The mitochondrial genome of Bottapotamon fukienense (Brachiura: Potamidae) is fragmented into two chromosomes.

BACKGROUND: China is the hotspot of global freshwater crab diversity, but their wild populations are facing severe pressures associated with anthropogenic factors, necessitating the need to map their taxonomic and genetic diversity and design conservation policies. RESULTS: Herein, we sequenced the mitochondrial genome of a Chinese freshwater crab species Bottapotamon fukienense, and found that it is fragmented into two chromosomes. We confirmed that fragmentation was not limited to a single specimen or population. Chromosome 1 comprised 15,111 base pairs (bp) and there were 26 genes and one pseudogene (pseudo-nad1) encoded on it. Chromosome 2 comprised 8,173 bp and there were 12 genes and two pseudogenes (pseudo-trnL2 and pseudo-rrnL) encoded on it. Combined, they comprise the largest mitogenome (23,284 bp) among the Potamidae. Bottapotamon was the only genus in the Potamidae dataset exhibiting rearrangements of protein-coding genes. Bottapotamon fukienense exhibited average rates of sequence evolution in the dataset and did not differ in selection pressures from the remaining Potamidae. CONCLUSIONS: This is the first experimentally confirmed fragmentation of a mitogenome in crustaceans. While the mitogenome of B. fukienense exhibited multiple signs of elevated mitogenomic architecture evolution rates, including the exceptionally large size, duplicated genes, pseudogenisation, rearrangements of protein-coding genes, and fragmentation, there is no evidence that this is matched by elevated sequence evolutionary rates or changes in selection pressures.

Bacterial recognition and inhibition activities of an LRR-only protein in the Chinese mitten crab, Eriocheir sinensis.

LRR-only protein (LRRop) is an important class of immune molecules that function as pattern recognition receptor in invertebrates, however, the bacterial inhibitory activity of this proteins remain largely unknown. Herein, a novel LRRop was cloned from Eriocheir sinensis and named as EsLRRop2. The EsLRRop2 consists of six LRR motifs and formed a horseshoe shape three-dimension structure. EsLRRop2 was mainly expressed in intestine and hepatopancreas. The transcripts of EsLRRop2 in the intestine and hepatopancreas were induced by Vibrio parahaemolyticus and Staphylococcus aureus, and displayed similar transcriptional profiles. The expression levels of EsLRRop2 responded more rapidly and highly to V. parahaemolyticus than S. aureus in the intestine and hepatopancreas. Although the basal expression level of EsLRRop2 in hemocytes was relatively low, its transcripts in hemocytes were significantly induced by V. parahaemolyticus and S. aureus. The recombinant proteins of EsLRRop2 (rEsLRRop2) displayed a wide range of binding spectrum against vibrios, including V. parahaemolyticus, V. alginolyticus, and V. harveryi. The rEsLRRop2 showed dose- and time-dependent inhibitory activity against V. parahaemolyticus and S. aureus, and it could agglutinate the two bacteria. Furthermore, the inhibitory activities of rEsLRRop2 against V. parahaemolyticus, V. alginolyticus, V. harveryi and S. aureus was slightly affected by pH and salinity, and the rEsLRRop2 displayed the strongest inhibitory activity against all the three vibrios when the salinity was 20 ‰ and pH was 8.0. Collectively, these results elucidate the bacterial binding and inhibitory activities of EsLRRop2, and provide theoretical foundations for the application of rEsLRRop2 in prevention and control of vibrio diseases in aquaculture.

Mitochondrial, nuclear and morphological differentiation in the swimming crab Liocarcinus depurator along the Atlantic-Mediterranean transition.

