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

Myosin Gene Expression and Protein Abundance in Different Castes of the Formosan Subterranean Termite (Coptotermes formosanus)

The Formosan subterranean termite (Coptotermes formosanus) is an important worldwide pest, each year causing millions of dollars in structural damage and control costs. Termite colonies are composed of several phenotypically distinct castes. Termites utilize these multiple castes to efficiently perform unique roles within the colony. During the molting/caste differentiation process, multiple genes are believed to be involved in the massive reorganization of the body plan. The objective of this research was to analyze the muscle gene, myosin, to further understand the role it plays in C. formosanus development. We find that comparing worker vs. solider caste myosin gene expression is up-regulated in the soldier and a myosin antibody-reactive protein suggests changes in splicing. Comparison of body regions of mature soldier and worker castes indicates a greater level of myosin transcript in the heads. The differential expression of this important muscle-related gene is anticipated considering the large amount of body plan reorganization and muscle found in the soldier caste. These results have a direct impact on our understanding of the downstream genes in the caste differentiation process and may lead to new targets for termite control

Genetic and transcriptomic dissection of the fiber length trait from a cotton (Gossypium hirsutum L.) MAGIC population

Abstract Background Improving cotton fiber length without reducing yield is one of the major goals of cotton breeding. However, genetic improvement of cotton fiber length by breeding has been a challenge due to the narrow genetic diversity of modern cotton cultivars and negative correlations between fiber quality and yield traits. A multi-parent advanced generation inter-cross (MAGIC) population developed through random mating provides an excellent genetic resource that allows quantitative trait loci (QTL) and causal genes to be identified. Results An Upland cotton MAGIC population, consisting of 550 recombinant inbred lines (RILs) derived from eleven different cultivars, was used to identify fiber length QTLs and potential genes that contribute to longer fibers. A genome wide association study (GWAS) identified a cluster of single nucleotide polymorphisms (SNPs) on chromosome (Chr.) D11 that is significantly associated with fiber length. Further evaluation of the Chr. D11 genomic region among lines of the MAGIC population detected that 90% of RILs have a D11 haplotype similar to the reference TM-1 genome (D11-ref), whereas 10% of RILs inherited an alternative haplotype from one of the parents (D11-alt). The average length of fibers of D11-alt RILs was significantly shorter compared to D11-ref RILs, suggesting that alleles in the D11-alt haplotype contributed to the inferior fiber quality. RNAseq analysis of the longest and shortest fiber length RILs from D11-ref and D11-alt populations identified 949 significantly differentially expressed genes (DEGs). Gene set enrichment analysis revealed that different functional categories of genes were over-represented during fiber elongation between the four selected RILs. We found 12 genes possessing non-synonymous SNPs (nsSNPs) significantly associated with the fiber length, and three that were highly significant and were clustered at D11:24-Mb, including D11G1928, D11G1929 and D11G1931. Conclusion The results of this study provide insights into molecular aspects of genetic variation in fiber length and suggests candidate genes for genetic manipulation for cotton improvement

Cross-reaction between Formosan termite (Coptotermes formosanus) proteins and cockroach allergens.

Cockroach allergens can lead to serious allergy and asthma symptoms. Termites are evolutionarily related to cockroaches, cohabitate in human dwellings, and represent an increasing pest problem in the United States. The Formosan subterranean termite (Coptotermes formosanus) is one of the most common species in the southern United States. Several assays were used to determine if C. formosanus termite proteins cross-react with cockroach allergens. Expressed sequence tag and genomic sequencing results were searched for homology to cockroach allergens using BLAST 2.2.21 software. Whole termite extracts were analyzed by mass-spectrometry, immunoassay with IgG and scFv antibodies to cockroach allergens, and human IgE from serum samples of cockroach allergic patients. Expressed sequence tag and genomic sequencing results indicate greater than 60% similarity between predicted termite proteins and German and American cockroach allergens, including Bla g 2/Per a 2, Bla g 3/Per a 3, Bla g 5, Bla g 6/Per a 6, Bla g 7/Per a 7, Bla g 8, Per a 9, and Per a 10. Peptides from whole termite extract were matched to those of the tropomyosin (Bla g 7), arginine kinase (Per a 9), and myosin (Bla g 8) cockroach allergens by mass-spectrometry. Immunoblot and ELISA testing revealed cross-reaction between several proteins with IgG and IgE antibodies to cockroach allergens. Several termite proteins, including the hemocyanin and tropomyosin orthologs of Blag 3 and Bla g 7, were shown to crossreact with cockroach allergens. This work presents support for the hypothesis that termite proteins may act as allergens and the findings could be applied to future allergen characterization, epitope analysis, and clinical studies

Termite proteins cross-react with IgE from human cockroach allergic sera.

