Identifying the genes involved in the egg-carrying ovigerous hair development of the female blue crab Callinectes sapidus: transcriptomic and genomic expression analyses.

BACKGROUND: Crustacean female sex hormone (CFSH) controls gradually developing adult female-specific morphological features essential for mating and brood care. Specifically, ovigerous hairs are developed during the prepuberty molt cycle of the blue crab Callinectes sapidus that are essential for carrying the eggs until they finish development. Reduced CFSH transcripts by CFSH-dsRNA injections result in fewer and shorter ovigerous hairs than the control. This study aimed to identify the specific genes responsible for ovigerous hair formation using transcriptomic, genomic and expression analyses of the ovigerous setae at three stages: prepuberty at early (OE) and late premolt (OL), and adult (AO) stages. RESULTS: The de novo Trinity assembly on filtered sequence reads produced 96,684 Trinity genes and 124,128 transcripts with an N50 of 1,615 bp. About 27.3% of the assembled Trinity genes are annotated to the public protein sequence databases (i.e., NR, Swiss-Prot, COG, KEGG, and GO databases). The OE vs. OL, OL vs. AO, and OE vs. AO comparisons resulted in 6,547, 7,793, and 7,481 differentially expressed genes, respectively, at a log2-fold difference. Specifically, the genes involved in the Wnt signaling and cell cycle pathways are positively associated with ovigerous hair development. Moreover, the transcripts of ten cuticle protein genes containing chitin-binding domains are most significantly changed by transcriptomic analysis and RT-qPCR assays, which shows a molt-stage specific, down-up-down mode across the OE-OL-AO stages. Furthermore, the expression of the cuticle genes with the chitin-binding domain, Rebers and Riddiford domain (RR)-1 appears at early premolt, followed by RR-2 at late premolt stage. Mapping these 10 cuticle protein sequences to the C. sapidus genome reveals that two scaffolds with a 549.5Kb region and 35 with a 1.19 Mb region harbor 21 RR1 and 20 RR2 cuticle protein genes, respectively. With these findings, a putative mode of CFSH action in decapod crustaceans is proposed. CONCLUSIONS: The present study describes a first step in understanding the mechanism underlying ovigerous hair formation in C. sapidus at the molecular level. Overall, demonstrating the first transcriptome analysis of crustacean ovigerous setae, our results may facilitate future studies into the decapod female reproduction belonging to the suborder Pleocyemata.

Twelve quick steps for genome assembly and annotation in the classroom.

Eukaryotic genome sequencing and de novo assembly, once the exclusive domain of well-funded international consortia, have become increasingly affordable, thus fitting the budgets of individual research groups. Third-generation long-read DNA sequencing technologies are increasingly used, providing extensive genomic toolkits that were once reserved for a few select model organisms. Generating high-quality genome assemblies and annotations for many aquatic species still presents significant challenges due to their large genome sizes, complexity, and high chromosome numbers. Indeed, selecting the most appropriate sequencing and software platforms and annotation pipelines for a new genome project can be daunting because tools often only work in limited contexts. In genomics, generating a high-quality genome assembly/annotation has become an indispensable tool for better understanding the biology of any species. Herein, we state 12 steps to help researchers get started in genome projects by presenting guidelines that are broadly applicable (to any species), sustainable over time, and cover all aspects of genome assembly and annotation projects from start to finish. We review some commonly used approaches, including practical methods to extract high-quality DNA and choices for the best sequencing platforms and library preparations. In addition, we discuss the range of potential bioinformatics pipelines, including structural and functional annotations (e.g., transposable elements and repetitive sequences). This paper also includes information on how to build a wide community for a genome project, the importance of data management, and how to make the data and results Findable, Accessible, Interoperable, and Reusable (FAIR) by submitting them to a public repository and sharing them with the research community.

Diet composition and long-term starvation do not affect crustacean hyperglycemic hormone (CHH) transcription in the burrowing crab Neohelice granulata (Dana, 1851).