Environmental gradients in the sea may coincide with phenotypic or genetic gradients resulting from an evolutionary balance between selection and dispersal. The population differentiation of the swimming crab, Liocarcinus depurator, an important by-catch species in the Mediterranean Sea and North-East Atlantic, was assessed using both genetic and morphometric approaches. A total of 472 specimens were collected along its distribution area, and 17 morphometric landmarks, one mitochondrial gene (COI) and 11 polymorphic microsatellite markers were scored in 350, 287 and 280 individuals, respectively. Morphometric data lacked significant differences, but genetic analyses showed significant genetic differentiation between Atlantic and Mediterranean populations, with a steeper gradient in COI compared to microsatellite markers. Interestingly, nuclear differentiation was due to an outlier locus with a gradient in the Atlantic-Mediterranean transition area overlapping with the mtDNA gradient. Such overlapping clines are likely to be maintained by natural selection. Our results suggest a scenario of past isolation with local adaptation and secondary contact between the two basins. Local adaptation during the process of vicariance may reinforce genetic differentiation at loci maintained by environmental selection even after secondary contact.

Effect of endocrine-disrupting chemicals on the expression of a calcium ion channel receptor (ryanodine receptor) in the mud crab (Macrophthalmus japonicus).

Endocrine-disrupting chemicals (EDCs) are toxic pollutants generated by artificial activities. Moreover, their hormone-like structure induces disturbances, such as mimicking or blocking metabolic activity. Previous studies on EDCs have focused on the adverse effect of the endocrine system in vertebrates, with limited investigations conducted on ion channels in invertebrates. Thus, in this study, we investigated the potential adverse effects of exposure to bisphenol-A (BPA) and di-(2-ethylhexyl) phthalate (DEHP) at the molecular level on the ryanodine receptor (RyR), a calcium ion channel receptor in Macrophthalmus japonicus. In the phylogenetic analysis, the RyR amino acid sequences in M. japonicus clustered with those in the Crustacean and formed separated branches for RyR in insects and mammals. When exposed to 1 µg L-1 BPA, a significant increase in RyR mRNA expression was observed in the gills on day 1, although a similar level to the control group was observed from day 4 to day 7. However, the RyR expression due to DEHP exposure decreased on days 1 and 4, although it increased on day 7 following exposure to 10 µg L-1. The RyR expression pattern in the hepatopancreas increased for up to 4 days, depending on the BPA concentration. However, there was a tendency for the expression to decrease gradually after the statistical significance increased during the early stage of DEHP exposure (D1). Hence, the transcriptional alterations in the M. japonicus RyR gene observed in the study suggest that exposure toxicities to EDCs, such as BPA and DEHP, have the potential to disrupt calcium ion channel signaling in the gills and hepatopancreas of M. japonicus crabs.

Integrative Analysis of Hepatopancreas Transcriptome and Proteome in Female Eriocheir sinensis under Thermal Stress.

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.

ERK/CREB and p38 MAPK/MMP14 Signaling Pathway Influences Spermatogenesis through Regulating the Expression of Junctional Proteins in Eriocheir sinensis Testis.

The hemolymph-testis barrier (HTB) is a reproduction barrier in Crustacea, guaranteeing the safe and smooth process of spermatogenesis, which is similar to the blood-testis barrier (BTB) in mammals. The MAPK signaling pathway plays an essential role in spermatogenesis and maintenance of the BTB. However, only a few studies have focused on the influence of MAPK on crustacean reproduction. In the present study, we knocked down and inhibited MAPK in Eriocheir sinensis. Increased defects in spermatogenesis were observed, concurrently with a damaged HTB. Further research revealed that es-MMP14 functions downstream of ERK and p38 MAPK and degrades junctional proteins (Pinin and ZO-1); es-CREB functions in the ERK cascade as a transcription factor of ZO-1. In addition, when es-MMP14 and es-CREB were deleted, the defects in HTB and spermatogenesis aligned with abnormalities in the MAPK. However, JNK impacts the integrity of the HTB by changing the distribution of intercellular junctions. In summary, the MAPK signaling pathway maintains HTB integrity and spermatogenesis through es-MMP14 and es-CREB, which provides insights into the evolution of gene function during barrier evolution.

