On the wave of the crustin antimicrobial peptide family: From sequence diversity to function.

Crustins represent the largest and most diverse family of antimicrobial peptides (AMPs) found in crustaceans. They are classically defined as disulfide-rich peptides/polypeptides holding a typical Whey Acidic Protein (WAP) domain at the C-terminal end. This WAP domain has eight cysteine residues forming a tightly packed structure, the four-disulfide core (4DSC) motif, that is also found in other proteins displaying protease inhibitory properties. Crustins are highly diverse in terms of primary structure, size and biochemical features, thus exhibiting a series of biological functions beyond their antimicrobial properties. In order to better categorize the distinct crustin members, different classification systems have been proposed. In this review, we discuss the current classification systems and explore the biological implication of the impressive molecular diversity of this unique AMP family. We also summarize the recent findings on the role of these effectors in crustacean immunity and homeostasis as well as in host-microbe interactions.

Exploring the Impact of the Biofloc Rearing System and an Oral WSSV Challenge on the Intestinal Bacteriome of Litopenaeus vannamei.

We provide a global overview of the intestinal bacteriome of Litopenaeus vannamei in two rearing systems and after an oral challenge by the White spot syndrome virus (WSSV). By using a high-throughput 16S rRNA gene sequencing technology, we identified and compared the composition and abundance of bacterial communities from the midgut of shrimp reared in the super-intensive biofloc technology (BFT) and clear seawater system (CWS). The predominant bacterial group belonged to the phylum Proteobacteria, followed by the phyla Bacteroidetes, Actinobacteria, and Firmicutes. Within Proteobacteria, the family Vibrionaceae, which includes opportunistic shrimp pathogens, was more abundant in CWS than in BFT-reared shrimp. Whereas the families Rhodobacteraceae and Enterobacteriaceae accounted for almost 20% of the bacterial communities of shrimp cultured in BFT, they corresponded to less than 3% in CWS-reared animals. Interestingly, the WSSV challenge dramatically changed the bacterial communities in terms of composition and abundance in comparison to its related unchallenged group. Proteobacteria remained the dominant phylum. Vibrionaceae was the most affected in BFT-reared shrimp (from 11.35 to 20.80%). By contrast, in CWS-reared animals the abundance of this family decreased from 68.23 to 23.38%. Our results provide new evidence on the influence of both abiotic and biotic factors on the gut bacteriome of aquatic species of commercial interest.

An immune-related gene expression atlas of the shrimp digestive system in response to two major pathogens brings insights into the involvement of hemocytes in gut immunity.

Much of our current knowledge on shrimp immune system is restricted to the defense reactions mediated by the hemocytes and little is known about gut immunity. Here, we have investigated the transcriptional profile of immune-related genes in different organs of the digestive system of the shrimp Litopenaeus vannamei. First, the tissue distribution of 52 well-known immune-related genes has been assessed by semiquantitative analysis in the gastrointestinal tract (foregut, midgut and hindgut) and in the hepatopancreas and circulating hemocytes of shrimp stimulated or not with heat-killed bacteria. Then, the expression levels of 18 genes from key immune functional categories were quantified by fluorescence-based quantitative PCR in the midgut of animals experimentally infected with the Gram-negative Vibrio harveyi or the White spot syndrome virus (WSSV). Whereas the expression of some genes was induced at 48 h after the bacterial infection, any of the analyzed genes showed to be modulated in response to the virus. Whole-mount immunofluorescence assays confirmed the presence of infiltrating hemocytes in the intestines, indicating that the expression of some immune-related genes in gut is probably due to the migratory behavior of these circulating cells. This evidence suggests the participation of hemocytes in the delivery of antimicrobial molecules into different portions of the digestive system. Taken all together, our results revealed that gut is an important immune organ in L. vannamei with intimate association with hemocytes.

Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis.