The burrowing crab Neohelice granulata is a key omnivorous species in intertidal areas along the southwestern Atlantic from southern Brazil to northern Argentinean Patagonia. This crab is adapted to starvation and can endure natural periods of food deprivation. The metabolic adjustments during starvation depend on the type of diet the crabs were fed previously. Since eyestalk-crustacean hyperglycemic hormone (CHH) is the principal regulator of glucose homeostasis in decapods, we investigated whether CHH transcription was affected by diet composition and starvation. Crabs were maintained in the laboratory for two weeks and subsequently divided in two groups. One received a high carbohydrate (HC) diet, and the other was fed a high protein (HP) diet. After this period, they were starved for four weeks. The full-length cDNA sequence of N. granulata CHH was determined and aligned with CHH sequences of other crabs. Levels of circulating glucose and glycogen were higher in the hepatopancreas and muscle of the HC-fed group and decreased after starvation. Glucose and glycogen concentrations were not altered by starvation in the HP group. Triglyceride levels within the hemolymph were not altered by diet or starvation. However, triglycerides concentration was higher in the hepatopancreas of HC compared to HP-fed group. Long-term starvation and diet composition did not affect CHH transcription.

The presence of an insulin-like androgenic gland factor (IAG) and insulin-like peptide binding protein (ILPBP) in the ovary of the blue crab, Callinectes sapidus and their roles in ovarian development.

Insulin-like androgenic gland factor (IAG) that is produced by the male androgenic gland (AG), plays a role in sexual differentiation and maintenance of male secondary sex characteristics in decapod crustaceans. With an earlier finding of IAG expression in a female Callinectes sapidus ovary, we aimed to examine a putative role of IAG during the ovarian development of this species. To this end, the full-length cDNA sequence of the ovarian CasIAG (termed CasIAG-ova) has been isolated. The predicted mature peptide sequence of CasIAG-ova is identical to that of the IAG from the AG, except in their signal peptide regions. The CasIAG-ova contains an alternative initiation codon (UUG) as the start codon, which suggests that the translational regulation of CasIAG-ova may differ from that of the IAG from AG. To define the function of CasIAG-ova, the expressions of CasIAG-ova as well as its putative binding protein, insulin-like peptide binding protein (ILPBP), are measured in the ovaries at various developmental stages obtained from different seasons. Season affects both CasIAG and ILPBP expression in the ovary. Overall, summer females at earlier ovarian stages contain high levels of CasIAG and ILPBP than spring or fall females. These findings indicate that CasIAG-ova and CasILPBP may be involved in the ovarian development. When comparing the levels of CasIAG and CasILPBP in the ovary, the latter are much higher (∼10-10000 fold) than the former. Expression patterns of CasILPBP differ from those of CasIAG-ova during ovarian development and by season, suggesting that ILPBP may have an additional role in ovarian development rather than a function of a putative binding protein of IAG.

Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?

Insulin-like peptides (ILPs) have regulatory roles in reproduction, development and metabolism in invertebrates. The mode of ILP actions has not been well studied in invertebrates in regard to the role of binding partners, i.e., ILP binding protein (ILPBP). In this study, the full-length cDNA of Callinectes sapidus ILPBP (Cas-ILPBP, 960 bp) has been isolated using RACE cloning, having short 5' and 3' UTRs of 30 and 162 bp, respectively. The predicted precursor of Cas-ILPBP (255 aa) contains, in order a signal peptide (23 aa), an insulin-like growth factor (IGF) binding (IB) domain (79 aa), a kazal-type serine protease inhibitor (KI) domain (36 aa) and an immunoglobulin (Ig) domain (101 aa). Phylogenetic analysis shows that Cas-ILPBP is grouped with the ILPBPs of other crustacean species, and it shares the closest relationship with the ILPBP from another crab species, Scylla paramamosain. Transcripts of Cas-ILPBP are found in all examined tissues, with the highest levels in the nervous tissues (eyestalk ganglia, brain and thoracic ganglia complex) and followed by midgut, the pericardial organ, abdominal muscle and the heart. As Cas-ILPBP contains a putative Ig domain, it is hypothesized that this protein may be involved in immunity, particularly in the adult females infected with a reo-like virus (CsRV1). The expression levels of Cas-ILPBP are examined in several tissues (hemocytes, midgut, eyestalk ganglia) from the animals carrying varying levels of CsRV1 at 17 and 23 °C water temperatures. Cas-ILPBP levels in the midgut are most significantly affected by high levels of CsRV1 infection. Reduction in Cas-ILPBP levels in the midguts is noted from the animals infected with high levels of CsRV1 that show reduced or stop feeding activity, indicating that it may play an important role in midgut functions such as digestion and nutrient absorption.