Genome-wide identification and expressional profile of the Dmrt gene family in the swimming crab (Portunus trituberculatus).

The swimming crab, Portunus trituberculatus is one of crucial aquaculture crabs with significant differences in growth and economic performance between male and female swimming crabs. Consequently, the culture of female populations presents higher economic value. The doublesex and mab-3 related transcription factor (Dmrt) gene family are known to play crucial role in gonad differentiation and development. However, there is limited information about this gene family in Portunus trituberculatus. In this study, we identified seven members of the Dmrt gene family in P. trituberculatus based on the published transcriptome and genome data and designated as Ptdmrt-1, Ptdoublesex (Ptdsx), Ptidmrt-1, Ptdmrt-11E, Ptidmrt-2, Ptdmrt-99B, and Ptdmrt-3 based on the homology analysis results, respectively. These Ptdmrt genes distributed across 6 chromosomes and were predicted to encode 283 aa, 288 aa, 529 aa, 436 aa, 523 aa, 224 aa, and 435 aa protein precursors, respectively. The expression patterns of these dmrt genes were characterized by qRT-PCR and gonad transcriptome data. The results showed that five members (Ptdmrt-99B, Ptdsx, Ptdmrt-1, Ptdmrt-3, and Ptdmrt-11E) were differentially expressed between the testis and ovary. In addition, their expression patterns from zoea 2 to juvenile 1 were characterized by published transcriptome data and the results showed that they were lowly expressed and did not exhibit notable difference except for Ptdsx during early development. Overall, majority of Ptdmrt genes were involved in gonad differentiation in the swimming crab. Current findings provide a solid foundation for further exploration of the roles of these genes in gonad development and differentiation in P. trituberculatus.

NinaB and BCO Collaboratively Participate in the ß-Carotene Catabolism in Crustaceans: A Case Study on Chinese Mitten Crab Eriocheir sinensis.

Carotenoid cleavage oxygenases can cleave carotenoids into a range of biologically important products. Carotenoid isomerooxygenase (NinaB) and ß, ß-carotene 15, 15'-monooxygenase (BCO1) are two important oxygenases. In order to understand the roles that both oxygenases exert in crustaceans, we first investigated NinaB-like (EsNinaBl) and BCO1-like (EsBCO1l) within the genome of Chinese mitten crab (Eriocheir sinensis). Their functions were then deciphered through an analysis of their expression patterns, an in vitro ß-carotene degradation assay, and RNA interference. The results showed that both EsNinaBl and EsBCO1l contain an RPE65 domain and exhibit high levels of expression in the hepatopancreas. During the molting stage, EsNinaBl exhibited significant upregulation in stage C, whereas EsBCO1l showed significantly higher expression levels at stage AB. Moreover, dietary supplementation with ß-carotene resulted in a notable increase in the expression of EsNinaBl and EsBCO1l in the hepatopancreas. Further functional assays showed that the EsNinaBl expressed in E. coli underwent significant changes in its color, from orange to light; in addition, its ß-carotene cleavage was higher than that of EsBCO1l. After the knockdown of EsNinaBl or EsBCO1l in juvenile E. sinensis, the expression levels of both genes were significantly decreased in the hepatopancreas, accompanied by a notable increase in the redness (a*) values. Furthermore, a significant increase in the ß-carotene content was observed in the hepatopancreas when EsNinaBl-mRNA was suppressed, which suggests that EsNinaBl plays an important role in carotenoid cleavage, specifically ß-carotene. In conclusion, our findings suggest that EsNinaBl and EsBCO1l may exhibit functional co-expression and play a crucial role in carotenoid cleavage in crabs.

Metal binding feature of copper‒induced metallothionein from freshwater crab Sinopotamon henanense reveals its Cu‒thionein character.