We have performed here a gene expression analysis to determine the developmental stage at the main genes involved in crustacean immune response begin to be expressed and their changes in mRNA abundance during shrimp development. By using a quantitative PCR-based approach, we have measured the mRNA abundance of 24 immune-related genes from different functional categories in twelve developmental stages ranging from fertilized eggs to larval and postlarval stages and also in juveniles. We showed for the first time that the main genes from the RNAi-based post-transcriptional pathway involved in shrimp antiviral immunity are transcribed in all developmental stages, but exhibit a diverse pattern of gene expression during shrimp ontogenesis. On the other hand, hemocyte-expressed genes mainly involved in antimicrobial defenses appeared to be transcribed in larval stages, indicating that hematopoiesis initiates early in development. Moreover, transcript levels of some genes were early detected in fertilized eggs at 0-4 h post-spawning, suggesting a maternal contribution of immune-related transcripts to shrimp progeny. Altogether, our results provide important clues regarding the ontogenesis of hemocytes as well the establishment of antiviral and antimicrobial defenses in shrimp.

Virulence genes of Rickettsia rickettsii are differentially modulated by either temperature upshift or blood-feeding in tick midgut and salivary glands.

BACKGROUND: Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever, is transmitted to humans by ticks. During tick feeding, R. rickettsii is exposed to both temperature elevation and components of the blood meal, which have previously been associated with the reactivation of its virulence. These environmental stimuli were also reported to modulate virulence genes of R. rickettsii infecting a set of organs of adult females of its natural vector, Amblyomma aureolatum. METHODS: In this study, we determined the effects of a temperature upshift, blood-feeding, and both stimuli simultaneously on the expression of 85 selected genes of R. rickettsii infecting either the midgut (MG) or salivary glands (SG) of male and female A. aureolatum by microfluidic high-throughput RT-qPCR. These two organs are key for acquisition of this bacterium by the tick and transmission to the vertebrate host, respectively. RESULTS: Data showed that these environmental stimuli exert distinct effects on rickettsial transcription depending on the colonized organ and gender of the vector. Temperature upshift induced the majority of differentially expressed genes of R. rickettsii in tick SG, including tRNA synthetases encoding genes. On the contrary, blood-feeding downregulated most of differentially expressed genes in both organs, but induced type IV secretion system components and OmpB in tick MG. The combined effects of both stimuli resulted in a merged gene expression profile representing features of each stimulus analyzed independently, but was more similar to the profile induced by blood-feeding. CONCLUSION: The upregulation of the majority of differentially expressed genes in tick SG by temperature upshift suggests that this stimulus is important to prepare R. rickettsii for transmission to the vertebrate host. Blood-feeding, on the other hand, induced important virulence genes in the tick MG, which might be associated with colonization of the tick and transmission to the vertebrate host. The role of the proteins identified in this study must be addressed and might help to define future targets to block tick infection, thereby preventing RMSF. To our knowledge, this is the first transcriptional tissue-specific study of a virulent strain of R. rickettsii infecting a natural tick vector.

Exploring the immune signalling pathway-related genes of the cattle tick Rhipicephalus microplus: From molecular characterization to transcriptional profile upon microbial challenge.

In dipteran insects, invading pathogens are selectively recognized by four major pathways, namely Toll, IMD, JNK, and JAK/STAT, and trigger the activation of several immune effectors. Although substantial advances have been made in understanding the immunity of model insects such as Drosophila melanogaster, knowledge on the activation of immune responses in other arthropods such as ticks remains limited. Herein, we have deepened our understanding of the intracellular signalling pathways likely to be involved in tick immunity by combining a large-scale in silico approach with high-throughput gene expression analysis. Data from in silico analysis revealed that although both the Toll and JAK/STAT signalling pathways are evolutionarily conserved across arthropods, ticks lack central components of the D. melanogaster IMD pathway. Moreover, we show that tick immune signalling-associated genes are constitutively transcribed in BME26 cells (a cell lineage derived from embryos of the cattle tick Rhipicephalus microplus) and exhibit different transcriptional patterns in response to microbial challenge. Interestingly, Anaplasma marginale, a pathogen that is naturally transmitted by R. microplus, causes downregulation of immune-related genes, suggesting that this pathogen may manipulate the tick immune system, favouring its survival and vector colonization.

An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes.

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due to their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Molecular signatures at imminent death: hemocyte gene expression profiling of shrimp succumbing to viral and fungal infections.