Elevated water temperature increases the levels of reo-like virus and selected innate immunity genes in hemocytes and hepatopancreas of adult female blue crab, Callinectes sapidus.

Seasonal changes in water temperature directly affect the aquatic ecosystem. The blue crab, Callinectes sapidus, inhabiting the Chesapeake Bay has been adapted to seasonal changes of the environmental conditions. In this, the animals halt their physiological process of the growth and reproduction during colder months while they resume these processes as water temperatures increase. We aimed to understand the effect of the elevated temperatures on a disease progression of reo-like virus (CsRLV) and innate immunity of adult female C. sapidus. Following a rise in water temperature from 10 to 23 °C, CsRLV levels in infected crabs rose significantly in hemocytes and multiple organs. However, in hemocytes, the elevated temperature had no effect on the levels of three innate immune genes: Cas-ecCuZnSOD-2, CasPPO and CasLpR three carbohydrate metabolic genes: CasTPS, CasGlyP; and CasTreh and the total hemocyte counts (THC). Interestingly, the hemocytes of CsRLV infected animals exposed to 23 °C for 10 days had significantly elevated levels of Cas-ecCuZnSOD-2 and CasTPS, compared to those of the uninfected ones also exposed to the same condition and compared to hatchery-raised females kept at 23 °C. Despite the lack of changes in THC, the types of hemocytes from the animals with high CsRLV levels differed from those of uninfected ones and from hatchery animals kept at 23 °C: CsRLV-infected crabs had hemocytes of smaller size with less cytosolic complexity than uninfected crabs. It therefore appears that the change in temperature influences rapid replication of CsRLV in all internal tissues examined. This implies that CsRLV may have broad tissue tropism. Interestingly, the digestive tract (mid- and hindgut) contains significantly higher levels of CsRLV than hemocytes while hepatopancreas and ovary have lower levels than hemocytes. Innate immune responses differ by tissue: midgut and hepatopancreas with upregulated Cas-ecCuZnSOD-2 similar to that found in hemocytes. By contrast, hepatopancreas showed a down-regulated CasTPS, suggesting carbohydrate stress during infection.

Crustacean hyperglycemic hormones of two cold water crab species, Chionoecetes opilio and C. japonicus: isolation of cDNA sequences and localization of CHH neuropeptide in eyestalk ganglia.

Crustacean hyperglycemic hormone (CHH) is primarily known for its prototypical function in hyperglycemia which is induced by the release of CHH. The CHH release takes place as an adaptive response to the energy demands of the animals experiencing stressful environmental, physiological or behavioral conditions. Although >63 decapod CHH nucleotide sequences are known (GenBank), the majority of them is garnered from the species inhabiting shallow and warm water. In order to understand the adaptive role of CHH in Chionoecetes opilio and Chionoecetes japonicus inhabiting deep water environments, we first aimed for the isolation of the full-length cDNA sequence of CHH from the eyestalk ganglia of C. opilio (ChoCHH) and C. japonicus (ChjCHH) using degenerate PCR and 5' and 3' RACE. Cho- and ChjCHH cDNA sequences are identical in 5' UTR and ORF with 100% sequence identity of the putative 138aa of preproCHHs. The length of 3' UTR ChjCHH cDNA sequence is 39 nucleotides shorter than that of ChoCHH. This is the first report in decapod crustaceans that two different species have the identical sequence of CHH. ChoCHH expression increases during embryogenesis of C. opilio and is significantly higher in adult males and females. C. japonicus males have slightly higher ChjCHH expression than C. opilio males, but no statistical difference. In both species, the immunostaining intensity of CHH is stronger in the sinus gland than that of X-organ cells. Future studies will enable us to gain better understanding of the comparative metabolic physiology and endocrinology of cold, deep water species of Chionoecetes spp.

