Characterization of microRNAs in Mud Crab Scylla paramamosain under Vibrio parahaemolyticus Infection

Background

Infection of bacterial Vibrio parahaemolyticus is common in mud crab farms. However, the mechanisms of the crab’s response to pathogenic V. parahaemolyticus infection are not fully understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs that function as regulators of gene expression and play essential roles in various biological processes. To understand the underlying mechanisms of the molecular immune response of the crab to the pathogens, high-throughput Illumina/Solexa deep sequencing technology was used to investigate the expression profiles of miRNAs in S . paramamosain under V. parahaemolyticus infection.

Methodology/Principal Findings

Two mixed RNA pools of 7 tissues (intestine, heart, liver, gill, brain, muscle and blood) were obtained from V. parahaemolyticus infected crabs and the control groups, respectively. By aligning the sequencing data with known miRNAs, we characterized 421 miRNA families, and 133 conserved miRNA families in mud crab S . paramamosain were either identical or very similar to existing miRNAs in miRBase. Stem-loop qRT-PCRs were used to scan the expression levels of four randomly chosen differentially expressed miRNAs and tissue distribution. Eight novel potential miRNAs were confirmed by qRT-PCR analysis and the precursors of these novel miRNAs were verified by PCR amplification, cloning and sequencing in S . paramamosain . 161 miRNAs (106 of which up-regulated and 55 down-regulated) were significantly differentially expressed during the challenge and the potential targets of these differentially expressed miRNAs were predicted. Furthermore, we demonstrated evolutionary conservation of mud crab miRNAs in the animal evolution process.

Conclusions/Significance

In this study, a large number of miRNAs were identified in S . paramamosain when challenged with V. parahaemolyticus, some of which were differentially expressed. The results show that miRNAs might play some important roles in regulating gene expression in mud crab under V. parahaemolyticus infection, providing a basis for further investigation of miRNA-modulating networks in innate immunity of mud crab.     

锯缘青蟹精巢发育的组织学观察

组织学观察表明,锯缘青蟹（Scylla serrata)精巢发育周期可以分为精原细胞期,精母细胞期,精子细胞期,精子期和休止期5个时期。在划分精巢发育期的基础上,结合生精小管直径的成熟节段比例这2个指标,可以较准确地反映锯缘青解精巢的发育状况。而成熟节段比例可以作为锯缘青蟹精巢发育的表征指标。     

锯缘青蟹复眼的形态和超微结构

对锯缘青蟹(Scylla serrata)的复眼做了电镜观察。扫描电镜下,半球形复眼的背面有一拇指状的无小眼区。透射电镜下,小眼为十足目短尾类特有的长六边形;复眼内小眼的感光系统包括了11个小网膜细胞(RCs),4个RCs位于感光部分的远端,7个RCs构成了感光系统的近端主体;上下两群细胞连接处清晰显示“4 7”且有局部交错的结构。这与三疣梭子蟹和罗氏沼虾溞状幼体的文献记述相似,而与蜘蛛蟹和中华绒螯蟹的“1 7”结构不同,可能与它们对视觉依赖程度差异有关。     

Genetic Identification of all Four Mangrove Mud Crab Species (genus Scylla) Using Multiple Molecular Markers

Biochemical Genetics - Misleading identification and subsequent publications on biological, molecular, and aquaculture data of mangrove mud crab (genus Scylla de Hann 1833) is a major concern in...     

Functional Assessment of Residues in the Amino- and Carboxyl-Termini of Crustacean Hyperglycemic Hormone (CHH) in the Mud Crab Scylla olivacea Using Point-Mutated Peptides

To assess functional importance of the residues in the amino- and carboxyl-termini of crustacean hyperglycemic hormone in the mud crab Scylla olivacea (Sco-CHH), both wild-type and point-mutated CHH peptides were produced with an amidated C-terminal end. Spectral analyses of circular dichroism, chromatographic retention time, and mass spectrometric analysis of the recombinant peptides indicate that they were close in conformation to native CHH and were produced with the intended substitutions. The recombinant peptides were subsequently used for an in vivo hyperglycemic assay. Two mutants (R13A and I69A rSco-CHH) completely lacked hyperglycemic activity, with temporal profiles similar to that of vehicle control. Temporal profiles of hyperglycemic responses elicited by 4 mutants (I2A, F3A, D12A, and D60A Sco-CHH) were different from that elicited by wild-type Sco-CHH; I2A was unique in that it exhibited significantly higher hyperglycemic activity, whereas the remaining 3 mutants showed lower activity. Four mutants (D4A, Q51A, E54A, and V72A rSco-CHH) elicited hyperglycemic responses with temporal profiles similar to those evoked by wild-type Sco-CHH. In contrast, the glycine-extended version of V72A rSco-CHH (V72A rSco-CHH-Gly) completely lost hyperglycemic activity. By comparing our study with previous ones of ion-transport peptide (ITP) and molt-inhibiting hormone (MIH) using deleted or point-mutated mutants, detail discussion is made regarding functionally important residues that are shared by both CHH and ITP (members of Group I of the CHH family), and those that discriminate CHH from ITP, and Group-I from Group-II peptides. Conclusions summarized in the present study provide insights into understanding of how functional diversification occurred within a peptide family of multifunctional members.     