Sinopotamon Henanense expresses two metal‒induced metallothioneins (MTs), Cd‒induced MT and Cu‒induced MT (ShCuMT). The Cd‒induced MT has been characterized as a Cd‒thiolate MT. However, it is unknown whether ShCuMT is a Cu‒thiolate MT. In the present study, ShCuMT was expressed heterologously in Escherichia coli and purified by Ni‒NTA column and superdex‒75 column. And its metal‒binding feature was evaluated by DTNB reaction, circular dichroism spectroscopy (CD), isothermal microtitration (ITC), electrospray flight mass spectrometry (ESI‒TOF‒MS), and matrix‒assisted laser desorption ionization flight mass spectrometry (MALDI‒TOF‒MS). Bioinformatics analysis demonstrated that ShCuMT possessed the cysteine‒triplet motif of a Cu‒specific MT. Expression and purification of ShCuMT illustrated that SUMO tag used as the production system for ShCuMT resulted in a high production yield. The stability order of ShCuMT binding metal ions were Cu (Ⅰ) > Cd (Ⅱ) > Zn (Ⅱ). The CD spectrum indicated that ShCuMT binding with Cu (I) exhibited a compact thiol metal clusters structure. Besides, there emerged no a visible nickel‒thiol absorption after Ni‒NTA column affinity chromatography. The ITC results implied that Cu‒ShCuMT possessed the optimal thermodynamic conformation and the highest stoichiometric number of Cu (Ⅰ). Overall, the results suggested that SUMO fusion system is a robust and inexpensive approach for ShCuMT expression and Ni‒NTA column had no influence on metal binding of ShCuMT and Cu(Ⅰ) was considered its cognate metal ion, and ShCuMT possessed canonical Cu‒thiolate characteristics. The metal binding feature of ShCuMT reported here contributes to elucidating the structure‒function relationship of ShCuMT in S. Henanense.

Identification of toll-like receptor family and the immune function of new Sptlr-6 gene of Scylla paramamosain.

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different SpTLRs were analyzed. It was found that different SpTLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish, myd88, dorsal, anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain.

The involvement of VEGF and VEGFR in bacterial recognition and regulation of antimicrobial peptides in Eriocheir sinensis.

Vascular endothelial growth factor (VEGF) and VEGF reporter (VEGFR) are essential molecules in VEGF signalling pathway. Although the functions of VEGF and VEGFR have been well reported in vertebrates, their functions are still poorly understood in invertebrates. In this study, the open reading frame sequences of EsVEGF1 and EsVEGFR4 were cloned from Eriocheir sinensis, and their corresponding proteins shared typical structure characteristics with their counterparts in other species. EsVEGF1 were predominantly expressed in hepatopancreas and muscle while EsVEGFR4 mainly expressed in hemocytes and intestine. The expression levels of EsVEGF1 in hemocytes were rapidly induced by Staphylococcus aureus and Vibrio parahaemolyticus, and it also increased rapidly in hepatopancreas after being challenged with V. parahaemolyticus. The expression levels of EsVEGFR4 only increased in hepatopancreas of crabs injected with S. aureus. The extracellular immunoglobulin domain of EsVEGFR4 could bind with Gram-negative and Gram-positive bacteria as well as lipopolysaccharide and peptidoglycan. EsVEGF1 could act as the ligand for EsVEGFR4 and Toll-like receptor and regulate the expression of crustins and lysozyme with a tissue-specific manner, while have no regulatory function on that of anti-lipopolysaccharide factors. This study will provide new insights into the immune defense mechanisms mediated by VEGF and VEGFR in crustaceans.

Whole transcriptome RNA sequencing provides novel insights into the molecular dynamics of ovarian development in mud crab, Scylla paramamosain after mating.