Seasonal variations in reproductive activity of the blue crab, Callinectes sapidus: Vitellogenin expression and levels of vitellogenin in the hemolymph during ovarian development.

In general, season affects the physiology and behavior of most animals. Warmer temperatures accelerate growth and reproduction of ectotherms, whereas these processes are slowed or halted in colder temperatures. Female blue crabs, Callinectes sapidus inhabiting the Chesapeake Bay, exhibit a seasonal migratory behavior that is closely tied with spawning and the release of larvae. To better understand reproductive activities of the migratory adult females, we examined two reproductive parameters of these crabs sampled monthly (April-December, 2006): the levels of vitellogenin (VtG) in the hemolymph and VtG expression in the hepatopancreas and ovary. The full-length cDNA of VtG (CasVtG-ova) has been isolated from the ovary. The putative CasVtG sequence found in the ovary is >99% identical to that of the hepatopancreas and is related most closely to the sequences reported in other crab species. In female C. sapidus, the hepatopancreas produces over 99% of the total VtG toward the ovarian development. Ovarian stages 2 and 3 in the sampled females are characterized by significant high levels of VtG in hemolymph and VtG expression in both the hepatopancreas and ovary. However, during the southbound migration in fall, females at ovarian stages 2 and 3 have decreased VtG levels, compared to those in spring and summer. The decreased vitellogenesis activity during the fall migration suggests seasonal adaptation to ensure successful spawning and the larval release.

An insulin-like growth factor found in hepatopancreas implicates carbohydrate metabolism of the blue crab Callinectes sapidus.

Hyperglycemia that is caused by the release of crustacean hyperglycemic hormone (CHH) from the sinus gland to hemolymph is one of the hallmark physiological phenomena, occurring in decapod crustaceans experiencing stressful conditions. However, the mechanism(s) by which such elevated glucose levels return to resting levels is still unknown. Interestingly, noted is a difference in the clearance rate of hemolymph glucose between adult females and adult males of the blue crab, Callinectes sapidus: the former with more rapid clearance than the latter. The presence of an endogenous-insulin-like molecule is suggested in C. sapidus because an injection of bovine insulin, significantly reduces the levels of hemolymph glucose that were previously elevated by emersion stress or the glucose injection. Using 5' and 3' RACE, the full-length cDNA of an insulin-like molecule is isolated from the hepatopancreas of an adult female C. sapidus and shows the same putative sequence of an insulin-like androgenic gland factor (IAG) but differs in 5' and 3' UTR sequences. A knock-down study using five injections of double-stranded RNA of CasIAG-hep (dsRNA-CasIAG-hep, 10µg/injection) over a 10-day period reduces CasIAG-hep expression by ∼50%. The levels of hemolymph glucose are also kept higher in dsRNA-CasIAG-hep injected group than those treated with dsRNA-green fluorescent protein (dsRNA-IAG-hep) or saline. Most importantly, the hepatopancreas of dsRNA-CasIAG-hep injected animals contains amounts of carbohydrate (glucose, trehalose, and glycogen) significantly lower than those of control groups, indicating that the function of CasIAG-hep in carbohydrate metabolism in crustaceans is similar to carbohydrate metabolism in vertebrates.

A brief and updated introduction to the neuroendocrine system of crustaceans.

The neuroendocrine system of crustaceans is complex and regulates many processes, such as development, growth, reproduction, osmoregulation, behavior, and metabolism. Once stimulated, crustaceans' neuroendocrine tissues modulate the release of monoamines, ecdysteroids, and neuropeptides that can act as hormones or neurotransmitters. Over a few decades, research has unraveled some mechanisms governing these processes, substantially contributing to understanding crustacean physiology. More aspects of crustacean neuroendocrinology are being comprehended with molecular biology, transcriptome, and genomics analyses. Hence, these studies will also significantly enhance the ability to cultivate decapods, such as crabs and shrimps, used as human food sources. In this review, current knowledge on crustacean endocrinology is updated with new findings about crustacean hormones, focusing mainly on the main neuroendocrine organs and their hormones and the effects of these molecules regulating metabolism, growth, reproduction, and color adaptation. New evidence about vertebrate-type hormones found in crustaceans is included and discussed. Finally, this review may assist in understanding how the emerging chemicals of environmental concern can potentially impair and disrupt crustacean's endocrine functions and their physiology.

