Screening of Multiple Potential Control Genes for use in Caste and Body Region Comparisons Using RT-qPCR in Coptotermes formosanus

Formosan subterranean termites, Coptotermes formosanus, are a significant worldwide pest. Molecular gene expression is an important tool for understanding the physiology of organisms. The recent advancement of molecular tools for Coptotermes formosanus is leading to the advancement of the understanding of termite physiology. One of the first steps in analyzing gene expression is the normalization to constant reference genes. Stable reference genes that have constant expression across multiple treatments are important for accurately comparing target genes' expression. The objective of this investigation was to analyze and validate a set of potential reference genes including 8SrRNA; Glyceraldehyde 3-phosphate dehydrogenase (Gadphd); ribosomal protein L7 (RPL); β-actin (BA1); α-tubulin (Atube); α-actin (Aactin); and elongation factor (Elong) as standards for analysis of transcriptional changes in the termite Coptotermes formosanus, across two phenotypic castes, body regions, and colonies. We also compared the expression of hexamerin-1 and 2 using stable and unstable reference genes to demonstrate the importance of consistent control genes. Our results demonstrate that 18S and RPL can serve as reliable expression standards when comparing these different castes and body regions, and we show that C. formosanus Hex-1 and Hex-2 have expression patterns similar to that previously described in R. flavipes

Examining the interplay between Streptococcus agalactiae, the biopolymer chitin and its derivative

Abstract Streptococcus agalactiae is a highly pathogenic bacterium of aquatic species and terrestrial animals worldwide, whereas chitin and its derivative chitosan are among the most abundant biopolymers found in nature, including the aquatic milieu. The present investigation focused on the capability of S. agalactiae to degrade and utilize these polymers. Growth of S. agalactiae in the presence of colloid chitin, chitosan, or N‐acetyl‐glucosamine (GlcNAc) was evaluated. Chitosanase production was measured daily over 7 days of growth period and degraded products were evaluated with thin later chorography. Chitin had no effect on the growth of S. agalactiae. Degraded chitin, however, stimulated the growth of S. agalactiae. S. agalactiae cells did not produce chitinase to degrade chitin; however, they readily utilize GlcNAc (product of degraded chitin) as sole source of carbon and nitrogen for growth. Chitosan at high concentrations had antibacterial activities against S. agalactiae, while in the presence of lower than the inhibitory level of chitosan in the medium, S. agalactiae secrets chitosanase to degrade chitosan, and utilizes it to a limited extent to benefit growth. The interaction of S. agalactiae with chitin hydrolytes and chitosan could play a role in the diverse habitat distribution and pathogenicity of S. agalactiae worldwide

Treatment of Trichodina sp reduced load of Flavobacterium columnare and improved survival of hybrid tilapia

Bacterium Flavobacterium columnare and protozoan Trichodina spp are common pathogens of cultured fish. Studies of parasite–bacterium interaction show evidence that concurrent infections increase severity of some infectious diseases, especially bacterial diseases. The effect of parasite treatment on F. columnare infection in tilapia is currently unknown. This study evaluated whether treatment of Trichodina sp parasitized hybrid tilapia (Oreochromis niloticus × Oreochromis aureus) with formalin would improve fish survival and reduce F. columnare infection in fish after F. columnare exposure. Hybrid tilapia parasitized by Trichodina sp were divided into 3 treatment groups. The first group of fish received no parasite treatment. The second group of fish were bath treated with 150 mg L−1 formalin for 1 h. The third group of fish treated twice with 150 mg L−1 formalin bath for 1 h each at 2 day intervals. All fish were then exposed to F. columnare by immersion challenge. The tilapia not treated with formalin showed significantly higher mortality (37.5%) than those treated with formalin (≤16.7%) after exposure to F. columnare. Fish treated twice showed lower mortality (6.37%) than those treated only once (16.7%). The non-treated fish showed significantly higher load of F. columnare in gill, kidney and liver compared to those treated with formalin following exposure to F. columnare. The bacterial load of non-treated fish was 27075 genome equivalents per mg of gill tissue (GEs/mg), 12 fold higher than those treated once with formalin (2250 GEs/mg) or 39 fold higher than those treated twice with formalin (699 GEs/mg) after exposure to F. columnare. This study demonstrated that formalin treatment for Trichodina sp parasitism reduced bacterial infection as suggested by reduced loads of bacteria in fish tissues and subsequently decreased fish mortality

Identification and Characterization of Differentially Expressed IgM Transcripts of Channel Catfish Vaccinated with Antigens of Virulent Aeromonas hydrophila

