Robert W. Chapman

Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the Pacific White Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp, L. setiferus.

A pilot program was undertaken in immune gene discovery in two sister species of litopenaeid shrimp, the Pacific white shrimp, Litopenaeus vannamei and the Atlantic white shrimp, L. setiferus. RNA from the hemocytes and hepatopancreas of single individuals from each species was recovered, 4 cDNA libraries (one from each tissue/species) were made by a PCR-based method and a total of approximately 2045 randomly selected clones were sequenced. A total of 268 expressed sequence tags (ESTs) were found that corresponded to 44 immune function genes. The most common immune-function ESTs (172) were antimicrobial peptides, which were restricted to the hemocyte libraries. Lectins were the largest group of immune-function ESTs found in the hepatopancreas. Analysis of these libraries indicates that EST approaches are effective for immune gene discovery in shrimp and that the diversity of these PCR-generated libraries would support full-scale EST collection.

Double-Stranded RNA Induces Sequence-Specific Antiviral Silencing in Addition to Nonspecific Immunity in a Marine Shrimp: Convergence of RNA Interference and Innate Immunity in the Invertebrate Antiviral Response?

ABSTRACT Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.

Crustins, homologues of an 11.5-kDa antibacterial peptide, from two species of penaeid shrimp, Litopenaeus vannamei and Litopenaeus setiferus.

The response of crustaceans to pathogens is believed to depend solely on innate, nonadaptive immune mechanisms, including phagocytosis, encapsulation, clotting, and a variety of soluble antimicrobial activities. Arthropod antimicrobial peptides, while characterized primarily from insects, also have been isolated from crustaceans. Expressed sequence tag analysis of hemocyte complementary DNA libraries from 2 species of shrimp, Litopenaeus vannamei and Litopenaeus setiferus, revealed transcripts with strong sequence similarity to an 11.5-kDa antibacterial peptide (crustin Cm1) found in Carcinus maenas. Crustins were also observed to contain motifs common to proteinase inhibitors. Analysis of these cDNA libraries yielded at least 3 different isoforms of this peptide in L. vannamei (crustin Lv1–Lv3) and 3 in L. setiferus (crustin Ls1–Ls3). Further analysis of a second L. vannamei cDNA library revealed the presence of 3 more possible isoforms (crustin Lv4–Lv6), which differed from those seen in the first L. vannamei cDNA library. Genomic Southern blot analysis revealed a complex family of crustin-related sequences. However, full-length crustin appears to be encoded by a much more restricted subset of sequences within this family.

The transcriptomic responses of the eastern oyster, Crassostrea virginica, to environmental conditions

Understanding the mechanisms by which organisms adapt to environmental conditions is a fundamental question for ecology and evolution. In this study, we evaluate changes in gene expression of a marine mollusc, the eastern oyster Crassostrea virginica, associated with the physico‐chemical conditions and the levels of metals and other contaminants in their environment. The results indicate that transcript signatures can effectively disentangle the complex interactive gene expression responses to the environment and are also capable of disentangling the complex dynamic effects of environmental factors on gene expression. In this context, the mapping of environment to gene and gene to environment is reciprocal and mutually reinforcing. In general, the response of transcripts to the environment is driven by major factors known to affect oyster physiology such as temperature, pH, salinity, and dissolved oxygen, with pollutant levels playing a relatively small role, at least within the range of concentrations found in the studied oyster habitats. Further, the two environmental factors that dominate these effects (temperature and pH) interact in a dynamic and nonlinear fashion to impact gene expression. Transcriptomic data obtained in our study provide insights into the mechanisms of physiological responses to temperature and pH in oysters that are consistent with the known effects of these factors on physiological functions of ectotherms and indicate important linkages between transcriptomics and physiological outcomes. Should these linkages hold in further studies and in other organisms, they may provide a novel integrated approach for assessing the impacts of climate change, ocean acidification and anthropogenic contaminants on aquatic organisms via relatively inexpensive microarray platforms.

Diversity of the penaeidin antimicrobial peptides in two shrimp species.

Abstract. Penaeidins, a unique family of antimicrobial peptides (AMPs) with both proline and cysteine-rich domains, were initially identified in the hemolymph of the Pacific white shrimp, Litopenaeus vannamei. Described here are the results of an investigation of penaeidin diversity in individual shrimp from two species, L. vannamei and L. setiferus (Atlantic white shrimp). We report the discovery of a novel penaeidin class, designated penaeidin 4 present in both L. vannamei and L. setiferus, and that all penaeidin classes were expressed in a single individual. In addition, nearly all penaeidins, regardless of class, shared an identical leader sequence while differing dramatically in the remainder of the peptide. Several new class 3 isoforms were identified, as well as sequence variants of Lv3a, which differ in the 3′ untranslated region. Penaeidin sequence variability (especially of class 3), within and between individuals, is not interpretable as simple allelic polymorphism and may reflect alternate transcriptional mechanisms. Penaeidins are encoded by a small number of genetic loci and are not likely representatives of a large gene family produced by whole gene duplication, but rather may be products of a multi-component locus. Based on phylogenetic analysis, penaeidins fall into three classes where 1 and 2 are combined while classes 3 and 4 remain distinct. Phylogenetic analysis indicates that all classes of penaeidin were likely present in both species prior to speciation.