Cloning of prophenoloxidase from hemocytes of the blue crab, Callinectes sapidus and its expression and enzyme activity during the molt cycle.

The arthropods cuticle undergoes dramatic morphological and biochemical changes from being soft to hardness through each molting process. Prophenoloxidase (PPO) known as a key enzyme in the arthropod innate immune system involved in the melanization reaction, has been related with the initial shell-hardening process, specifically in the sclerotization of the protein matrix in the new cuticle. Since hemocytes have been reported as the main PPO source in arthropods, the transport of hemocyte PPO into the newly laid, soft cuticle has been proposed for shell-hardening occurring during and immediately after ecdysis. In order to define the role of hemocyte PPO in the shell-hardening of crustaceans, the full-length cDNA sequence (2806 nt) of hemocytes PPO of the blue crab Callinectes sapidus (CasPPO-hemo) is isolated using degenerate PCR and 5'-3' RACE. CasPPO-hemo encodes a putative PPO (672 aa) showing three hemocyanin domains: N, M, and C in order and two copper binding sites (CuA & CuB). The sequence analysis identifies the putative CasPPO-hemo as zymogen which requires the cleavage at the N-terminus for its activation. Hemocyte extract (CasHLS) contains the PO, the activity of which depends on the in vitro activation of trypsin. The expression levels of CasPPO-hemo are kept constant during the molt cycle. The increase in the number of hemocytes at early premolt correlates with the elevated PO activity, while at late premolt, the increment in hemocyte numbers does not reflect on the PO activity. The functional importance of the changes in the levels of CasHLS-PO activity during molt cycle is discussed in relation to cuticle hardening process.

A novel putative lipoprotein receptor (CasLpR) in the hemocytes of the blue crab, Callinectes sapidus: cloning and up-regulated expression after the injection of LPS and LTA.

The full-length cDNA encoding a putative lipoprotein receptor (CasLpR) was isolated from the hemocytes of Callinectes sapidus using 5' and 3' RACEs. The open reading frame for CasLpR contains a precursor of putative CasLpR consisting of 1710 amino acid residues including 22 amino acid residues of the signal peptide (22 amino acids). Mature CasLpR (1688 amino acids with 5.6% of phosphorylation sites) has multiple, putative functional domains: five low-density lipoprotein receptor domains in the N-terminus, and a G-protein-coupled receptor proteolysis site domain and a 7 transmembrane receptor (secretin family) domain in the C-terminus. To date, there are no proteins with a similar domain structure in the GenBank. The expression pattern of CasLpR was exclusive in hemocytes among all tested tissues obtained from a juvenile female at intermolt stage: brain, eyestalk ganglia, pericardial organs, and thoracic ganglia complex (nervous system); hepatopancreas (digestive system); heart, artery and hemocytes (circulatory system); gill and antennal gland (excretory system), hypodermis; and Y-organ (endocrine organ). There was no CasLpR expression in the ovary of an adult female. A putative function of CasLpR was examined after challenges of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in vivo using qRT-PCR assays. Animals at 24 h after injection of LPS or LTA up-regulated the expression of CasLpR in hemocytes by ∼3.5 and 1.4 folds, respectively, compared to the controls that received saline injection. LPS challenge also caused the greatest increment (∼55 folds) of heat shock protein 90 (Hsp90) expression in these samples. These data indicate that putative CasLpR and CasHsp90 may be involved in the defense system or the stress response of C. sapidus.

Stimulation of molt by RNA interference of the molt-inhibiting hormone in the crayfish Cherax quadricarinatus.

