Normal Crabs Research Articles

The forkhead box O transcription factor regulates lipase and anti-microbial peptide expressions to promote lipid catabolism and improve innate immunity in the Eriocheir sinensis with hepatopancreatic necrosis disease

Eriocheir sinensis is a crustacean with great economic value, but the occurrence of hepatopancreatic necrosis disease (HPND) severely restricts the development of crab aquaculture. Study on the survival mechanism of crabs with HPND is beneficial to provide new strategies for disease prevention and control. The Forkhead box O (FOXO) transcription factor family is involved in various key biological processes of organisms. In this study, a FOXO gene (named as EsFOXO) from E. sinensis was cloned. The full-length cDNA of EsFOXO is 2592 bp containing a 2133 bp open reading frame that encodes 710 amino acids. EsFOXO was widely distributed in multiple immune tissues. Further study found that the expression of EsFOXO in the intestine of crabs with HPND was significantly upregulated compared with that in the normal crabs. However, whether EsFOXO is involved in the immune and metabolic regulation of crabs remains unknown. RNA interference analysis showed that EsFOXO participates in the positive regulation of the expression of two pancreatic lipases, three anti-lipopolysaccharide factors, and three crustins. Results from our research suggest that two strategies are adopted by crabs with HPND for survival under starvation: on the one hand, the synthesis of antimicrobial peptides is increased to improve the innate immunity; on the other hand, the expression of enzymes correlated with lipid catabolism is up-regulated that mobilizes the fat in the crab, going through catabolism. Our study provides more evidence for an in-depth understanding of the survival mechanism of crabs with HPND.

Identification and function analysis of an immune deficiency homolog in swimming crab, Portunus trituberculatus

The immune deficiency (IMD) pathway is involved in both antiviral and antibacterial immune responses in Drosophila. IMD protein is the key adaptor to link the extracellular signal and the intracellular reaction to initiate the signal transduction in IMD pathway. In present study, the cDNA of the IMD (Pt-IMD) was identified from a marine crab, Portunus trituberculatus. The Pt-IMD is predicted to encode 170 amino acids with a death domain. Real-Time quantitative PCR analysis showed that Pt-IMD was constitutively expressed in hemocytes, intestine, gill, heart, muscle and hepatopancreas in normal crab. Moreover, the transcript of Pt-IMD in large-granule hemocytes is approximately 6-fold higher than semi-granular cells and agranular cells. Intracellular localization showed Pt-IMD was distributed mainly in the cytoplasm when it was over-expressed in Drosophila Schneider 2 (S2) cell. Functionally, over-expression of Pt-IMD could activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. Furthermore, Pt-IMD expression was also knock-down by RNAi to determine the function of Pt-IMD on regulation of the expression of different antimicrobial peptides (AMPs) in crab. In the primary cultured hemocytes challenged with or without Vibrio alginolyticus, after Pt-IMD was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), ALF3, crustin1, crustin3, arasin2, hyastatin1and hyastatin3 have been significantly inhibited in normal cell or bacterial infected cell, while the expression of lysozyme was normal in non-infected cells and was significantly induced in bacterial infected cells, which compared to the non-specific dsRNA treated cells.

Comparison of the Nutritional Quality of Three Edible Tissues from Precocious and Normal Adult Female Chinese Mitten Crabs (Eriocheir sinensis)

ABSTRACT We investigated and evaluated the nutritional composition of the hepatopancreas, gonads, and muscle from two types (precocious/normal) of adult female Chinese mitten crabs (Eriocheir sinensis). Our results showed that normal crabs had the highest gonadosomatic index, meat yield, and hepatosomatic index. There was no considerable difference in the proximate composition of the edible parts of normal vs precocious crabs, particularly in the muscles and gonads. A balanced amino acid composition and high amino acid score was reported in the muscle and gonads of the precocious crabs. The normal crabs had less amino acid contents than the precocious crabs in gonads and muscle. Regarding the mineral element profile, in the muscle of the precocious crabs, the contents of Mg, Ca, and Mn were significantly higher than those in the normal crabs. Regarding the fatty acid profile, the hepatopancreas and gonads of the precocious crabs had more polyunsaturated fatty acids (PUFA) but less monounsaturated fatty acids (MUFA) than in the normal crabs. The muscle of precocious crabs contained significantly higher unsaturated fatty acids (UFA) but lower saturated fatty acids (SFA). Precocious and adult female Chinese mitten crabs were found to have a higher nutritional value and can contribute to a healthy diet.

