SpALF4: A newly identified anti-lipopolysaccharide factor from the mud crab Scylla paramamosain with broad spectrum antimicrobial activity

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with binding and neutralizing activities to lipopolysaccharide (LPS) in crustaceans. This study identified and characterized a novel ALF homolog (SpALF4) from the mud crab Scylla paramamosain. The complete cDNA of SpALF4 had 756 bp with a 381 bp open reading frame encoding a protein with 126 aa. The deduced protein contained a signal peptide and a LPS-binding domain. SpALF4 shared the highest identity with PtALF5 at amino acid level but exhibited low similarity with most of other crustacean ALFs. Furthermore, different from the previously identified three SpALF homologs and most of other ALFs, SpALF4 had a low isoelectric point (pI) for the mature peptide and the LPS-binding domain with the values of 6.93 and 6.74, respectively. These results indicate that SpALF4 may be a unique ALF homolog with special biological function in the mud crab. Similar to the spatial structure of ALFPm3, SpALF4 contains three α-helices packed against a four-strand β-sheet, and an amphipathic loop formed by a disulphide bond between two conserved cysteine residues in LPS-binding domain. SpALF4, mainly distributed in hemocytes, could be upregulated by Vibrio harveyi, Staphylococcus aureus, or white spot syndrome virus. Recombinant SpALF4 could inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio anguillarum, Vibrio alginolyticus, Aeromonas hydrophila, Pseudomonas putida), Gram-positive bacteria (S. aureus and Bacillus megaterium), and a fungus Candida albicans to varying degrees. Further study showed that it could also bind to all the aforementioned microorganisms except S. aureus. These results demonstrate that SpALF4 is a unique ALF homolog with potent antimicrobial activity against bacteria and fungi. This characteristic suggests SpALF4 plays an essential function in immune defense against pathogen invasion in mud crab.     

Inhibitory effects of the androgenic gland on ovarian development in the mud crab Scylla paramamosain

Isolation and characterization of androgenic hormone in decapod crustaceans depend on an effective bioassay of its action. In the present study, the effect of androgenic gland on ovarian development in the mud crab Scylla paramamosain was investigated with a view to develop a bioassay for androgenic hormone. Ovarian regression with degeneration of oocytes occurred in some female crabs implanted with androgenic gland in vivo. In vitro incubation of ovarian tissues at secondary vitellogenesis in extract of androgenic gland resulted in a significant decrease in amino acid uptake by the tissues. We propose that this inhibitory effect could be established as an effective bioassay for the isolation of androgenic hormone in the mud crab.     

Production and characterization of monoclonal antibodies to the hemocytes of mud crab, Scylla serrata

Monoclonal antibodies (MAbs) to hemocytes of mud crab, Scylla serrata, were produced by immunizing mice with formalin-fixed hemocytes. Of the six MAbs produced, two (MAb 1D11 and MAb 1A2) reacted specifically with hemocyte proteins in western blot. MAb 1A2 showed strong immunofluorescent reaction with granular hemocytes and a weak reaction with semigranular cells. However, hyaline cells did not show any reaction. The MAbs also showed strong cross-reactivity with the hemocytes of different crab species but not with other crustaceans. The MAbs produced can be used as a marker for granular hemocytes of crabs.     

低温胁迫下两种不同形态群体拟穴青蟹转录组分析

为研究拟穴青蟹(Scylla paramamosain)不同形态群体对温度变化的适应差异,以Sp1群体(腕节外缘中部都不具有发达的刺)和Sp2群体(至少一只螯足腕节外缘中部具有发达的刺)为研究对象,选用低温胁迫组(8℃)和对照组(20℃)拟穴青蟹肝胰腺为实验材料,进行转录组测序。结果显示,测序共获得81853个Unigene,平均长度为420 bp, N₅₀为1460 bp,其中22.33%为已知基因。差异表达基因筛选发现Sp1群体在低温胁迫下共有15773个Unigene显著差异表达,其中15628个Unigene表达下调, 145个Unigene表达上调,这些Unigene显著富集于细胞质基质等生物学过程和RNA结合、核糖体结构组分等分子功能,并进一步富集在核糖体、剪接体和内质网蛋白质加工等信号通路。Sp2群体在低温胁迫下共有323个Unigene显著差异表达,其中上调114个,下调209个。这些Unigene显著富集于DNA整合等生物学过程和甾体羟化酶活性、碳酸氢盐跨膜转运体活性等分子功能,并进一步富集在赖氨酸降解、甘氨酸、丝氨酸和苏氨酸的代谢、精氨酸和脯氨...     