In crustaceans, molting is known to be under the control of neuropeptide hormones synthesized and secreted from the eyestalk ganglia. While the role of molt-inhibiting hormone (MIH) in regulating molting has been described in several species using classical methods, an in vivo specific MIH targeted manipulation has not been described yet. In the present study, an MIH cDNA was isolated and sequenced from the eyestalk ganglia of the Australian freshwater red claw crayfish Cherax quadricarinatus (Cq) by 5' and 3' RACE. We analyzed the putative Cq-MIH based on sequence homology, a three dimensional structure model and transcript's tissue specificity. We further examined the involvement of Cq-MIH in the control of molt in the crayfish through RNAi by in vivo injections of Cq-MIH double-stranded RNA, which resulted in, similarly to eyestalk ablation, acceleration of molt cycles. This acceleration was reflected by a significant reduction (up to 32%) in molt interval and an increased rate in molt mineralization index (MMI), which correlated with the induction of ecdysteroid hormones compared to control. Altogether, this study provides a proof of function for the involvement of the Cq-MIH gene in molt regulation in the crayfish.

17ß-Estradiol (E2) may be involved in the mode of crustacean female sex hormone (CFSH) action in the blue crab, Callinectes sapidus.

17ß-estradiol (E2) has been proved to control reproduction, sexual differentiation, and the development of the secondary sexual characteristics of vertebrate females. In decapod crustacean species, crustacean female sex hormone (CFSH), a protein hormone, is required for developing adult-specific ovigerous setae for embryo brooding and gonophores for mating at the blue crab Callinectes sapidus puberty molting. However, it is unclear that whether the mode of CFSH action involves a vertebrate-type sex steroid hormone in crustaceans. To this end, E2 levels were first measured using a competitive ELISA in the hemolymph and the potential CFSH target tissues from both prepuberty and adult females; the presence of E2 was further confirmed with a liquid chromatography tandem mass spectrometry method. Then, the cDNAs of the following genes known to be associated with vertebrate steroidogenic pathways were isolated: StAR-related lipid transfer protein 3 (StAR3); 3ß-hydroxysteroid dehydrogenase (3ßHSD); two isoforms of 17ß-hydroxysteroid dehydrogenase 8 (17ßHSD8); and, estradiol-related receptor (ERR). RT-PCR analysis revealed that these genes were widely distributed in the eyestalk ganglia, hepatopancreas, brain, ovary, spermathecae, ovigerous and plumose setae tissues of adult females. The 17ßHSD8 transcripts were localized in the follicle cells, the periphery of the nuclear membrane of primary oocytes, and yolk granules of the vitellogenic oocytes using in situ hybridization, and the corresponding protein was detected in the follicle cells and ooplasm of primary oocytes using immunohistochemistry. Furthermore, the adult females injected with CFSH-dsRNA (n = 30 times) had E2 and StAR3 transcripts levels lower in the ovigerous and plumose setae, spermathecae than controls. These results suggested that the mode of CFSH action in C. sapidus might involve E2 in these adult-female-specific tissues.

Seasonal changes in the expression of insulin-like androgenic hormone (IAG) in the androgenic gland of the Jonah crab, Cancer borealis.

Harvesting the adult male Jonah crab, Cancer borealis, mainly based on the size, has become an economically significant fishery, particularly in the Southern New England region of the US since 2000. Many decapod crustacean fisheries including C. borealis rely on harvesting adult males. Understanding the size related-sexual maturity and the seasonal changes in male reproductive activity is critical for sustainable management. In other decapods, an insulin-like hormone produced by the male-specific androgenic gland (AG), called insulin-like androgenic gland factor (IAG), plays an essential role in sexual maturity. Specifically IAG is involved in developing male primary and secondary sexual characteristics including spermatogenesis. This study aimed first to identify the IAG, then examine if season influences IAG expression in C. borealis males. Finally, the AG transcriptome was used to test if eyestalk neuropeptides regulate IAG levels via an endocrine axis between the two endocrine tissues as established in other crustaceans. The full-length CabIAG sequence is 928 nucleotides long, encoding a 151 amino acid deduced sequence. The CabIAG identified from the AG transcriptome after eyestalk ablation was the most highly expressed gene and accounted for up to 25% of transcripts, further confirming the presence of an endocrine axis between the androgenic gland and eyestalk ganglia. This gene expression was exclusive in male C. borealis AG. The transcriptomic analysis also revealed strong upregulation of the PPOAE transcript and downregulation of proteolytic enzymes. The CabIAG levels differ by season, increasing AG activity in fall and possibly coinciding with high mating activity. The timing of increased AG activity correlating to mating with females should be considered for better stock management for the C. borealis population.

