Role Of Crustaceans Research Articles

Insights into lipid function for ovarian development in the swimming crab (Portunus trituberculatus): A comparison of lipid mobilization and deposition in hepatopancreas and ovary

Ovarian development is a complex and dynamical process regulated by the coordinated actions of the hepatopancreas and ovary. In mammals, lipids provide necessary energy for oocytes to mature, whereas their regulatory role in crustaceans is not fully understood. This study compares lipid mobilization and deposition in hepatopancreas and ovary of the swimming crab at different stages of ovarian maturation. The results revealed that cholesterol (CHO) and triglyceride (TG) content in hepatopancreas increased with ovary development increased from stage I to Ⅲ, and then decreased at stage V, while those content in ovary significantly increased from stage I to V. Additionally, apolipoprotein (ApoA and ApoB) contents in hepatopancreas and ovary increased significantly during ovary development. Moreover, the highest expression levels of cholesterol transport related genes (srb, ldlr, lrp2 and abcg1) were observed in the hepatopancreas at stage Ⅲ and in the ovary at stage Ⅳ. Furthermore, as ovary development form stage I to Ⅳ, the hepatopancreas up-regulated the expression levels of genes related to lipid anabolism (fas, acc, g6pd, and 6pgd) and fatty acid transport (fabp9 and fatp4). Concurrently, genes related to lipid catabolism (il) and fatty acid β-oxidation (acox2 and acox3) in the hepatopancreas were down-regulated. Differently, with the ovary development increased from stage I to Ⅳ, the ovary up-regulated gene expression levels involved in lipid anabolism (acc, 6pgd, g6pd, dgat1, and gpat3), fatty acid synthesis (elovl and elovl4) and fatty acid β-oxidation (cpt1, cpt2, acox1, and acox3). The mRNA expression levels of lipolysis (tgl) and fatty acid transport (fabp1, fbap9, and fatp4) in ovary were down-regulated. Overall, these findings demonstrate that the hepatopancreas and ovary exhibit different responses to lipid metabolism during ovarian development.

Corazonin Stimulates Ecdysteroid Synthesis during the Molting Process of the Swimming Crab, Portunus trituberculatus.

The neuropeptide corazonin (Crz) exerts diverse physiological effects in insects, yet its role in crustaceans remains elusive. The abundant expression of Crz receptor (CrzR) in the Y-organs of several crustaceans suggests a potential involvement of Crz in regulating ecdysteroid synthesis. In this study, we examined the effects of PtCrz on ecdysteroid synthesis during the molting period of Portunus trituberculatus through PtCrz treatments and PtCrzR silencing. Our results showed that PtCrz peptide stimulates ecdysteroid levels and the gene expression involved in ecdysteroidogenesis both in vitro and in vivo, whereas dsPtCrzR treatments had opposite effects on ecdysteroid levels and associated gene expression. Thus, our study suggests that PtCrz may modulate ecdysteroid synthesis via Y-organ-expressed PtCrzR. Furthermore, we also discovered the involvement of PtCrz/PtCrzR signaling in regulating PtETH expression. Notably, the inhibitory effect of dsPtCrzR on ecdysteroid synthesis or PtETH expression can be reversed by PtCrz treatment, suggesting the potential existence of multiple receptors for PtCrz. This study provides new insights into the function of crustacean Crz and, for the first time, elucidates the presence of a neuropeptide that can stimulate ecdysteroid synthesis in crustaceans.

Identification of ETH receptor and its possible roles in the mud crab Scylla paramamosain

