Structural and functional differences of Litopenaeus vannamei crustins

Penaeid crustins were described in Litopenaeus vannamei and L. setiferus as proteins belonging to an antibacterial peptide family with similar sequences but different sizes. Six crustin-coding clones were isolated from a cDNA library from L. vannamei hemocytes, sequenced and compared. Two different isoforms (named I and P) were found, based on two nucleotide differences that produce one change in amino acid sequence (Ile/Pro). Other single differences in nucleotide sequences were also noted, but they did not change the translated product. The mRNA steady state levels of crustin I, but not of crustin P, were down regulated by Vibrio alginolyticus inoculation. Thus, the differences among penaeid crustins seem to be associated with one amino acid substitution, which affects their expression after bacterial inoculation. By structural similarity, shrimp crustins seem to belong to an antibacterial WAP-domain containing protein family.     

Cloning and characterisation of cDNA sequences encoding for anti-lipopolysaccharide factors (ALFs) in Brazilian palaemonid and penaeid shrimps

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides found in limulids and crustaceans that have a potent and broad range of antimicrobial activity. We report here the identification and molecular characterisation of new sequences encoding for ALFs in the haemocytes of the freshwater prawn Macrobrachium olfersi and also in two Brazilian penaeid species, Farfantepenaeus paulensis and Litopenaeus schmitti. All obtained sequences encoded for highly cationic peptides containing two conserved cysteine residues flanking a putative LPS-binding domain. They exhibited a significant amino acid similarity with crustacean and limulid ALF sequences, especially with those of penaeid shrimps. This is the first identification of ALF in a freshwater prawn.     

Molecular phylogeny of western Atlantic Farfantepenaeus and Litopenaeus shrimp based on mitochondrial 16S partial sequences

Partial sequences for the 16S rRNA mitochondrial gene were obtained from 10 penaeid shrimp species: Farfantepenaeus paulensis, F. brasiliensis, F. subtilis, F. duorarum, F. aztecus, Litopenaeus schmitti, L. setiferus, and Xiphopenaeus kroyeri from the western Atlantic and L. vannamei and L. stylirostris from the eastern Pacific. Sequences were also obtained from an undescribed morphotype of pink shrimp (morphotype II) usually identified as F. subtilis. The phylogeny resulting from the 16S partial sequences showed that these species form two well-supported monophyletic clades consistent with the two genera proposed in a recent systematic review of the suborder Dendrobranchiata. This contrasted with conclusions drawn from recent molecular phylogenetic work on penaeid shrimps based on partial sequences of the mitochondrial COI region that failed to support recent revisions of the Dendrobranchiata based on morphological analysis. Consistent differences observed in the sequences for morphotype II, coupled with previous allozyme data, support the conclusion that this is a previously undescribed species of Farfantepenaeus.     

Cationic Antimicrobial Peptides in Penaeid Shrimp

Penaeid shrimp aquaculture has been consistently affected worldwide by devastating diseases that cause a severe loss in production. To fight a variety of harmful microbes in the surrounding environment, particularly at high densities (of which intensive farming represents an extreme example), shrimps have evolved and use a diverse array of antimicrobial peptides (AMPs) as part of an important first-line response of the host defense system. Cationic AMPs in penaeid shrimps composed of penaeidins, crustins, and anti-lipopolysaccharide factors are comprised of multiple classes or isoforms and possess antibacterial and antifungal activities against different strains of bacteria and fungi. Shrimp AMPs are primarily expressed in circulating hemocytes, which is the main site of the immune response, and hemocytes expressing AMPs probably migrate to infection sites to fight against pathogen invasion. Indeed, most AMPs are produced as early as the nauplii developmental stage to protect shrimp larvae from infections. In this review, we discuss the sequence diversity, expression, gene structure, and antimicrobial activities of cationic AMPs in penaeid shrimps. The information available on antimicrobial activities indicates that these shrimp AMPs have potential therapeutic applications in the control of disease problems in aquaculture.     