Characterization, subcellular localization and function analysis of myeloid differentiation factor 88 (Pt-MyD88) in swimming crab, Portunus trituberculatus

Myeloid differentiation factor 88 (MyD88) is a universal and essential adaptor protein required for the Toll-like receptors (TLRs) pathway activation in invertebrates as well as in vertebrates. Herein, we characterized a MyD88 (Pt-MyD88) cDNA sequence in the swimming crab (Portunus trituberculatus). The Pt-MyD88 ORF is predicted to encode 469 peptides with an N-terminal death domain and a typical C-terminal TIR domain. Real-Time quantitative PCR analysis showed that the Pt-MyD88 transcriptions were constitutively expressed in hemocytes, gill, intestine, heart and muscle in normal crab. The expressions of Pt-MyD88 would be down-regulated by V. alginolyticus or LPS challenge, and be up-regulated by WSSV infection in hemocytes. Intracellular localization showed Pt-MyD88 was distributed mainly in the cytoplasm when it was over-expressed in human cell HEK293T or in Drosophila Schneider 2 (S2). Functionally, over-expression of Pt-MyD88 could either activate the NF-κB in HEK293T cells or activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. In primary cultured hemocytes of swimming crab, after Pt-MyD88 was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), hyastatin3, crustin1 and crustin3 have been significantly inhibited, while the expression of other AMPs is normal compared to non-specific dsRNA treated cells.

Nutritional qualities of normal and precocious adult male Chinese mitten crabs (Eriocheir sinensis)

The nutritional values of edible gonad, hepatopancreas and muscle tissues from normal and precocious adult male Chinese mitten crabs (Eriocheir sinensis) were investigated herein. The results showed that the average weight and meat yield (MY) of normal crabs were significantly higher than those of precocious crabs, while the hepatopancreas indexes (HSI) and gonadosomatic indexes (GSI) had no significant differences. Precocious and normal crabs had greater protein and lipid contents, respectively, and the differences were mainly reflected in gonads and hepatopancreas. While assessing the amino acid profiles, both precocious and normal crabs could be high-quality protein sources, particularly the precocious crabs which showed higher levels of essential amino acids. In fatty acid profiling experiments, precocious crabs revealed higher EPA, ∑n-3 polyunsaturated fatty acids (PUFA) and ∑n-3/∑n-6 ratios than normal crabs; however, DHA and ∑n-6 PUFA contents were considerably lower. Additionally, contents of nutritious minerals K and Ca in precocious crabs were significantly higher than those in normal crabs, while Zn and Fe were significantly lower. In short, our data indicated that precocious Chinese mitten crabs are also a nutritious food, and even in some respects their nutritional value is higher than normal crabs.

Enzymes involved in immunity and characteristics of hemolymph in red sternum syndrome mud crabs (Scylla serrata)

The activities were compared of the immunological enzymes phenoloxidase (PO) and superoxide dismutase (SOD) and the concentration of proteins and urea nitrogen (UN) in the hemolymph of red sternum syndrome mud crabs, Scylla serrata, (Forskål, 1755) (Decapoda: Portunidae), with those of normal crabs. Twenty-five red sternum mud crabs and 10 normal mud crabs were randomly collected from crab farms in Samut Songkram province, Thailand. Statistical analysis used the mean ± SD and Student's t test at a significance level of α = 0.05. Levels of PO were 0.032 ± 0.013 units/mg protein and 0.075 ± 0.028 units/mg protein, while SOD levels were 0.493 ± 0.079 units/mg protein and 1.464 ± 0.783 units/mg protein, in normal and red sternum mud crabs, respectively. There was a significant difference in enzyme activities between the two groups. The hemolymph protein concentration in normal crabs was 1.03 ± 0.20 mg/mL which was significantly higher than in red sternum syndrome crabs (0.66 ± 0.23 mg/mL). The UN level in the hemolymph of normal mud crabs was significantly higher (10.78 ± 3.96 mg/dL) compared with that of red sternum mud crabs (4.59 ± 1.57 mg/dL). The findings revealed that the protein and UN levels in the hemolymph of red sternum mud crabs had decreased significantly. The red sternum syndrome was inversely related to the accumulated amount of protein and UN in crabs and had an extreme effect on protein metabolism.

