. The hepatopancreas of E. sinensis from the HS and NS groups were used for transcriptome and proteomic analyses. A total of 2350 upregulated and 1081 downregulated differentially expressed genes (DEGs) were identified between the HS and NS groups. In addition, 126 differentially expressed proteins (DEPs) were upregulated and 35 were downregulated in the two groups. An integrated analysis showed that 2641 identified genes were correlated with their corresponding proteins, including 25 genes that were significantly differentially expressed between the two omics levels. Ten Gene Ontology terms were enriched in the DEGs and DEPs. A functional analysis revealed three common pathways that were significantly enriched in both DEGs and DEPs: fluid shear stress and atherosclerosis, leukocyte transendothelial migration, and thyroid hormone synthesis. Further analysis of the common pathways showed that MGST1, Act5C, HSP90AB1, and mys were overlapping genes at the transcriptome and proteome levels. These results demonstrate the differences between the HS and NS groups at the two omics levels and will be helpful in clarifying the mechanisms underlying the thermal tolerance of E. sinensis.

Background Arthropods require periodic molting (ecdysis) for growth. While the neuroendocrine orchestration of ecdysis is well characterized in insects, it remains comparatively poorly understood in crustaceans. In insects, ecdysis-triggering hormone (ETH) from epitracheal cells and eclosion hormone (EH) from the brain initiate and coordinate ecdysis. ETH triggers pre-ecdysis behaviors, while EH amplifies ETH release and promotes neuropeptide secretion of crustacean cardioactive peptide (CCAP), myoinhibitory peptide (MIP) for exuviation. Definitive evidence for functional ETH and EH in crustaceans is lacking. Here, we investigate ETH and EH in the crab Carcinus maenas by functionally characterizing the ETH receptor, comprehensively mapping transcript and peptide localization of ETH and EH, and quantifying ETH neurohormone and transcript throughout the molt cycle. Results We identified a single CamETH GPCR with high specificity for arthropod ETHs, signaling via calcium and cAMP. ETHR mRNA is expressed in multiple tissues, but ETH is restricted to the central nervous system (CNS) with a complex neuroarchitecture in pericardial organs, adjacent to the branchiocardiac veins. EH is limited to CNS, notably in the eyestalk ganglia. Of note, complex EH immunopositive fibers in the abdominal ganglion, adjacent to CCAP/allatostatin-C (AST-C)/bursicon cells, represent a novel putative neurohemal site. Measurements of ETH mRNA and peptide through the molt cycle showed that transcription peaks in late premolt, while peptide is released during active ecdysis, later than observed in insects. Conclusions While crustacean and insect ecdysis share neuroendocrine components, reflecting common ancestry, there is clear divergence in terms of functionality. We propose a new crustacean ecdysis cascade: ETH released from pericardial organs may stimulate EH to initiate CCAP/AST-C and bursicon release from adjacent neurons.