Infectious diseases represent the most serious threat to shrimp farming worldwide. Understanding the molecular mechanisms driving shrimp-pathogen interactions is necessary for developing strategies to control disease outbreaks in shrimp production systems. In the current study, we experimentally reproduced mortality events using standardized infections to characterize the hemocyte transcriptome response of the shrimp Litopenaeus vannamei succumbing to infectious diseases. By using a high-throughput microfluidic RT-qPCR approach, we identified molecular signatures in shrimp during lethal infections caused by the White Spot Syndrome Virus (WSSV) or the filamentous fungus Fusarium solani. We successfully identified gene expression signatures shared by both infections but also pathogen-specific gene responses. Interestingly, whereas lethal WSSV infection induced the expression of antiviral-related genes, the transcript abundance of many antimicrobial effectors was reduced by lethal F. solani infection. To our knowledge, this is the first report of the immune-gene repertoire of infected shrimp at the brink of death.

Detection of infectious myonecrosis virus in penaeid shrimps using immunoassays: usefulness of monoclonal antibodies directed to the viral major capsid protein.

Despite the economic impact of the infectious myonecrosis virus (IMNV) on shrimp farms in several countries, no method for immunological detection is currently available. With the aim of developing immunodiagnostic methods for IMNV detection in infected shrimps, a recombinant fragment of the IMNV major capsid protein gene encoding amino acids 105-297 (rIMNV105₋297 was heterologously expressed in Escherichia coli and used to immunize Balb/c mice, generating monoclonal antibodies (MAbs). Six hybridomas were obtained, and four of these recognized the presence of IMNV in tissue homogenates from naturally infected shrimps by immunodot blot assay. Among these MAbs, three were able to detect a ~100-kDa protein, which corresponds to the predicted mass of the IMNV major capsid protein, as well as viral inclusion bodies in muscle fibroses by western blot and immunohistochemistry. Two MAbs showed high specificity and sensitivity, showing no cross-reaction with healthy shrimp tissues in any assays, indicating their usefulness for IMNV detection.

Detection of major capsid protein of infectious myonecrosis virus in shrimps using monoclonal antibodies.

Infectious myonecrosis virus (IMNV) has been causing a progressive disease in farm-reared shrimps in Brazil and Indonesia. Immunodiagnostic methods for IMNV detection, although reliable, are not employed currently because monoclonal antibodies (MAbs) against this virus are not available. In this study, a fragment of the IMNV major capsid protein gene, comprising amino acids 300-527 (IMNV(300-527)), was cloned and expressed in Escherichia coli. The nucleotide sequence of the recombinant IMNV(300-527) fragment displayed a high degree of identity to the major capsid protein of IMNV isolates from Brazil (99%) and Indonesia (98%). Ten MAbs were generated against the expressed fragment, and eight of these, mostly IgG(2a) or IgG(2b), were able to bind to IMNV in tissue extracts from shrimps infected naturally in immunodot-blot assays. Six of these MAbs recognized a approximately 100 kDa protein in a Western-blot, which is the predicted mass of IMNV major capsid protein, and also bound to viral inclusions present in muscle fibroses and in coagulative myonecrosis, as demonstrated by immunohistochemistry. Among all those MAbs created, four did not cross-react with non-infected shrimp tissues; this observation supports their applicability as a sensitive and specific immunodiagnosis of IMNV infection in shrimps.

A novel monoclonal antibody that binds to hemocytes from shrimps and oysters.

The monoclonal antibody (MAb) LITO-1 was produced from a stable hybridoma cell line generated by the fusion of NS1 myeloma cells with spleen cells isolated from Balb/c mice immunized with a paraformaldehyde-fixed hemocyte suspension of Litopenaeus vannamei. This MAb reacted with all three hemocyte subtypes, but no reaction was observed with components of plasma. Immunohistochemistry assays demonstrated that LITO-1 was very effective in specifically distinguishing hemocytes infiltrated in several tissues such as striated muscle, brain, and hepatopancreas. Moreover, this antibody was able to recognize hemocytes from two shrimp species, Litopenaeus schmitti and Farfantepenaeus paulensis, as well as hemocytes of the oyster Crassostrea gigas. No reaction was observed against hemocytes from the terrestrial insect Triatoma klugi or with mammalian RAW cells. This novel MAb can be useful in revealing the presence and function of a conservative epitope in hemocytes of marine crustaceans and mollusks.