β-actin gene expression is variable among individuals and not suitable for normalizing mRNA levels in Portunus trituberculatus

The housekeeping gene encoding β-actin appears to be the most widely-used internal reference for gene expression studies in experimental animals or their cell lines. However, the effectiveness of β-actin to normalize mRNA levels expression in many crustacean species is still object of debate. To date, it is still unclear if β-actin is suitable to be utilized as the internal reference in qualitative real-time gene expression study in crab species. To address this concern, we evaluated 5 candidate reference genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A, elongation factor 1-α (EF1-α), and 18 S ribosomal RNA (18 S rRNA) in the swimming crabs (Portunus trituberculatus) models. Our data showed that the β-actin gene expression varied significantly across individual swimming crab individuals in gills or hemocytes and the expression of 18 S rRNA, EF1-α, cyclophilin or GAPDH gene were relatively stable compared to that of β-actin. Moreover, the expression stability of the reference genes among different tissues in normal crabs or after WSSV challenge was also tested by geNorm and NormFinder software. Among tissues, 18 S rRNA was most stably expressed in different tissues, followed by cyclophilin A and EF1-α, compared to β-actin and GAPDH. Upon to viral simulation, GAPDH was found to be the most stable internal control gene in gills and cyclophilin A was ranked as the most stable gene in hemocytes.

Does the wild-caught Chinese mitten crab megalopae perform better than the hatchery-produced seed during the juvenile culture?

Although wild Yangtze Eriocheir sinensis juveniles (coin‐sized seed) have better culture performance than the pond‐reared juveniles during the adult crab culture stage, the culture performance of wild‐caught megalopae (WC) remains unknown. Therefore, this study was designed to compare the growth, survival rate, crab yield, feed conversion rate (FCR), precocity, final weight and size distribution between WC and hatchery‐produced megalopae (HP) during the juvenile culture stage. Wild‐caught megalopae had significantly higher body weight, weight gain rate (WGR) and specific growth rate (SGR) during 0–4 weeks but lower WGR and SGR than HP during 4–8 weeks (p < .05). Although WC had a significantly lower survival rate and yield of normal juvenile crabs than HP, total yields of normal juveniles and precocious crabs were similar. Wild‐caught megalopae had a significantly higher precocious rate and yield of precocious crabs than HP (p < .05). Regardless of sex, no significant differences were found in the gonadosomatic index (GSI), hepatosomatic index (HSI) and final weight of normal juveniles and precocious crabs between WC and HP. As for the size distribution of normal crabs, WC had a higher percentage of small female juveniles (<3 g) but a lower proportion of middle size ranges (3–5.99 g and 6–8.99 g) than HP for females (p < .05), and there was no significant difference for males. In conclusion, wild megalopae of the Yangtze E. sinensis had worse culture performance than the hatchery‐produced seeds; therefore, wild megalopae would not be recommended for commercially juvenile culture of E. sinensis.

SpCrus3 and SpCrus4 share high similarity in mud crab (Scylla paramamosain) exhibiting different antibacterial activities