Experimental effects of cadmium on physiological response of Callinectes danae (Crustacea, Portunidae) from environments with different levels of Cd contamination.

Cadmium (Cd) can adversely affect aquatic life, altering reproductive and molting processes in crustaceans. The objective of this study was to evaluate the influence of Cd on reproduction and molting in the crab Callinectes danae. Adult females were obtained from environments with different levels of pollution: low (LC), medium (MC), and high contaminated (HC) areas. Animals from LC, MC, and HC areas were exposed to 0, 0.5, and 2 mg L-1 of CdCl2 for 3 h. Cd bioaccumulation, oxidative stress (evaluated by antioxidant enzymes activity), and lipid peroxidation (LPX) were analyzed in mature ovaries (stage II), gills, and hepatopancreas. The expression levels of crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) genes were quantified in the eyestalks, while 17ß-estradiol (E2) and melatonin concentration were measured in the hemolymph. Cd bioaccumulated mainly in the hepatopancreas and gills, with increased E2, LPX, and antioxidant enzymes in HC compared to the LC region. Decreased CHH and MIH transcripts were observed in the animals from HC regions compared to LC and MC areas. Physiological differences were recorded, especially for bioaccumulation, oxidative stress, and hormone levels, in animals sampled in HC areas compared to LC and MC regions. In conclusion, the physiological damage triggered by Cd could be reduced due to higher levels of melatonin and antioxidant enzymes in HC areas.

Cloning of an insulin-like androgenic gland factor (IAG) from the blue crab, Callinectes sapidus: implications for eyestalk regulation of IAG expression.

In malacostracan crustaceans, sex differentiation is uniquely regulated by a hormone secreted by the male-specific androgenic gland (AG). An isopod AG hormone was the first to be structurally elucidated and was found to belong to the insulin superfamily of proteins. Recently, it has been found that the AGs of several decapod crustaceans express insulin-like androgenic gland factors (IAGs), whose function is believed to be similar to that of the isopod AG hormone. Here we report the isolation from the blue crab Callinectes sapidus of the full-length cDNA encoding a candidate insulin-like AG hormone, termed Cas-IAG. The predicted protein Cas-IAG was encoded as a precursor consisting of a signal peptide, the B chain, the C peptide, and the A chain in that order. While the AG was the main source of Cas-IAG expression, as found in other decapod species, the hepatopancreas of male Callinectes sapidus crabs displayed minor Cas-IAG expression. Eyestalk ablation confirmed the presence of a possible endocrine axis between the eyestalk ganglia and the AG, implying that Cas-IAG expression is negatively regulated by (a) substance(s) present in the eyestalk ganglia.

Understanding molt control switches: Transcriptomic and expression analysis of the genes involved in ecdysteroidogenesis and cholesterol uptake pathways in the Y-organ of the blue crab, Callinectes sapidus.