Cationic Antimicrobial Peptides in Penaeid Shrimp

Penaeid shrimp aquaculture has been consistently affected worldwide by devastating diseases that cause a severe loss in production. To fight a variety of harmful microbes in the surrounding environment, particularly at high densities (of which intensive farming represents an extreme example), shrimps have evolved and use a diverse array of antimicrobial peptides (AMPs) as part of an important first-line response of the host defense system. Cationic AMPs in penaeid shrimps composed of penaeidins, crustins, and anti-lipopolysaccharide factors are comprised of multiple classes or isoforms and possess antibacterial and antifungal activities against different strains of bacteria and fungi. Shrimp AMPs are primarily expressed in circulating hemocytes, which is the main site of the immune response, and hemocytes expressing AMPs probably migrate to infection sites to fight against pathogen invasion. Indeed, most AMPs are produced as early as the nauplii developmental stage to protect shrimp larvae from infections. In this review, we discuss the sequence diversity, expression, gene structure, and antimicrobial activities of cationic AMPs in penaeid shrimps. The information available on antimicrobial activities indicates that these shrimp AMPs have potential therapeutic applications in the control of disease problems in aquaculture.     

Adaptive evolution of crustin antimicrobial peptides in decapods

Antimicrobial peptides (AMPs) are present in a wide range of taxonomic groups and played a crucial role in host adaptation to a diverse array of ever-changing pathogens. Crustin, a cysteine-rich cationic AMP, is known to exhibit antimicrobial activity against Gram-positive and Gram-negative bacteria in decapods. Given their important role in host-immune defense, a large proportion of amino acid substitutions in crustin AMPs are expected to be fixed by natural selection. Utilizing the complete coding nucleotide sequence data of crustin, the present study revealed the pervasive role of positive Darwinian selection in the evolution and divergence of crustin AMPs in decapods. Approximately, 20–35?% of codons in two phylogenetically distinct groups of closely related crustins in Penaeid shrimps are shown to have evolved under positive selection. Interestingly, several of these positively selected sites are located at the carboxyl-terminal region, the region that directly interacts with the invading pathogens. Pathogen-mediated selection pressure could be the likely cause for such an accelerated rate of amino acid substitutions and could have contributed to the structural and functional diversification of crustin AMPs in several taxa.     

Discovery of immune molecules and their crucial functions in shrimp immunity

Several immune-related molecules in penaeid shrimps have been discovered, most of these via the analysis of expressed sequence tag libraries, microarray studies and proteomic approaches. These immune molecules include antimicrobial peptides, serine proteinases and inhibitors, phenoloxidases, oxidative enzymes, clottable protein, pattern recognition proteins, lectins, Toll receptors, and other humoral factors that might participate in the innate immune system of shrimps. These molecules have mainly been found in the hemolymph and hemocytes, which are the main sites where immune reactions take place, while some are found in other immune organs/tissues, such as the lymphoid organs, gills and intestines. Although the participation of some of these immune molecules in the shrimp innate immune defense against invading pathogens has been demonstrated, the functions of many molecules remain unclear. This review summarizes the current status of our knowledge concerning the discovery and functional characterization of the immune molecules in penaeid shrimps.     

Temporal variation of the population structure and genetic diversity of Farfantepenaeus notialis assessed by allozyme loci

Population genetic studies carried out on penaeid shrimps have disclosed different patterns of population subdivision, revealing new aspects of shrimp biology as well as the effects of historical contingency molding those patterns. However, the stability of observed allele frequencies over time still remains untested. The objective of this article is to show the analysis of the temporal variation of allozymes in a shrimp species inhabiting Cuba which proves that the genetic structure of this species could significantly change in time. The study involves four populations of Farfantepenaeus notialis sampled in a period of 8 years. The significant statistics obtained from partitions observed in 1995 were not detected in 2003 (as suggested by AMOVA and F(ST)), whereas temporal genetic differentiation and heterozygosity became highly significant. The results strongly suggest that the effect of migrations could be the cause for the loss of F. notialis genetic structure in 2003. It is therefore imperative to call attention on the vulnerability of these populations when facing unstable environmental and habitat conditions.     

Phylogeny of whey acidic protein (WAP) four-disulfide core proteins and their role in lower vertebrates and invertebrates

Proteins containing WAP (whey acidic protein) domains with a characteristic WFDC (WAP four-disulfide core) occur not only in mammals (including marsupials and monotremes) but also in birds, reptiles, amphibians and fish. In addition, they are present in numerous invertebrates, from cnidarians to urochordates. Many of those from non-mammalian groups are poorly understood with respect to function or phylogeny. Those well characterized so far are waprins from snakes, perlwapins from bivalves and crustins from decapod crustaceans. Waprins are venom proteins with a single WAP domain at the C-terminus. They display antimicrobial, rather than proteinase inhibitory, activities. Perlwapins, in contrast, possess three WAP domains at the C-terminus and are expressed in the shell nacre of abalones. They participate in shell formation by inhibiting the growth of calcium crystals in the shell. The crustin group is the largest of all WFDC-containing proteins in invertebrates with the vast majority being highly expressed in the haemocytes. Most have a single WAP domain at the C-terminus. The presence and type of the domains between the signal sequence and the C-terminus WAP domain separate the different crustin types. Most of the Type?I and II crustins are antimicrobial towards Gram-positive bacteria, whereas the Type?III crustins tend to display protease inhibition. Expression studies show that at least some crustins have other important biological effects, as levels change with physiological stress, wound repair, tissue regeneration or ecdysis. Thus WAP domains are widely distributed and highly conserved, serving in diverse physiological processes (proteinase inhibition, bacterial killing or inhibition of calcium transport).     