Type I crustins are crucial effectors of crustacean immune system. Various type I crustins with high sequence diversity possess different antimicrobial activities. To date, the mechanism on how the sequence diversity of type I crustins affects their antimicrobial activities is largely unclear, and how different crustins function together against bacterial invasion still remains unknown. In this study, we identified two novel type I crustins, namely, SpCrus3 and SpCrus4, from an economically important crab, Scylla paramamosain. Either SpCrus3 or SpCrus4 was highly expressed in gill. After challenges with Vibrio parahemolyticus or Staphylococcus aureus, SpCrus4 was up-regulated, whereas SpCrus3 was down-regulated. No significant expression change of SpCrus3 and SpCrus4 was observed after white spot syndrome virus injection, suggesting that these two genes may not participate in the antiviral immune responses. SpCrus3 and SpCrus4 had the common 5′ terminus and high similarity of 66.06%, but SpCrus4 exhibited stronger antimicrobial activity than that of SpCrus3. Microorganism-binding assay results revealed that both SpCrus3 and SpCrus4 exhibited binding ability to all tested microorganisms. Furthermore, the polysaccharide-binding assay showed that these two proteins exhibited strong binding activity to bacterial polysaccharides, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN). SpCrus3 and SpCrus4 exhibited stronger binding activity to LPS or LTA than to PGN. Moreover, SpCrus4 showed stronger binding activity to LTA than that of SpCrus3, which may be responsible for the significantly distinct antimicrobial activity between these two proteins. In addition, SpCrus4 displayed stronger agglutination activity against several kinds of microorganisms than that of SpCrus3. This increased agglutination activity may also contribute to the strong antibacterial activity of SpCrus4. On the basis of all these results, a possible antibacterial mode exerted by SpCrus3 and SpCrus4 was proposed as follows. SpCrus3 was highly expressed in normal crabs to maintain low-level antibacterial activity without bacterial challenges. When crabs were challenged with bacteria, large amount of SpCrus4 was generated to exhibit strong antibacterial activity against bacterial invasion. This study provides new insights to understand the antibacterial functions and mechanisms of type I crustins.

The parasitic barnacle Sacculina lata Boschma, 1933 (Cirripedia, Rhizocephala, Sacculinidae) infecting the commercial swimming crab Charybdis miles (De Haan, 1835) (Decapoda, Portunidae) in the Beibu Gulf, South China Sea

AbstractA sacculinid rhizocephalan parasite,Sacculina lataBoschma, 1933, was discovered in the Beibu Gulf (= Gulf of Tonkin) west of Hainan Island, China, occurring in 7.4% of 363Charybdis miles(De Haan, 1835) collected by 30 trawls in July 2017. This is the first time thatS. latahas been recorded in Beibu Gulf and in the Chinese Exclusive Economic Zones (EEZs). Of the sampled crabs from 9 stations, 8 males and 19 females were found bearing externae ofS. lataon the abdomen, a prevalence of 4.1 and 11.2% in male and female crabs, respectively. The dominant size group of infected males was larger than that of females. There was no significant relationship between water depth and infection rate of the parasite. A positive correlation was found both between volume and weight of externae, and between surface area of externae and abdomen width of the host crabs. Carapace width and wet weight of infected crabs were significantly less than those of uninfected crabs. In male crabs, the first pleopods of infected individuals were shorter than normal, but their pleons were wider. The propodus of the chela of a parasitized crab was significantly larger than that of normal crabs, both in males and females. No rhizocephalan-infected female crab was found bearing eggs.

Cytotoxic effect of crab shell extracts on different tumor cell lines

Crab, a kind of marine animal that has a unique status in evolution, has many bioactive substances with special functions. In the present study, we investigated the antitumor effects of four different extracts derived from crab shell (aqueous and methanolic extracts of burned and normal crab shell powder). Aqueous and methanolic extracts of burned crab shell powder were shown to be cytotoxic and inhibit the in vitro proliferation of human laryngeal carcinoma tumor cell lines (Hep-2) and human Rhabdomyosarcoma cell line (RD) and one murine mammary adenocarcinoma tumor cell line (AMN3), in dose and time dependant manner. These results suggest that the burned crab shell powder have an antitumor effect that needs more investigations.

Infestation of parasitic rhizocephalan barnacles Sacculina beauforti (Cirripedia, Rhizocephala) in edible mud crab, Scylla olivacea.

Screening of mud crab genus Scylla was conducted in four locations (Marudu Bay, Lundu, Taiping, Setiu) representing Malaysia. Scylla olivacea with abnormal primary and secondary sexual characters were prevalent (approximately 42.27% of the local screened S. olivacea population) in Marudu Bay, Sabah. A total of six different types of abnormalities were described. Crabs with type 1 and type 3 were immature males, type 2 and type 4 were mature males, type 5 were immature females and type 6 were mature females. The abdomen of all crabs with abnormalities were dented on both sides along the abdomen’s middle line. Abnormal crabs showed significant variation in their size, weight, abdomen width and/or gonopod or pleopod length compared to normal individuals. The mean body weight of abnormal crabs (type 1–5) were higher than normal crabs with smaller body size, while females with type 6 abnormality were always heavier than the normal counterparts at any given size. Sacculinid’s externa were observed in the abdomen of crabs with type 4 and type 6 abnormalities. The presence of embryos within the externa and subsequent molecular analysis of partial mitochondrial COI region confirmed the rhizocephalan parasite as Sacculina beauforti. Future in-depth descriptions of the life cycle and characteristics of S. beauforti are recommended as it involves a commercially important edible crab species and the effect on human health from the consumption of crabs is of crucial concern.