Ecdysis-triggering hormone (ETH) is a neuropeptide hormone characterized by a conserved KxxKxxPRx amide structure widely identified in arthropods. While its involvement in the regulation of molting and reproduction in insects is well-established, its role in crustaceans has been overlooked. This study aimed to de-orphanise a receptor for ETH in the mud crab Scylla paramamosain and explore its potential impact on ovarian development. A 513-amino-acid G protein-coupled receptor for ETH (SpETHR) was identified in S. paramamosain, exhibiting a dose-dependent activation by SpETH with an EC50 value of 75.18 nM. Tissue distribution analysis revealed SpETH was in the cerebral ganglion and thoracic ganglion, while SpETHR was specifically expressed in the ovary, hepatopancreas, and Y-organ of female crabs. In vitro experiments demonstrated that synthetic SpETH (at a concentration of 10−8 M) significantly increased the expression of SpVgR in the ovary and induced ecdysone biosynthesis in the Y-organ. In vivo experiments showed a significant upregulation of SpEcR in the ovary and Disembodied and Shadow in the Y-organ after 12 h of SpETH injection. Furthermore, a 16-day administration of SpETH significantly increased 20E titers in hemolymph, gonadosomatic index (GSI) and oocyte size of S. paramamosain. In conclusion, our findings suggest that SpETH may play stimulatory roles in ovarian development and ecdysone biosynthesis by the Y-organ.

A new type II CHH neuropeptide involves ovarian development in the peppermint shrimp, Lysmata vittata.

Type II crustacean hyperglycemic hormone (CHH) neuropeptides play diverse roles in crustaceans. In the hermaphrodite shrimp Lysmata vittata, two transcripts of type II CHHs (molt-inhibiting hormone/gonad-inhibiting hormone, MIH/GIH1 and MIH/GIH2) were identified by transcriptome sequencing, and MIH/GIH1 was later named Lvit-GIH1 for its inhibitory effect on ovarian development. Based on the high similarity of MIH/GIH2 to Lvit-GIH1, we named tentatively MIH/GIH2 as Lvit-GIH2 and explored the role of Lvit-GIH2 in ovarian development. The open reading frame (ORF) of Lvit-GIH2 was 333 bp in length, encoding a precursor consisted of a 32-aa signal peptide and a 78-aa mature peptide, which shared high sequence similarity with the type II subfamily peptides in crustaceans. Notably, Lvit-GIH2 was widely expressed in multiple tissues. The qRT-PCR findings indicated a rising trend in the expression of Lvit-GIH2 from the male phase to the euhermaphrodite phase. Both RNA interference and addition of GIH2 recombinant proteins (rGIH2) experiments showed that Lvit-GIH2 suppressed Lvit-Vg expression in hepatopancreas and Lvit-VgR expression in ovary. To further investigate the role of Lvit-GIH2 in ovarian development, the RNA-sequence analysis was performed to examine the changes in ovary after addition of rGIH2. The results showed that the pathways (Cysteine and methionine metabolism, Apoptosis-multiple species, etc.) and the genes (17bHSD8, IGFR, CHH, etc.) related to ovarian development were negatively regulated by rGIH2. In brief, Lvit-GIH2 might inhibit the ovarian development in L. vittata.

Large-scale lysine crotonylation analysis reveals the role of TRAF6-Ecsit complex in endoplasmic reticulum stress in mud crab (Scylla paramamosain)

Lysine crotonylation (Kcr) is a newly discovered type of post-translational modification. Although Kcr has been reported in several species, its role in crustaceans remains largely unknown. In this study, Kcr in hemocytes of mud crab (Scylla paramamosain) was characterized using pan anti-crotonyllysine antibody enrichment and high-resolution liquid chromatogram-mass spectrometry analysis after SpTRAF6 or SpEcsit silencing. Altogether, 1,800 Kcr sites with six conserved motifs were identified from 512 proteins. Subcellular localization analysis showed that the identified Kcr proteins were mainly localized to the cytoplasm, nucleus, and mitochondria. The cellular components analysis showed that the ‘chromosomal region’ was enriched in the hemocytes of SpTRAF6-or SpEcsit-silenced mud crabs. The KEGG and PPI analyses showed that the identified Kcr proteins in the hemocytes SpTRAF6-or SpEcsit-silenced mud crabs were related to the ‘protein processing in endoplasmic reticulum’; of which the marker of endoplasmic reticulum stress (Bip) was identified to be crotonylated. These datasets present the first comprehensive analysis of the crotonylome in mud crab hemocytes, providing invaluable insights into the regulatory functions of SpTRAF6 and SpEcsit in Kcr. Additionally, our findings shed light on the potential role of these proteins in activating marker proteins during endoplasmic reticulum stress in invertebrates.