Ovarian development in animals is a complicated biological process, requiring the simultaneous coordination among various genes and pathways. To understand the dynamic changes and molecular regulatory mechanisms of ovarian development in mud crab (Scylla paramamosain), both histological observation and whole transcriptome sequencing of ovarian tissues at different mating stages were implemented in this study. The histological results revealed that ovarian development was delayed in unmated females (60 days after courtship behavior but not mating), who exhibited an oocyte diameter of 56.38 ± 15.17 µm. Conversely, mated females exhibited accelerated the ovarian maturation process, with females reaching ovarian stage III (proliferative stage) 23 days after mating and attained an average oocyte diameter of 132.19 ± 15.07 µm. Thus, mating process is essential in promoting the rapid ovarian development in mud crab. Based on the whole transcriptome sequencing analysis, a total of 518 mRNAs, 1502 lncRNAs, 18 circRNAs and 151 miRNAs were identified to be differentially expressed between ovarian tissues at different mating stages. Notably, six differentially expressed genes (DEGs) associated with ovarian development were identified, including ovary development-related protein, red pigment concentrating hormone receptor, G2/mitotic-specific cyclin-B3-like, lutropin-chorio gonadotropic hormone receptor, renin receptor, and SoxB2. More importantly, both DEGs and targets of differentially expressed non-coding RNAs (DEncRNAs) were enriched in renin-angiotensin system, TGF-ß signaling, cell adhesion molecules, MAPK signaling pathway, and ECM-receptor interaction, suggesting that these pathways may play significant roles in the ovarian development of mud crabs. Moreover, competition endogenous RNA (ceRNA) networks were constructed while mRNAs were differentially expressed between mating stages were involved in Gene Ontology (GO) biological processes such as developmental process, reproduction, and growth. These findings could provide solid foundations for the future development of female mud crab maturation enhancement strategy, and improve the understanding of the ovarian maturation process in crustaceans.

Allergenicity and Linear Epitope Analysis of Scy p 8, an Allergen from Mud Crab.

Scy p 8 (triosephosphate isomerase) as a crab allergen in inducing distinct T-helper (Th) cell differentiation and a linear epitope associated with allergenicity remain elusive. In this study, mice sensitized with Scy p 8 exhibited significantly upregulated levels of IgE, IgG1, and IL-4 release, inducing a Th2 immune response. Moreover, the release of IFN-γ (Th1) and the levels of Treg cells were downregulated, while IL-17A (Th17) was upregulated, indicating that Scy p 8 disrupted the Th1/Th2 balance and Th17/Treg balance in mice. Furthermore, bioinformatics prediction and serum samples from crab-allergic patients and mice enabled the discovery of 8 linear epitopes of Scy p 8. Meanwhile, the analysis of peptide similarity and tertiary superposition revealed that 8 epitopes of Scy p 8 exhibited conservation across various species, potentially resulting in cross-reactivity. These findings possess the potential to enhance the comprehension of crab allergens, thereby establishing a foundation for investigating cross-reactivity.

Identification and functional characterization of laminin receptor in the mud crab, Scylla paramamosain, in response to MCDV-1 challenge.

Laminin receptor (LR), which mediating cell adhesion to the extracellular matrix, plays a crucial role in cell signaling and regulatory functions. In the present study, a laminin receptor gene (SpLR) was cloned and characterized from the mud crab (Scylla paramamosain). The full length of SpLR contained an open reading frame (ORF) of 960 bp encoding 319 amino acids, a 5' untranslated region (UTR) of 66 bp and a 3' UTR of 49 bp. The predicted protein comprised two Ribosomal-S2 domains and a 40S-SA-C domain. The mRNA of SpLR was highly expressed in the gill, followed by the hepatopancreas. The expression of SpLR was up-regulated after mud crab dicistrovirus-1(MCDV-1) infection. Knocking down SpLR in vivo by RNA interference significantly down-regulated the expression of the immune genes SpJAK, SpSTAT, SpToll1, SpALF1 and SpALF5. This study shown that the expression level of SpToll1 and SpCAM in SpLR-interfered group significantly increased after MCDV-1 infection. Moreover, silencing of SpLR in vivo decreased the MCDV-1 replication and increased the survival rate of mud crabs after MCDV-1 infection. These findings collectively suggest a pivotal role for SpLR in the mud crab's response to MCDV-1 infection. By influencing the expression of critical innate immune factors and impacting viral replication dynamics, SpLR emerges as a key player in the intricate host-pathogen interaction, providing valuable insights into the molecular mechanisms underlying MCDV-1 pathogenesis in mud crabs.