Molecular cloning and expression of a 2-Cys peroxiredoxin gene in the crustacean Eurypanopeus depressus induced by acute hypo-osmotic stress

Peroxiredoxins (Prxs) are a family of ubiquitous proteins that help minimize the harmful effects of oxidative stress by catalyzing the reduction of hydrogen peroxide (H₂O₂) and organic hydroperoxides to less harmful forms. A full-length cDNA corresponding to a 2-Cys Prx gene was isolated from the flatback mud crab Eurypanopeus depressus and designated as EdPrx-1 (GenBank accession no. EU684547). EdPrx-1 has a major open-reading frame of 594 bp and is capable of encoding a polypeptide of 198 amino acid residues. Like other 2-Cys Prxs, EdPrx-1 protein possesses two conserved cysteine residues that play an essential role for the antioxidant activity of the proteins. The EdPrx-1 protein, as deduced from the cDNA sequence, shows a high level (74–93%) of sequence similarity to the 2-Cys Prxs from other crustaceans as well as those from many arthropod species (73–76% similarity). It shares about 70% sequence similarity with homologs from mammalian species. EdPrx-1 gene is expressed at low level in the gill, hypodermis, and hepatopancreas tissues of the crab under non-stressed condition; however, its expression is elevated about three-fold in the gills under hypo-osmotic stress. This suggests a possible role in protecting against oxidative stress caused by the increased metabolic activities associated with hyperosmoregulation.     

Erk2 in ovarian development of green mud crab Scylla paramamosain

We identified extracellular signal-regulated kinase 2 (erk2) from green mud crab, Scylla paramamosain, in this article. It was originally identified from an expressed sequence tag fragment from a normalized gonadal cDNA library. 5' Rapid amplification of cDNA end (RACE) technique was used to obtain the 5' untranslated region (UTR). The full-length cDNA of Sp-erk2 is 1516 bp, including a 5'-terminal UTR of 19 bp, an open-reading frame of 1098 bp, and a 3'-terminal UTR of 399 bp. The translated protein is 365 amino acids in length with a predicted molecular weight of 42 kDa, which is the same as other species. It is the first time that the expression of Sp-erk2 in different stages of ovary development of crustacean was analyzed, and the result showed that the expression of Sp-erk2 increased gradually with ovarian development, with a peak in the mature phase. In situ hybridization histochemistry was used to clarify the detail of expression. Positive signals illustrated that Sp-erk2 mRNA is present in follicular cells when the ovary is in early stages, and in both follicular cells and oocytes when it is in mature phases. All above suggest that Sp-erk2 is important for ovarian development.     

Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean

It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.     

Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea

Molecular Biology Reports - Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this...     

WSSV对锯缘青蟹的致病性及血清酶指标影响

锯缘青蟹(Scylla serrata)俗称青蟹,是我国重要的海水养殖蟹类.近年来,浙江、福建、广东等青蟹主要养殖地区出现了严重的青蟹病害.对浙江省养殖青蟹的发病原因和流行病学调查发现,白斑综合征病毒(white spotsyndrome virus,WSSV)与青蟹发病存在较大相关性.为进一步研究WSSV对青蟹的致病性和发病机理,作者采用白斑综合征病毒的除菌过滤液,以1:10-1:10000稀释度注射感染青蟹,结果表明1:10、1:100感染组的青蟹死亡率达100%,1:1000感染组死亡率为66.7%,1:10000感染组死亡率为38.9%.根据攻毒悬液的病毒浓度计算出WSSV对青蟹的LD50为1.19×104拷贝/只(7.93×103拷贝/g组织);取WSSV感染青蟹血淋巴进行PCR检测,攻毒死亡青蟹的WSSV检出率为100%,表明WSSV对青蟹有很强的致病力.分析病毒感染濒死蟹的血清酚氧化酶(PO)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、碱性磷酸酶(ALP)、谷丙转氨酶(GPT)、符草转氨酶(GOT)等主要酶指标,发现病毒感染青蟹的PO、POD和SOD活力明显低于对照组,而ALP、GPT和GOT的活力则明显高于对照组;用WSSV单克隆抗体对感染蟹进行免疫组化分析,发现WSSV主要侵染青蟹的鳃、甲壳下表皮、心脏、肠、胃等组织的上皮细胞,尤其以鳃上皮细胞损害最为严重.