Putative role of corazonin in the ovarian development of the swimming crab Portunus trituberculatus

Corazonin (Crz) is a neuropeptide that widely distributed in insects and crustaceans. The Crz is proposed to have pleiotropic functions in insects, but its physiological roles in crustaceans are poorly understood. In the present study, Crz and its putative receptor (CrzR) were identified from the swimming crab, Portunus trituberculatus, and their interaction was validated using the Dual-Luciferase reporter assay system. Tissue distribution analysis showed the PtCrz was mainly derived from center nerve system, while its receptor was highly expressed in Y-organ, the main site for ecdysteroids synthesis. Exposure of YO to synthetic Crz and CrzR dsRNA respectively led to the transcriptional changes of two ecdysteroidogenesis genes, further indicating a putative role of Crz signaling on ecdysteroids synthesis. During the ovarian development, the mRNA levels of PtCrz and PtCrzR increased significantly in vitellogenic stages, suggesting a potential role of Crz signaling in vitellogenesis. The hypothesis was further strengthened by in vitro experiments that the expression of vitellogenin (Vg), Vg receptor (VgR), cyclinB, and Cdc2 in ovary explants could be induced by synthetic Crz, whereas reduced by CrzR dsRNA. In addition, since 20-hydroxyecdysone also showed a stimulating effect on Vg expression, an indirect regulation of Crz signaling on ovarian development via ecdysteroids might also exist.

Gene cloning and transcriptional regulation of the alkaline and acid phosphatase genes in Scylla paramamosain.

In crustaceans, innate immunity serves as the frontline of defense against microbes. Alkaline phosphatases (ALPs) and acid phosphatases (ACPs) are essential enzymes that play a significant role in crustaceans' immune defenses. However, the function and transcriptional regulation of the alp and acp genes in the Scylla paramamosain, an important aquaculture species in China, have not been elucidated. In this study, the full-length cDNAs of Spalp and Spacp were identified, which consist of 2,718bp and 3,768bp, encoding 579 and 452 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis showed that these two genes were conserved among different species and shared high homology with crustaceans. The mRNA expression of Spalp and Spacp were examined in eight tested tissues, with the highest levels in the hepatopancreas. The 5'-flanking regions of Spalp and Spacp were cloned and sequenced. The core promoter region of the Spalp and Spacp was -39bp∼+8 bp and -39bp∼+10bp, respectively. Potential binding sequences for SOX-2, c-fos, SP1, NF-κB, GATA-1, YY1, and AP-1 transcription factors were found in the 5'-flanking regions of Spalp and Spacp. The NF-κB binding site located between -1,223bp and -972bp in Spalp while SP1 and AP-1 binding sites located between -1,249bp and -514bp in Spacp. Mutation analysis confirmed that NF-κB negatively regulated the expression of Spalp gene, and SP1 and AP-1 positively regulated Spacp gene expression. These results provide us with essential information to elucidate the function of the Spalp and Spacp in S. paramamosain. This study is the first one to analyze the activity of Spalp and Spacp promoters.

Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt-/- larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox-/- mutants, which synthesized MF but no JH, completed the entire life cycle. While epox-/- adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.

Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress

Chitinase is a multi-gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase-4 gene (PmChi-4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi-4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi-4 protein was 67.7 kDa and shared 61%–88% identity with the type of Chi-4s from other crustaceans. Quantitative real-time (qRT-PCR) analysis indicated that PmChi-4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi-4 was expressed throughout the whole larvae stages, and the highest level of PmChi-4 transcripts was detected at Mysis3 stage, which indicated that PmChi-4 may be involved in larval metamorphosis. In order to know whether PmChi-4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi-4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi-4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi-4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon.