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.

Mitogenome selection shaped the terrestrial adaptation of Grapsidae (Decapoda: Brachyura).

The colonization of aquatic to terrestrial habitats by brachyuran crabs requires genetic innovations as well as morphological adaptations to adapt to terrestrial environments. The genetic basis of such adaptive evolution, however, is largely unknown. This study focuses on terrestrialization in Geograpsus (Grapsidae) the only highly terrestrial genus in this family, which represents a notable example of terrestrial adaptive radiation. Here, we sequenced the mitogenomes of two Geograpsus species and used the mitogenomes of 215 representative crabs to construct phylogenetic and time frameworks that we used to infer terrestrial origins and evolution. Using mitochondrial genomic data, we demonstrated that marine crab ancestors began to settle on land during the early Eocene. Ocean acidification, the Paleocene-Eocene Thermal Maximum (PETM), and mangrove expansion at that time may have driven the diversification and ecological expansion of these terrestrial crabs. Evolutionary analyses reveal strong positive selection signals on monophyletic lineages of Grapsidae, especially the terrestrial species of Geograpsus. Positively selected sites in functionally important regions of ND5 and ND4 may imply enhanced energy metabolism in Grapsidae compared to other crabs, and may have played an important role in their terrestrial adaptation. Overall, our work provides valuable resources and opportunities to reveal the adaptation of crabs to complex terrestrial environments.

Cytoplasmic tail of transmembrane dscam controls antibacterial responses by regulating cell proliferation-related genes in hemocytes of Chinese mitten crab (Eriocheir sinensis).

In arthropods, the involvement of Dscam (Down syndrome cell adhesion molecule) in innate immunity has been extensively demonstrated. Its cytoplasmic tail contains multiple conserved functional sites, which indicates its involvement in different intracellular signaling pathways. In this study, we focused on the role of the cytoplasmic tail of Dscam in the Chinese mitten crab (Eriocheir sinensis) immune defense. In the group with cytoplasmic tail knockdown (the site was located on constant exons 37 and 38), 3885 differentially expressed genes (DEGs) were identified. The DEGs were enriched in small molecule binding, protein-containing complex binding, and immunity-related pathways. The expression of selected genes were validated using quantitative real-time reverse transcription PCR. We identified key Cell cycle, Janus kinase (JAK)-signal transducer, activator of transcription (STAT) and mitogen-activated protein kinase (MAPK) signaling pathway genes, the results indicated that the cytoplasmic tail of Dscam controls antibacterial responses by regulating cell proliferation-related genes in hemocytes.

Invasive European green crab (Carcinus maenas) predation in a Washington State estuary revealed with DNA metabarcoding.