Reproductive performance, offspring quality, proximate and fatty acid composition of normal and precocious Chinese mitten crab Eriocheir sinensis

Precocity is a common phenomenon among farmed Chinese mitten crab Eriocheir sinensis that significantly affect culture productivity and profitability. This study compared the reproductive performance, offspring quality, proximate and fatty acid composition of pond-reared precocious and normal sexual matured females of E. sinensis. While the average gonado-somatic index (GSI) of the normal female crabs was significantly lower than that of the precocious crabs, fecundity, egg fertilization and hatching rate were significantly higher in the former. On the other hand, comparing various larval quality parameters measured with newly hatched larvae produced by the two types of females, only significant difference detected was dry weight with larvae produced by the normal females having significant higher dry weight. Biochemical analysis showed that eggs produced by precocious females contained a higher water content, but lower DHA, n−3 PUFA and n−3 HUFA contents than those produced by normal crabs and egg fertilization and hatching rates were, in general, correlated positively with the biochemical and fatty acid composition of the eggs. However, interestingly, such a correlation was not identified in newly hatched larvae. The results of hepatopancreas and ovarian proximate and fatty acid composition analysis suggested that lower reproductive performance of precocious females is likely related to the deficiencies in PUFA and general poorer nutrient conditions of these crabs. Statement of relevanceThe Chinese mitten crab, Eriocheir sinensis, is currently farmed throughout China. Precocity, commonly accounts for 15–30% of cultured crab populations, is a major problem for E. sinensis aquaculture. The precocious crabs reach terminal moult at small sizes, hence have low market value. To date, it is unknown whether the precocious crabs are reproductive functional. This study confirmed that precocious E. sinensis could produce viable offspring, and subsequently compared reproductive performance and offspring quality between normal and precocious E. sinensis, providing useful information for aquaculture of the species.

Effects of limb autotomy on growth, feeding and regeneration in the juvenile Eriocheir sinensis

Eriocheir sinensis, as an emerging aquaculture species, has become more popular in Chinese pond culture. However, high density stocking has usually caused high limb autotomy rate among juvenile crabs, and there are still very few reports about the effects of autotomy on both survival and growth rates in the juvenile E. sinensis. Therefore, we aimed at the effects of limb autotomy on the growth of E. sinensis through the individual cultivation method. The results showed that almost all injured juvenile crabs could feed and molt as normal crabs, and could manage to regenerate new limbs. The duration of the 1st molt had a negative correlation with the number of autotomized limbs. After the 2nd molt, the size of the new limb was comparable to that of the normal limb and the weight growth rate decreased with the increasing number of autotomized limbs. However, a positive correlation was found between the specific growth rate and the number of autotomized limbs during the 1st molting period. In this period, the feeding rate decreased slightly, and the crabs with more limb loss tended to have the lower feeding rates, but this had no significant difference (P>0.05). Besides, feed efficiencies in experimental sets were slightly higher than the control, and those crabs that autotomized more limbs correspondingly had the higher feed efficiencies (P<0.05). Meanwhile, the results also revealed that limb autotomy had no significant effects on the duration of the 2nd molting, the weight growth rate, the specific growth rate as well as the feeding rate in E. sinensis juveniles. Statement of relevanceEriocheir sinensis, as one emerging aquaculture species, has become more popular in Chinese pond culture. However, the high culture density has usually caused a high rate of autotomy among the juveniles and there are still few reports on the effects of autotomy on the survival and growth rate in E. sinensis. The present study was to assess the effect of autotomy on the growth of E. sinensis such as the molting period, weight growth rate, specific growth rate, feeding rate as well as feed efficiency in E. sinensis juveniles, through the individual cultivation method.