The crustacean molting process is regulated by an interplay of hormones produced by the eyestalk ganglia and Y-organs (YO). Molt-inhibiting hormone and crustacean hyperglycemic hormone released by the sinus gland of the eyestalk ganglia (EG) inhibit the synthesis and secretion of ecdysteroid by the YO, hence regulating hemolymph levels during the molt cycle. The purpose of this study is to investigate the ecdysteroidogenesis pathway, specifically genes linked to changes in ecdysteroid levels occurring at early premolt (ePM). To this end, a reference transcriptome based on YO, EG, and hepatopancreas was de novo assembled. Two genes (cholesterol 7-desaturase Neverland and cytochrome p450 307a1-like Spook) involved in ecdysteroidogenesis were identified from the YO transcriptome using sequence comparisons and transcript abundance. Two other candidates, Hormone receptor 4 and probable cytochrome p450 49a1 potentially involved in ecdysteroidogenesis were also identified. Since cholesterol is the ecdysteroid precursor, a putative cholesterol carrier (Apolipoprotein D-like) was also examined to understand if cholesterol uptake coincided with the increase in the ecdysteroid levels at the ePM stage. The expression level changes of the five candidate genes in the YO were compared between intermolt (IM) and induced ePM (iePM) stages using transcriptomic analysis. Expression analysis using qPCR were carried out at IM, iePM, and normal ePM. The increase in Spook and Neverland expression in the YO at the ePM was accompanied by a concomitant rise in ecdysteroid levels. The data obtained from iePM stage were congruent with those obtained from the normal ePM stage of intact control animals. The present findings support the role of Halloween genes in the ecdysteroidogenesis and molt cycle in the blue crab, Callinectes sapidus.

Chromosome-level genome assembly of the blue crab, Callinectes sapidus.

The blue crab, Callinectes sapidus (Rathbun, 1896) is an economically, culturally, and ecologically important species found across the temperate and tropical North and South American Atlantic coast. A reference genome will enable research for this high-value species. Initial assembly combined 200× coverage Illumina paired-end reads, a 60× 8 kb mate-paired library, and 50× PacBio data using the MaSuRCA assembler resulting in a 985 Mb assembly with a scaffold N50 of 153 kb. Dovetail Chicago and HiC sequencing with the 3d DNA assembler and Juicebox assembly tools were then used for chromosome scaffolding. The 50 largest scaffolds span 810 Mb are 1.5-37 Mb long and have a repeat content of 36%. The 190 Mb unplaced sequence is in 3921 sequences over 10 kb with a repeat content of 68%. The final assembly N50 is 18.9 Mb for scaffolds and 9317 bases for contigs. Of arthropod BUSCO, ∼88% (888/1013) were complete and single copies. Using 309 million RNAseq read pairs from 12 different tissues and developmental stages, 25,249 protein-coding genes were predicted. Between C. sapidus and Portunus trituberculatus genomes, 41 of 50 large scaffolds had high nucleotide identity and protein-coding synteny, but 9 scaffolds in both assemblies were not clear matches. The protein-coding genes included 9423 one-to-one putative orthologs, of which 7165 were syntenic between the two crab species. Overall, the two crab genome assemblies show strong similarities at the nucleotide, protein, and chromosome level and verify the blue crab genome as an excellent reference for this important seafood species.

N-glycan moieties of the crustacean egg yolk protein and their glycosylation sites.

Vitellogenin (Vg) is the precursor of the egg yolk glycoprotein of crustaceans. In the prawn Macrobrachium rosenbergii, Vg is synthesized in the hepatopancreas, secreted to the hemolymph, and taken up by means of receptor-mediated endocytosis into the oocytes. The importance of glycosylation of Vg lies in its putative role in the folding, processing and transport of this protein to the egg yolk and in the fact that the N-glycan moieties could provide a source of carbohydrate during embryogenesis. The present study describes, for the first time, the structure of the glycan moieties and their sites of attachment to the Vg of M. rosenbergii. Bioinformatics analysis revealed seven putative N-glycosylation sites in M. rosenbergii Vg; two of these glycosylation sites are conserved throughout the Vgs of decapod crustaceans from the Pleocyemata suborder (N 159 and N 660). The glycosylation of six putative sites of M. rosenbergii Vg (N 151, N 159, N ,168 N ,614 N 660 and N 2300) was confirmed; three of the confirmed glycosylation sites are localized around the N-terminally conserved N-glycosylation site N 159. From a theoretical three-dimensional structure, these three N-glycosylated sites N 151, N 159, and N 168 were localized on the surface of the Vg consensus sequence. In addition, an uncommon high mannose N-linked oligosaccharide structure with a glucose cap (Glc1Man9GlcNAc2) was characterized in the secreted Vg. These findings thus make a significant contribution to the structural elucidating of the crustacean Vg glycan moieties, which may shed light on their role in protein folding and transport and in recognition between Vg and its target organ, the oocyte.