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.     

Functional Analysis of TolC Homologs in Vibrio vulnificus

Gram-negative bacteria use tripartite pumps to transport antibacterial drugs and other toxic compounds across the inner and outer membranes, which are separated by the periplasmic space. The TolC protein is an outer membrane factor that participates in the formation of tripartite efflux pumps. The genome of Vibrio vulnificus encodes two E. coli TolC homologs, TolCV1 and TolCV2. Here, we show that both TolCV1 and TolCV2 are involved in the efflux of antimicrobial agents. Deletion of tolCV1 resulted in increased susceptibility of V. vulnificus to chemical detergents, DNA intercalating agents, and antibiotics including erythromycin, novobiocin, and tetracycline, whereas deletion of tolCV2 rendered V. vulnificus more susceptible to the above mentioned antibiotics only. We also observed that tolCV1 deletion resulted in reduced motility of V. vulnificus. Our results indicate active roles for TolCV1 and TolCV2 in the physiology of V. vulnificus.     

Identification and Functional Analysis of the Nocardithiocin Gene Cluster in Nocardia pseudobrasiliensis

Nocardithiocin is a thiopeptide compound isolated from the opportunistic pathogen Nocardia pseudobrasiliensis. It shows a strong activity against acid-fast bacteria and is also active against rifampicin-resistant Mycobacterium tuberculosis. Here, we report the identification of the nocardithiocin gene cluster in N. pseudobrasiliensis IFM 0761 based on conserved thiopeptide biosynthesis gene sequence and the whole genome sequence. The predicted gene cluster was confirmed by gene disruption and complementation. As expected, strains containing the disrupted gene did not produce nocardithiocin while gene complementation restored nocardithiocin production in these strains. The predicted cluster was further analyzed using RNA-seq which showed that the nocardithiocin gene cluster contains 12 genes within a 15.2-kb region. This finding will promote the improvement of nocardithiocin productivity and its derivatives production.     

Characterization and Functional Analysis of Four Glutathione S-Transferases from the Migratory Locust,Locusta migratoria

Glutathione S-transferases (GSTs) play an important role in detoxification of xenobiotics in both prokaryotic and eukaryotic cells. In this study, four GSTs (LmGSTd1, LmGSTs5, LmGSTt1, and LmGSTu1) representing different classes were identified from the migratory locust, Locusta migratoria. These four proteins were heterologously expressed in Escherichia coli as soluble fusion proteins, purified by Ni²⁺-nitrilotriacetic acid agarose column and biochemically characterized. LmGSTd1, LmGSTs5, and LmGSTu1 showed high activities with 1-chloro-2, 4-dinitrobenzene (CDNB), detectable activity with p-nitro-benzyl chloride (p-NBC) and 1, 2-dichloro-4-nitrobenzene (DCNB), whereas LmGSTt1 showed high activity with p-NBC and detectable activity with CDNB. The optimal pH of the locust GSTs ranged between 7.0 to 9.0. Ethacrynic acid and reactive blue effectively inhibited all four GSTs. LmGSTs5 was most sensitive to heavy metals (Cu²⁺ and Cd²⁺). The maximum expression of the four GSTs was observed in Malpighian tubules and fat bodies as evaluated by western blot. The nymph mortalities after carbaryl treatment increased by 28 and 12% after LmGSTs5 and LmGSTu1 were silenced, respectively. The nymph mortalities after malathion and chlorpyrifos treatments increased by 26 and 18% after LmGSTs5 and LmGSTu1 were silenced, respectively. These results suggest that sigma GSTs in L. migratoria play a significant role in carbaryl detoxification, whereas some of other GSTs may also involve in the detoxification of carbaryl and chlorpyrifos.     

Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris

Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BB^ΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BB^ΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications.     

Effects of Mesh Size and Escape Gaps on Discarding in an Australian Giant Mud Crab (Scylla serrata) Trap Fishery

In response to concerns over excessive discarding from Australian recreational round traps (with four funnel entrances) used to target giant mud crabs, Scylla serrata, an experiment was done to assess the independent and cumulative utility of paired, bottom-located horizontal escape gaps (46×120 mm) and increasing mesh size (from 51 to 101 mm). Compared to conventional traps comprising 51-mm mesh throughout, those with the same mesh size and escape gaps caught significantly fewer (by 95%) undersize (<85 mm carapace length – CL) crabs while maintaining legal catches. Traps made from 101-mm mesh (but with the same funnel entrances as conventional designs) and with and without escape gaps similarly retained fewer undersize crabs and also yellowfin bream Acanthopagrus australis (the key bycatch species) by up to 94%, but there were concomitant reductions in fishing power for legal sizes of S. serrata. Although there were no immediate mortalities among any discarded crabs, there was a greater bias towards wounding among post molts than late inter-molts and less damage to individuals in the 101-mm conventional than 51-mm conventional traps (without escape gaps). The results support retrospectively fitting escape gaps in conventional S. serrata traps as a means for reducing discarding, but additional work is required to determine appropriate mesh sizes/configurations that maximize species and size selectivity.