Molecular characterization and function of the Prohibitin2 gene in Litopenaeus vannamei responses to Vibrio alginolyticus

Prohibitin2 (PHB2), a potential tumor suppressor protein, plays important roles in inhibition of cell cycle progression, transcriptional regulation, apoptosis and the mitochondrial respiratory chain. To explore its potential roles in crustaceans' immune responses we have identified and characterized LvPHB2, a 891 bp gene encoding a 297 amino acids protein in the shrimp Litopenaeus vannamei. Expression analyses showed that LvPHB2 is expressed in all examined tissues, and largely present in cytoplasm, correlating with its known anti-oxidation function in mitochondria. Luciferase reporter assays showed that over-expression of LvPHB2 could activate the p53 pathway, indicating that it might participate in apoptosis regulation. Quantitative real-time PCR revealed that infection with Vibrio alginolyticus induces its up-regulation in hepatopancreas. Moreover, RNAi knock-down of LvPHB2 in vivo raises mortality rates of L. vannamei infected by V. alginolyticus, and affects expression of STAT3, Caspase3 and p53 genes. We found significantly higher reactive oxygen species production, DNA damage and apoptosis rates in LvPHB2-silenced shrimp challenged with V. alginolyticus than in controls injected with a Green Fluorescent Protein-silencing construct. Our results suggest that LvPHB2 plays a vital role in shrimp responses to V. alginolyticus infection through its participation in regulation of oxidants and apoptosis.

Visualization of ecdysteroid activity using a reporter gene in the crustacean, Daphnia

Ecdysone is a hormone known to play a pivotal role in crustaceans and insects. In order to evaluate the ecdysone activities in the environment and within the organism, we have developed a biomonitoring Daphnia strain by introducing a reporter gene. In this study, the ecdysone response element was inserted in the upstream region of a reporter gene, and the DNA construct was injected into Daphnia eggs. The expression of the reporter gene was detected during the early embryonic development stage. In addition, when the eggs expressing the reporter gene were exposed to ecdysone, there was enhanced expression of the reporter gene at detectable levels, while the presence of an antagonist led to its downregulation. These results suggested that this system could be potentially developed for monitoring ecdysone activities in media.

Six chitinases from oriental river prawn Macrobrachium nipponense: cDNA characterization, classification and mRNA expression during post-embryonic development and moulting cycle

Chitinase plays crucial physiological roles in crustaceans, including the digestion of chitin-containing food, moulting and the defense of shrimp against viruses. However, in contrast to insect species, no genome-wide analysis has been carried out in crustacean species and cDNAs encoding chitinase and chitinase-like proteins have been characterized in relatively few species. In this study, we identified six chitinase genes in the oriental river prawn, Macrobrachium nipponense, according to the established expressed sequence tag (EST) information using Rapid Amplification of the cDNA Ends (RACE) technique and homology cloning. We assigned these genes to three different chitinase groupings, which were designated MnCht1A, 1B, 3A, 3B, 3C and 4. The domain organization analysis of the six MnCht proteins revealed that only MnCht3C and MnCht4 possessed full structure, while MnCht1A, 1B, 3A and 3B lacked the serine/threonine (S/T)-rich linker and chitin-binding domains (CBDs). Their expression in different tissues and different developmental stages suggested that all of them have a function in the digestion of chitinous foods, modification of gut peritrophic membrane and degradation of the chitin exoskeleton. Analysis of the stage-specific moulting cycle and different temperature stimulation provided further evidence that MnCht1A, 1B and 3B have pivotal roles in the moulting cycle, while MnCht 4 only assists in the moulting process. This study provides important information for further investigations on the functions of chitinase in M. nipponense and other crustaceans.

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+, suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.

A Valanginian crustacean microcoprolite ichnofauna from the shallow-marine hydrothermal vent site of Zengővárkony (Mecsek Mts., Hungary)