<p>(A) Western blot of GCr (left lane of each panel) and Cf termite (right lane of each panel) extracts. The proteins were separated on SDS-PAGE gel followed by transfer onto PVDF membrane. After blocking for 2 hr the blot was incubated with four GCr allergic serum pools S1Cr and P1-4 (1:10). Specific IgE binding was determined following sequential addition of biotinylated goat anti-human IgE (1:1000), and HRP-conjugated streptavidin (1:10,000). (B). 96-well plates were coated with termite extract (100 μg/mL) overnight at 4°C. After blocking for 2 hr, GCr allergic serum pools (1:10) were added to each well. Specific IgE binding was determined following sequential addition of biotinylated goat anti-human IgE (1:1000), HRP-conjugated streptavidin (1:10,000), and TMB substrate. (C) Dose response binding of human IgE in human serum pool (S1Cr) with the indicated amount of Cf termite extract.</p

Termite proteins cross-react with anti-cockroach allergen scFvs in ELISA assays.

<p>(A) This assay was performed in sandwich format using two separate collections of detection agents. Serially diluted Cf termite extract was first added to a mixture of anti-cockroach scFvs either generated non-specifically using whole body GCr extract or specifically targeted against recombinant Bla g 1, 2, or 4, and one scFv from a naïve human library screened against whole GCr extract that were coupled to unique bead sets as capture agents in 96-well filter bottom plates. A mixture of rabbit anti-E6Cg cockroach extract polyclonal antibodies, anti Bla g 1, anti Bla g 2, and anti Bla g 4 IgGs (1:500) were used as detection antibodies, followed by biotinylated anti-rabbit (1:1000) and streptavidin-RPE (1:500). MFI detected for selected scFvs is on the y-axis. (B) Ninety six well plates were coated with serially diluted Cf termite extract overnight at 4°C. ScFvs (1.0 ug/mL) were added to each well after blocking. The binding was determined after addition of HRP-conjugated anti-HA antibodies. Samples were tested 2 times and mean absorbance values are reported. Serially diluted extract (log scale) is on the x-axis.</p

Termite proteins cross-react with anti-cockroach allergen antibodies.

<p>Cf termite extract (1 μg) or German cockroach extract (GCr, 1 μg) was added to the wells of a microtiter plate for ELISA and probed with rabbit anti-cockroach allergen antibodies (1:500) followed by IRdye 800 labeled anti-rabbit antibody (1:10000). Black bars represent GCr extract and white bars represent Cf termite extract signals. Samples were tested 4 times and mean values are shown with standard deviation included as error bars. Relative IRdye800 signal is shown on the y-axis and anti-cockroach allergen antibody designation is shown on the x-axis.</p

Immunoblots of termite extract with anti-cockroach allergen antibodies.

<p>(A) GCr (left lane of each panel) and Cf termite extract (right lane of each panel) (1 μg) were resolved by SDS-PAGE and stained to visualize protein or transferred to PVDF and probed with rabbit anti-cockroach allergen antibodies (1:500) followed by IRdye 800 labeled anti-rabbit secondary antibody (1:10000). Molecular weight markers are shown to the left of the SDS-PAGE gel. (B) The GCr (left lane of each panel) and Cf extracts (right lane of each panel) were probed with a monoclonal anti-Bla g 1 antibody and an IRdye800 labeled goat anti-mouse secondary antibody. Molecular weight markers are shown to the left of the PVDF membrane. (C) Western blots of Cf termite whole body extract. Purified scFvs were used as primary antibodies (1.0 μg/mL) and horseradish peroxidase-conjugated anti-hemagglutinin (1:1000) was used for detection using a chemiluminescent substrate.</p

Putative termite homologs of cockroach allergen genes.

<p>Putative termite homologs of cockroach allergen genes.</p