Potential Indicators of Stress Response Identified by Expressed Sequence Tag Analysis of Hemocytes and Embryos from the American Oyster, Crassostrea virginica

Abstract: A pilot program was initiated to identify genes from the American oyster, Crassostrea virginica, that are potentially involved in the stress response for use as bioindicators of exposure to environmental pollutants and to toxic and infectious agents. A PCR-based method was used to construct cDNA libraries from pooled embryos and the hemocytes of a single individual. A total of 998 randomly selected clones (expressed sequence tags, ESTs) were sequenced. Approximately 40% of the ESTs are novel sequences. Several potential biomarkers identified include an antimicrobial peptide, recognition molecules (lectin receptors), proteinases and proteinase inhibitors, and a novel metallothionein. Diversity analysis shows that 363 and 286 unique genes were identified from the hemocyte and embryo libraries, respectively, indicating that full-scale EST collection is a valuable approach for the discovery of new genes of potential significance in the molluscan stress response.

Stock Identification of Gag, Mycteroperca microlepis, Along the Southeast Coast of the United States

Abstract: The gag grouper Mycteroperca microlepis is an important component of commercial and recreational fisheries along the South Atlantic coast of the United States and in the Gulf of Mexico. Over the past two decades, this species has experienced significant declines in abundance and an increasing skew in sex ratios. Analysis of microsatellite DNA variation in this species shows mosaic patterns of population subdivision and significant departures from Hardy-Weinberg equilibrium in all sampling locations. Given the length of the pelagic stage (egg and larvae), the prevailing current patterns, and the migratory capabilities of the adults, it is unlikely that these observations are the result of restricted gene flow among genetically differentiated populations. The apparent structure of gag populations most likely reflects inbreeding in size-limited populations. Population declines, skewed sex ratios, and perhaps variance in female fecundity appear to have acted in concert to limited the number of individuals that contribute to a given year class. These data are reinforced by studies of other fish stocks that have experienced precipitous declines over the past two decades.

Non-specific activation of antiviral immunity and induction of RNA interference may engage the same pathway in the Pacific white leg shrimp Litopenaeus vannamei.

Many questions remain unanswered regarding RNAi-based mechanisms and dsRNA-induced antiviral immune responses in penaeid shrimp. In this study, we report the characterization in the white leg shrimp Litopenaeus vannamei of RNAi pathway associated proteins Lv-Ago 1 and Lv-Ago 2, two members of the Argonaute family of proteins, as well as Lv-sid 1, the first shrimp homologue of Sid-1, a membrane channel-forming protein implicated in the cellular import of dsRNA. To decipher their functional implication in RNAi-related phenomena, we monitored their relative expression following stimulation by specific and non-specific RNA duplexes of diverse length. The findings show that the length of small RNA duplexes plays a critical role in the activation of both RNAi-related and innate antiviral responses. They also suggest that these two mechanisms of antiviral response may activate the same pathway, requiring Lv-Sid 1 and Lv-Ago 2 induction.

A cDNA Microarray for Crassostrea virginica and C. gigas

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.

Molecular Characterization and Expression of Vitellogenin Receptor from White Perch (Morone americana)

Abstract A full-length (4021 base pair [bp]) cDNA encoding a polypeptide (844 amino acids) with a predicted mass of 93 kDa and other characteristic structural features of a vertebrate vitellogenin receptor (VgR) was isolated from a white perch (Morone americana) ovarian cDNA library. Northern blotting performed using a specific digoxygenin-labeled VgR cDNA probe revealed a distinct ∼4.1 kilobase (kb) hybridization signal in an mRNA preparation obtained from previtellogenic perch ovaries. The deduced amino acid sequence of the perch VgR was 89% and 82% identical, respectively, to that of the tilapia and rainbow trout. Because it possessed an eight-repeat ligand-binding domain (LR8) but lacked an O-linked sugar domain (−), the perch VgR was identified as a non-O-linked form of VgR (LR8−). Unlike the case in other vertebrates investigated, including tilapia and trout, no species of mRNA encoding an O-linked form of VgR (LR8+) could be detected when perch ovarian or liver mRNA reverse transcripts or cDNA libraries were screened by PCR using primer sets flanking the putative O-linked sugar domain. These novel findings call into question the assumptions that an LR8+ splice variant of the VgR always is dominantly present in somatic tissues and exists at lower levels in ovarian tissues to sequester lipoproteins distinct from Vg. A SYBR-green-based real-time reverse transcription-polymerase chain reaction assay was developed and used to quantitatively measure VgR expression in gonadal and somatic tissues, for the first time in any vertebrate. The main site of perch VgR mRNA expression was the ovary and the highest level of VgR mRNA expression was in ovaries whose largest follicles contained previtellogenic oocytes. Expression of VgR mRNA decreased with oocyte growth during vitellogenesis and was very limited in ovulated eggs. These quantitative results verify the concept that growing oocytes must extensively recycle LR8− forms of the VgR.