Bacterial flora of mud crab, Scylla olivacea, collected from different markets of Dhaka City

The present study was carried out to investigate the bacterial flora of mud crab (Scylla olivacea) meat collected from the five markets of Dhaka city. Faecal Coliform, Salmonella sp. and Shigella sp., E. coil, and Vibrio spp. were detected in most of the samples except the frozen ones from Agora super shop. The samples from Basabo and Karwan bazar had higher bacterial load than the others. The highest TBC counts were observed in the samples from Karwan bazar (38.1 ± 1.20 x 104cfu/g) and the lowest were from Agora super shop (4.48 ± 0.26 x104cfu/g). The total Vibrio spp. counts were found only in two markets, i.e. from Farmgate and Basabo. The total E. coli counts ranged from 1.25 ± 0.02 x 104cfu/g to 4.33 ± 0.56 x 104cfu/g. The Salmonella and Shigella counts varied between3.95 ± 0.28 x 104cfu/g for Karwan bazaar and 16.76 ± 1.26 x 104cfu/g for Basabo. Total Staphylococcus spp. counts ranged between 14.4 ± 1.1 x 104cfu/g and 20.01± 1.94 x 104cfu/g for normal crab samples and 0.37 ± 0.025 x 104cfu/g for frozen samples. The results of this study showed that the bacterial load was significantly higher in live crab than the frozen crab indicating that later are safe for human consumption.Bangladesh J. Zool. 43(1): 55-62, 2015

Hemolymph Responses of The Xanthid Crab Actaea hirsutissima to Rhizocephalan Sacculina ignorata From Hurghada, Red Sea

The effect of parasitic invasion by the Rhizocephala Sacculina ignorata on brachyuran crabs Actaea hirsutissima was studied. The organic constituents (glucose, total proteins, albumin, triglycerides and cholesterol) of the haemolymph of the infected and normal crab Actaea hirsutissima were estimated. A significant reduction in serum protein and albumin was observed in parasitized individuals; while serum glucose, cholesterol and triglycerides concentrations were significantly increased. Parasitism by S. ignorata dramatically affects the serum chemistry of infected crabs. T-test values were statistically highly significant between healthy and infected specimens.

Transcriptome profiling of the eyestalk of precocious juvenile Chinese mitten crab reveals putative neuropeptides and differentially expressed genes

Chinese mitten crabs that reach maturity 1year earlier than normal crabs are known as precocious juvenile crabs. The molecular mechanisms underlying the precocity of the Chinese mitten crab are poorly understood. To identify the genes that may be involved in the control of precocity in Chinese mitten crab, we measured the expression profile of eyestalk genes in precocious and normally developed juvenile crabs using high-throughput sequencing on an Illumina HiSeq 2500 platform. We obtained 56,446,284 raw reads from the precocious crabs and 58,029,476 raw reads from the normally developed juvenile crabs. Reads from the two libraries were combined into a single data set. De novo assembly of the combined read set yielded 78,777 unigenes with an average length of 1563bp. A total of 41,405 unigenes with predicted ORFs were selected for functional annotation. Among these genes, we identified three neuropeptide genes belonging to the crustacean hyperglycemic hormone family and two neuropeptide genes encoding the chromatophorotropic hormones. Transcriptome comparison between the two libraries revealed 42 genes that exhibited significant differential expression, of which 29 genes were up-regulated and 13 genes were down-regulated in the precocious crabs. To confirm the sequencing data, six differentially expressed genes with functional annotations were selected and validated by qRT-PCR. In conclusion, we obtained the comprehensive transcriptome of the eyestalk tissues of precocious juvenile crabs. The sequencing results may provide new insights into the biomolecular basis of precocity in the Chinese mitten crab.

Identification, characterization, and functional studies of a Pelle gene in the Chinese mitten crab, Eriocheir sinensis.