A rich and diverse crustacean microcoprolite ichnofauna is reported from the Valanginian of the Mecsek Mts., Hungary. Localities from the Bolondut and Dezső Rezső valleys near Zengővarkony (Eastern Mecsek Mts.) provided Favreina aff. dispentochetarius, Palaxius darjaensis, P. triochetarius,P. salataensis, P. tetraochetarius, and P. decaochetarius. They occur in limonitic and partly metasomatized limestones, limestone olistoliths, and ammonite body-chambers. The remarkable richness of this ichnofauna plus the previous records from the same environment (altogether 11 ichnospecies of four ichnogenera from a single stratigraphic level) make it the most diverse crustacean ichnofauna of the Mesozoic. The former ecosystem of the ichnofauna is considered a deeper shallow-marine (water depth <300 m) hydrothermal vent on which the producers of the microcoprolites lived. The related brachiopod fauna shows a remarkable size and is dominated by Lacunosella. This fauna is not similar to typical fossil deep-sea vent faunas; however, it offers a thorough understanding of other fossil shallow-marine hydrothermal vent faunas and the role of crustaceans in these ecosystems. This is the first record of crustacean coprolites from such a fossil site and documents that crustaceans were diverse and played an important role in shallow-marine, non photosynthesis-based ecosystems in the geological past.

Calcium accumulation and regulation in Daphnia magna: Links with feeding, growth and reproduction

Calcium is involved in a wide variety of biological processes and has an important structural role in crustaceans. The present study aimed at exploring the possible link between Ca body concentrations and the ingestion rate and the role of soft tissue vs. total tissue Ca accumulation in Daphnia magna. D. magna was cultured for 21 days at different water Ca concentrations ranging from 3.4 to 32.5 mg/L. Every week Ca body concentrations (soft and total tissues), ingestion rate, growth, survival and reproduction were measured. Daily, algal food that was not deficient in Ca was supplied. Ca in the soft tissues represented 8 to 26% of the total Ca body concentrations. The ratio Ca in soft tissue/Ca in total tissue was generally not influenced by the Ca exposure concentration but decreased with time, i.e., age (from an average of 0.24 at day 7 to 0.09 at day 21). During week 1, a 54% decrease in Ca body concentrations was observed in daphnids exposed in medium with 3.4 mg/L Ca compared to those exposed to 32.5 mg/L. The concurrent decrease in ingestion rate was 14%. No significant differences among Ca treatments were observed during week 2 for ingestion rate and week 3 for calcium body concentrations. Also, no effects on growth and reproduction were observed, although these were expected at the lowest Ca concentration tested. It is hypothesised that Ca absorption from food in combination with an increased ingestion rate is used to maintain Ca homeostasis under Ca limiting conditions.

Identification of a Juvenile Hormone-Like Compound in a Crustacean

Juvenile hormone (JH) has central roles in the regulation of insect development and reproduction but has not previously been identified in other arthropod classes. The hemolymph of a crustacean, Libinia emarginata (Leach), has now been analyzed for JH-like compounds. Samples contained 0.003 to 0.030 nanogram of JH III per milliliter and 10 to 50 nanograms of methyl farnesoate per milliliter; methyl farnesoate is a compound structurally related to JH III that has JH bioactivity. Several tissues were examined for synthesis and secretion of JH-like compounds. Of these tissues, only the mandibular organs produced and secreted JH III and methyl farnesoate. However, microchemical analysis revealed that this JH III was racemic, and thus likely an artifactual oxidation product of methyl farnesoate. Secretion of methyl farnesoate was related to reproduction in females, with the highest rates observed in Libinia near the end of the ovarian cycle when oocyte growth and vitellogenesis are greatest. These results indicate that JH-like compounds such as methyl farnesoate have regulatory roles in crustaceans.

Lobster muscle and synapse respond to cortisol, a vertebrate hormone.

Cortisol (0.28 mumol X L-1) applied to lobster (Homarus americanus) neuromuscular preparations produces a hyperpolarization in muscle fibers and an increase in amplitude of excitatory postsynaptic potentials. The effect appears to be surface-mediated, because of its rapid onset (within seconds). It is also Na+-K+ ATPase dependent, because ouabain blocks the effects. The effects are relatively short-lasting, and gradually subside within 15 min. The increase in excitatory postsynaptic potentials is attributed in part to increased quantal output of transmitter, and not to changes in muscle fiber membrane resistance. The effects of cortisol on neuromuscular transmission and membrane potential indicate that cortisol may have a physiological role in crustaceans.