Channel catfish (Ictalurus punctatus) is the top species produced in US aquaculture and motile Aeromonas septicemia, caused by virulent Aeromonas hydrophila (vAh), is one of the most severe diseases that afflict catfish farms. Previously, vaccination of fish with extracellular proteins (ECP) of vAh was shown to produce a robust antibody-mediated immune response against vAh infection. In this study, we analyzed IgM transcripts that were differentially expressed in the head kidney and liver of ECP-immunized and mock-immunized (control) fish with emphasis on a variable domain of heavy chain. Quantitative PCR analysis indicated that immunized fish produced significantly more IgM transcripts than control fish. Full-length IgM heavy chain cDNA was cloned, which encoded typical IgM peptide, including signal peptide, variable domain (VH), constant domain (CH), and carboxyl terminal peptide. Great sequence diversity was revealed in a VH segment, with the third complementarity diversity region (CDR3) being most variable. Using germline VH gene grouping method, variants (clones) of VH characterized in this study belonged to nine VH families. The most unique variants (approximately 49%) were found in the VH2 family. Vaccinated fish apparently had more unique variants than in the control fish. There were 62% and 79% of unique variants in the head kidney and liver of vaccinated fish, respectively, while 44% and 27% unique variants in the head kidney and liver of control fish, respectively. Among the unique variants in VH2 family, approximately 87% of them were found in vaccinated fish. Two-dimensional gel electrophoresis of semi-purified IgM protein confirmed that matured IgM protein was as variable as IgM transcripts identified in this study, with isoelectric points crossing from 6 to 10. Results of this study provided insight into the molecular and genetic basis of antibody diversity and enriched our knowledge of the complex interplay between antigens and antibodies in Ictalurid catfish

Cross-Serological Reaction of Glandless Cottonseed Proteins to Peanut and Tree Nut Allergic IgE

Food allergy is a potentially life-threatening health concern caused by immunoglobulin E (IgE) antibodies that mistakenly recognize normally harmless food proteins as threats. Peanuts and tree nuts contain several seed storage proteins that commonly act as allergens. Glandless cottonseed, lacking the toxic compound gossypol, is a new food source. However, the seed storage proteins in cottonseed may act as allergens. To assess this risk, glandless cottonseed protein extracts were evaluated for IgE binding by peanut and tree nut allergic volunteers. ELISA demonstrated that 25% of 32 samples had significant binding to cottonseed extracts. Immunoblot analysis with pooled sera indicated that IgE recognized a pair of bands migrating at approximately 50 kDa. Excision of these bands and subsequent mass-spectrometric analysis demonstrated peptide matches to cotton C72 and GC72 vicilin and legumin A and B proteins. Further, in silico analysis indicated similarity of the cotton vicilin and legumin proteins to peanut vicilin (Ara h 1) and cashew nut legumin (Ana o 2) IgE-binding epitopes among others. The observations suggest both the cotton vicilin and legumin proteins were recognized by the nut allergic IgE, and they should be considered for future allergen risk assessments evaluating glandless cottonseed protein products

Cross-Serological Reaction of Glandless Cottonseed Proteins to Peanut and Tree Nut Allergic IgE

Food allergy is a potentially life-threatening health concern caused by immunoglobulin E (IgE) antibodies that mistakenly recognize normally harmless food proteins as threats. Peanuts and tree nuts contain several seed storage proteins that commonly act as allergens. Glandless cottonseed, lacking the toxic compound gossypol, is a new food source. However, the seed storage proteins in cottonseed may act as allergens. To assess this risk, glandless cottonseed protein extracts were evaluated for IgE binding by peanut and tree nut allergic volunteers. ELISA demonstrated that 25% of 32 samples had significant binding to cottonseed extracts. Immunoblot analysis with pooled sera indicated that IgE recognized a pair of bands migrating at approximately 50 kDa. Excision of these bands and subsequent mass-spectrometric analysis demonstrated peptide matches to cotton C72 and GC72 vicilin and legumin A and B proteins. Further, in silico analysis indicated similarity of the cotton vicilin and legumin proteins to peanut vicilin (Ara h 1) and cashew nut legumin (Ana o 2) IgE-binding epitopes among others. The observations suggest both the cotton vicilin and legumin proteins were recognized by the nut allergic IgE, and they should be considered for future allergen risk assessments evaluating glandless cottonseed protein products

Identification and Characterization of Differentially Expressed IgM Transcripts of Channel Catfish Vaccinated with Antigens of Virulent <i>Aeromonas hydrophila</i>

Channel catfish (Ictalurus punctatus) is the top species produced in US aquaculture and motile Aeromonas septicemia, caused by virulent Aeromonas hydrophila (vAh), is one of the most severe diseases that afflict catfish farms. Previously, vaccination of fish with extracellular proteins (ECP) of vAh was shown to produce a robust antibody-mediated immune response against vAh infection. In this study, we analyzed IgM transcripts that were differentially expressed in the head kidney and liver of ECP-immunized and mock-immunized (control) fish with emphasis on a variable domain of heavy chain. Quantitative PCR analysis indicated that immunized fish produced significantly more IgM transcripts than control fish. Full-length IgM heavy chain cDNA was cloned, which encoded typical IgM peptide, including signal peptide, variable domain (VH), constant domain (CH), and carboxyl terminal peptide. Great sequence diversity was revealed in a VH segment, with the third complementarity diversity region (CDR3) being most variable. Using germline VH gene grouping method, variants (clones) of VH characterized in this study belonged to nine VH families. The most unique variants (approximately 49%) were found in the VH2 family. Vaccinated fish apparently had more unique variants than in the control fish. There were 62% and 79% of unique variants in the head kidney and liver of vaccinated fish, respectively, while 44% and 27% unique variants in the head kidney and liver of control fish, respectively. Among the unique variants in VH2 family, approximately 87% of them were found in vaccinated fish. Two-dimensional gel electrophoresis of semi-purified IgM protein confirmed that matured IgM protein was as variable as IgM transcripts identified in this study, with isoelectric points crossing from 6 to 10. Results of this study provided insight into the molecular and genetic basis of antibody diversity and enriched our knowledge of the complex interplay between antigens and antibodies in Ictalurid catfish