The toll-like receptor/NF-κB signaling pathways play an important role in the innate immune system. In the present study, one Pelle gene (named EsPelle) was identified for the first time from the Chinese mitten crab Eriocheir sinensis. The full-length cDNA of EsPelle is 3797 bp with a 3156 bp-long open reading frame that encodes a 1051 amino acid protein. EsPelle protein contains a death domain at the N-terminal and a serine/threonine kinase domain at the C-terminal. A neighbor joining phylogenetic tree showed that the EsPelle protein, which is closest to those of Scylla paramamosain Pelle and Litopenaeus vannamei Pelle, was clustered to a group of crustacean Pelle proteins. EsPelle was expressed in all tested tissues of normal crabs, and its expression was regulated in hemocytes and hepatopancreas of crabs challenged with lipopolysaccharide, peptidoglycan, Staphyloccocus aureus, Vibrio parahaemolyticus, and Aeromonas hydrophila. Overexpression of EsPelle in Drosophila Schneider 2 cells could upregulate the expression of Drosophila antimicrobial peptides, namely, metchnikowin (Mtk), attacinA (Atta), drosomycin (Drs), and cecropinA (CecA). Moreover, EsPelle silencing by siRNA reduced the transcription of anti-lipopolysaccharide factor 1 and 2, crustin 2, and lysozyme in crabs challenged with V. parahaemolyticus. From the results, we speculated that EsPelle was involved in innate immune defense against V. parahaemolyticus in E. sinensis.

Occurrence and effects of the rhizocephalan parasite Diplothylacus sinensis (Cirripedia: Rhizocephala: Thomsoniidae) in the swimming crab Portunus sanguinolentus (Decapoda: Portunidae) in Honghai Bay, South China Sea

A thompsoniid rhizocephalan parasite, Diplothylacus sinensis (Keppen, 1877), was discovered in the semi-open Honghai Bay in the South China Sea, occurring in 26.4% of 1852 (934 males and 918 females) Portunus sanguinolentus (Herbst, 1783) (Decapoda: Portunidae) collected monthly between November 2012, and October 2013. Of the sampled crabs, 243 males and 246 females were infected with externae of D. sinensis or bore attachment scars on the body surface, giving an overall prevalence of infection of 26.0% and 26.8% in male and female crabs, respectively. The infection rate changed monthly with the highest prevalence in December (50.6%) and the lowest in June (1.3%). New externae filled with eggs or embryos were observed throughout the year, which indicates a year-round reproductive cycle, but virgin externae were more commonly present in late spring or early summer. Carapace width and wet weight of infected crabs of both sexes were significantly smaller than those of uninfected crabs. Compared to normal crabs, the pleon of infected females was significantly wider and the first pleopod of parasitized males was shorter. On addition, crabs infected by the rhizocephalan parasite were more susceptible to other epibionts, which included Octolasmis warwickii Gray, 1825 (10.1%), Chelonibia patula (Ranzani, 1818) (6.8%), Obelia spp. (7.2%) and Membranipora membranacea (6.5%) on the carapace surface, and Octolasmis bullata (Aurivillius, 1894) (64.7%) on the gills.

Novel myeloid differentiation factor 88, EsMyD88, exhibits EsTube-binding activity in Chinese mitten crab Eriocheir sinensis

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter protein that participates in the activation of the Toll-like receptor/interleukin-1 receptor-mediated signaling pathway. In the present study, a new MyD88 gene (named EsMyD88) was identified in the Chinese mitten crab Eriocheir sinensis. The cDNA of EsMyD88 was 2210 bp long with a 1416 bp open reading frame that encoded a protein with 472 amino acids. Predicted EsMyD88 protein had a death domain at the N-terminal and a TIR domain at the C-terminal. BLASTP and phylogenetic analysis results showed that EsMyD88 was clustered in one group together with other crustaceans MyD88 (SpMyD88, FcMyD88, LvMyD88, and LvMyD88-1). EsMyD88 was detected in all the examined tissues of healthy crabs, and was mainly expressed in the hemocytes and nerves. When normal crabs were challenged with lipopolysaccharide, peptidoglycan, Staphylococcus aureus, Vibrio parahaemolyticus, or Aeromonas hydrophila, the expression levels of EsMyD88 significantly increased either in the hepatopancreas or hemocytes. Results of the pull-down assay showed that EsMyD88 could bind to downstream cytosolic adaptor EsTube. Overexpression of EsMyD88 protein in Drosophila Schneider 2 cells led to the activation of antimicrobial peptide genes. RNA interference assay showed that EsMyD88 is involved in regulating the transcription of ALF1 and ALF2, Cru1 and Cru2, and Lys in crab challenged with V. parahaemolyticus. All the results mentioned earlier indicated that EsMyD88 gene has a key function in antibacterial innate immune defense.