Purification and partial characterization of the plasma clotting protein from the pink shrimp Farfantepenaeus paulensis

A clotting protein (CP) was purified from the plasma of the pink shrimp Farfantepenaeus paulensis by sequential anion-exchange chromatography. The shrimp 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 present in shrimp hemocytes. Dansylcadaverine was incorporated into the shrimp CP in the presence of endogenous transglutaminase (hemocyte lysate), confirming that the shrimp purified CP is the substrate for the transglutaminase enzyme. The molecular mass of the CP was determined by gel filtration to be 341 kDa and 170 kDa by SDS-PAGE under reducing conditions. These results suggest that the shrimp CP consists of two identical subunits, covalently linked by disulphide bonds. The amino acid sequence at the N-terminus was 100% identical to that of the penaeids Litopenaeus vannamei and Penaeus monodon and 66% to 80% identical to the CPs of other decapods. This is the first report of a CP characterization in an Atlantic penaeid species. Further studies, including a molecular cloning approach would enable to detect which tissues express the gene of the clotting protein. It would be also useful to understand the mechanism by which the coagulation time is delayed in shrimps under stress conditions.     

Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus

The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 μM and 3.5-8 μM, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.     

Molecular cloning and characterization of three crustins from the Chinese white shrimp,Fenneropenaeus chinensis

Antibacterial peptides crustins are the effector molecules of innate immunity in decapods. In this study, three crustin cDNA sequences (Fc-crus 1, Fc-crus 2, and Fc-crus 3) were cloned from the Chinese white shrimp Fenneropenaeus chinensis. The full-length cDNAs of Fc-crus 2 and 3 are 473 bp and 574 bp, respectively. The deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 is 575 bp long and the deduced amino acids also contain a crustin domain. The expression profiles of these three crustins were studied with RT-PCR. Fc-crus 1 and Fc-crus 2 constitutively expressed in hemocytes with high levels, and the expression level is increased in the heart, stomach, intestine and ovaries when shrimp was challenged with Vibrio anguillarum, The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stage. Fc-crus 3 was constitutively expressed in the ovaries and induced as an expression in the stomach. Unlike Fc-crus 1 and Fc-crus 2, the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii and mysis to post-larvae. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in Escherichia coli and respectively purified. The antibacterial assays revealed that the recombinant mFc-crus could inhibit the growth of Gram-positive bacteria in vitro.     

Hemocytes of the palaemonids Macrobrachium rosenbergiiand M. acanthurus,and of the Penaeid Penaeus paulensis

The hemocytes of two palaemonids and one penaeid were characterized using light and transmission electron microscopy (TEM). The blood cells in all three species were classified as hyaline hemocytes (HH), small granule hemocytes (SGH), and large granule hemocytes (LGH). The HH are unstable hemocytes with a characteristic high nucleo-cytoplasmic ratio. Their cytoplasm appears particularly dense and has from few to numerous granules that often exhibit a typical striated substructure. In both palaemonids, the great majority of the HH contain numerous granules, whereas in Penaeus paulensis, a small number of these cells have few or no granules. The cytoplasm of some HH of the penaeid exhibits typical electron-dense deposits. The granulocytes, LGH and SGH, contain abundant electron-dense granules that are usually smaller in the SGH. In both hemocyte types, the cytosol, but not the granules, is rich in carbohydrates (PAS positive) and numerous vesicles contain acid phosphatase (Gomori reactive). In all studied shrimps, the SGH and LGH were actively phagocytic when examined on blood cell monolayers incubated with the yeast Saccharomyces cerevisiae. A few mitotic figures (less than 1%) were observed in the granulocytes of P. paulensis, but not in the palaemonids. SGH is the main circulating blood cell type in both palaemonids, whereas HH is predominant in the penaeid. Based on morphological and functional features, it appears that the hyaline and the granular hemocytes of the three shrimp species represent different cell lineages. J. Morphol. 236:209–221, 1998. © 1998 Wiley-Liss, Inc.     