Predation by invasive species can threaten local ecosystems and economies. The European green crab (Carcinus maenas), one of the most widespread marine invasive species, is an effective predator associated with clam and crab population declines outside of its native range. In the U.S. Pacific Northwest, green crab has recently increased in abundance and expanded its distribution, generating concern for estuarine ecosystems and associated aquaculture production. However, regionally-specific information on the trophic impacts of invasive green crab is very limited. We compared the stomach contents of green crabs collected on clam aquaculture beds versus intertidal sloughs in Willapa Bay, Washington, to provide the first in-depth description of European green crab diet at a particularly crucial time for regional management. We first identified putative prey items using DNA metabarcoding of stomach content samples. We compared diet composition across sites using prey presence/absence and an index of species-specific relative abundance. For eight prey species, we also calibrated metabarcoding data to quantitatively compare DNA abundance between prey taxa, and to describe an 'average' green crab diet at an intertidal slough versus a clam aquaculture bed. From the stomach contents of 61 green crabs, we identified 54 unique taxa belonging to nine phyla. The stomach contents of crabs collected from clam aquaculture beds were significantly different from the stomach contents of crabs collected at intertidal sloughs. Across all sites, arthropods were the most frequently detected prey, with the native hairy shore crab (Hemigrapsus oregonensis) the single most common prey item. Of the eight species calibrated with a quantitative model, two ecologically-important native species-the sand shrimp (Crangon franciscorum) and the Pacific staghorn sculpin (Leptocottus armatus)-had the highest average DNA abundance when detected in a stomach content sample. In addition to providing timely information on green crab diet, our research demonstrates the novel application of a recently developed model for more quantitative DNA metabarcoding. This represents another step in the ongoing evolution of DNA-based diet analysis towards producing the quantitative data necessary for modeling invasive species impacts.

The multi-omics analysis in the hepatopancreas of Eriocheir sinensis provides novel insights into the response mechanism of heat stress.

Under global warming, heat stress can induce the excessive production of reactive oxygen species, causing irreversible damage to aquatic animals. It is essential to predict potentially harmful impacts on aquatic organisms under heat stress. Eriocheir sinensis, a typical crustacean crab, is widely distributed in China, American and Europe. Parent E. sinensis need migrate to the estuaries to reproduce in winter, and temperature is a key environmental factor. Herein, we performed a comprehensive transcriptomic and proteomic analysis in the hepatopancreas of E. sinensis under heat stress (20 °C and 30 °C), focusing on heat shock protein family, antioxidant system, energy metabolism and immune defense. The results revealed that parent E. sinensis generated adaptative responses to maintain physiological function under 20 °C stress via the transcriptional up-regulation of energy metabolism enzymes, mRNA synthesis and heat shock proteins. The transcriptional inhibition of key enzymes related to energy metabolism implied that 30 °C stress may lead to the dysfunction of energy metabolism in parent E. sinensis. Meanwhile, parent E. sinensis also enhanced the expression of ferritin and phospholipase D at translational level, and the glutathione s-transferase and heat shock protein 70 at both transcriptional and translational levels, speculating that parent E. sinensis can strengthen antioxidant and immune capacity to resist oxidative stress under 30 °C stress. This study elucidated the potential molecular mechanism in response to heat stress of parent E. sinensis hepatopancreas. The preliminary selection of heat tolerance genes or proteins in E. sinensis can provide a reference for the population prediction and the study of evolutionary mechanism under heat stress in crabs.

Analysis of the Overlength Main Noncoding Region in Metacarcinus magister (Decapoda: Brachyura) and a Phylogenetic Study of the Cancroidea Species.

Complete mitochondrial genomes (mitogenomes) can provide important information regarding the molecular evolution and phylogenetic relationships of marine invertebrates, especially in Brachyura. Only one Cancroidea species of mitogenomes has been sequenced before; in this research, the mitogenomic characteristics of Metacarcinus magister (Cancridae: Cancroidea) are newly studied. The length of the M. magister mitogenome was 48,820 bp, and it contained the typical 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. We performed a series of analyses on the characteristics of the mNCR of M. magister. The phylogenetics, life circumstances, and selective pressures were all analyzed to explain the formation of this length, which revealed the length of the M. magister mitogenome to be approximately three times greater than the normal length of Brachyuran mitogenomes. Phylogenetic analyses based on a dataset of 215 Decapodan mitogenomes indicated that all Eriphioidea crabs were clustered together as a group. Moreover, the rearrangement mechanism of the Cancroidea species was predicted to provide stronger evidence for the phylogenetic analysis. In general, the results obtained in this study will contribute to a better understanding of the cause of the unusual length of the M. magister mitogenome and provide new insights into the phylogeny of Brachyura.