Molecular Characterization of a Newly Identified Subfamily Member of Penaeidin from two Penaeid Shrimps, <Emphasis Type="Italic">Fenneropenaeus indicus</Emphasis> and <Emphasis Type="Italic">Metapenaeus monoceros</Emphasis>

Penaeidins are a major group of antimicrobial peptides found in penaeid shrimps. This study reports a new isoform of penaeidin from the hemocytes of Indian white shrimp, Fenneropenaeus indicus (Fi-PEN, JX657680), and the pink shrimp, Metapenaeus monoceros (Mm-PEN, KF275674). Mm-PEN is also the first antimicrobial peptide to be identified from M. monoceros. The complete coding sequences of the newly identified Fi-PEN and Mm-PEN consisted of an ORF of 338 bp encoding 71 amino acids with a predicted molecular weight of 5.66 kDa and a pI of 9.38. The penaeidins had its characteristic signal peptide region (19 amino acids), which was followed by a mature peptide with a proline-rich domain (24 amino acids) at the N-terminal region and a cysteine-rich domain (28 amino acids) at the C-terminal region, designating it to penaeidin-3 subgroup. Structural analysis revealed an alpha-helix in its secondary structure and an extended structure at the proline-rich domain. The newly identified penaeidin isoform showed maximum similarity of 63 % to a penaeidin-3 isoform of P. monodon, which further proves it to be a new isoform. Phylogenetic analysis showed that it possessed similar evolutionary status like other penaeidins, which has subsequently diverged at different phases of evolution. The wide distribution of penaeidins in penaeid shrimps indicates the importance of these AMPs in the innate immunity.     

Isolation and characterization of new microsatellite loci in the Pacific white shrimp Litopenaeus vannamei and cross‐species amplification in other penaeid species

The goal of the present study was to obtain valuable microsatellite markers useful in genetic approaches of the shrimp Litopenaeus vannamei, an important aquaculture species of the world. Eight loci produced suitable polymorphic patterns with allele number ranging from two to 12 and expected heterozygosity from 0.18 to 0.89. Cross‐species amplification was successfully performed in five other penaeid species (Litopenaeus schmitti, Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Xiphopenaeus kroyeri and Rimapenaeus constrictus), indicating that some these loci can be useful in studies of other related species.     

Isolation and characterization of microsatellites in three overexploited penaeid shrimp species along the Brazilian coastline

Most Brazilian commercial species of penaeid shrimp are currently overexploited. Thirteen polymorphic microsatellite loci herein isolated and characterized in Farfantepenaeus brasiliensis, Rimapenaeus constrictus and Xiphopenaeus kroyeri could be very useful for population studies on these penaeid species and proved to be potentially functional in cross-amplification with other species of shrimp. These microsatellites may be very helpful tools for research programs aimed at the sustainable management and conservation of these important fishery resources.     

基于线粒体16S rRNA和COI基因序列探讨对虾属(Penaeus)物种系统发生关系

有关对虾属(Penaeus)的设置及其相互亲缘关系一直是分类学争论的焦点,利用线粒体16S rRNA基因片段及COI基因片段序列分析的方法,以长臂虾科的脊尾白虾(Exopalaemon carinicauda)为外群,对对虾属的6亚属23种对虾进行了分子系统学研究。经ClustalX多重比对和MEGA4.0软件分析,得到种间序列的遗传距离并构建了最大简约(MP)系统树。结果表明:分子系统学数据支持Perez F等将对虾属的6个亚属提升为属级阶元的观点。囊对虾属的日本囊对虾(Marsupenaeus japonicus)和沟对虾属(Melicertus)的深沟对虾(Melicertus canaliculatus)之间的16S遗传距离只有0.007,而且COI遗传距离仅有0.065,比深沟对虾与同为沟对虾属的其他虾类遗传距离还小,说明囊对虾亚属(Marsupenaeus)和沟对虾亚属之间亲缘关系较近。另外美对虾亚属的褐美对虾(Farfantepenaeus aztec-us)和巴西美对虾(Farfantepenaeus brasiliensis)之间的16S rRNA基因序列遗传距离仅为0.012,但是与其他同亚属的虾类遗传距离相对较大,推测美对虾亚属(Farfantepenaeus)中的虾类根据亲缘关系远近和地理分布可以分为2大类群:墨西哥湾类群和南美洲类群。可以为对虾属的6个亚属的分类问题及演化提供一定的分子生物学依据。