A deletion/duplication in the Ligon lintless-2 locus induces siRNAs that inhibit cotton fiber cell elongation.

Most cultivated cotton (Gossypium hirsutum L.) varieties have two types of seed fibers: short fuzz fiber strongly adhered to the seed coat, and long lint fiber used in the textile industry. The Ligon lintless-2 (Li2) cotton mutant has a normal vegetative phenotype but produces very short lint fiber on the seeds. The Li2 mutation is controlled by a single dominant gene. We discovered a large structural rearrangement at the end of chromosome D13 in the Li2 mutant based on whole-genome sequencing and genetic mapping of segregating populations. The rearrangement contains a 177-kb deletion and a 221-kb duplication positioned as a tandem inverted repeat. The gene Gh_D13G2437 is located at the junction of the inverted repeat in the duplicated region. During transcription such structure spontaneously forms self-complementary hairpin RNA of Gh_D13G2437 followed by production of small interfering RNA (siRNA). Gh_D13G2437 encodes a Ran-Binding Protein 1 (RanBP1) that preferentially expresses during cotton fiber elongation. The abundance of siRNA produced from Gh_D13G2437 reciprocally corresponds with the abundance of highly homologous (68%-98% amino acid sequence identity) RanBP1 family transcripts during fiber elongation, resulting in a shorter fiber phenotype in the Li2. Overexpression of Gh_D13G2437 in the Li2 mutant recovered the long lint fiber phenotype. Taken together, our findings revealed that siRNA-induced silencing of a family of RanBP1s inhibit elongation of cotton fiber cells in the Li2 mutant.

Mapping-by-sequencing the locus of EMS-induced mutation responsible for tufted-fuzzless seed phenotype in cotton.

Cotton fiber mutants are valuable resources for studying functions of altered genes and their roles in fiber development. The n4t is a recessive tufted-fuzzless seed mutant created through chemical mutagenesis with ethyl methanesulfonate. Genetic analysis indicated that the tufted-fuzzless phenotype is controlled by a single recessive locus. In this study, we developed an F2 population of 602 progeny plants and sequenced the genomes of the parents and two DNA bulks from F2 progenies showing the mutant phenotype. We identified DNA sequence variants between the tufted-fuzzless mutant and wild type by aligning the sequence reads to the reference TM-1 genome and designed subgenome-specific SNP markers. We mapped the n4t locus on chromosome D04 within a genomic interval of about 411 kb. In this region, seven genes showed significant differential expression between the tufted-fuzzless mutant and wild type. Possible candidate genes are discussed in this study. The utilization of the n4t mutant along with other fiber mutants will facilitate our understanding of the molecular mechanisms of cotton fiber cell growth and development.

Elucidation of sequence polymorphism in fuzzless-seed cotton lines.

Most commercially produced cotton cultivars have two types of fibers on the seed coat, short fuzz and long lint. Lint fiber is used in the textile industry, while fuzz is considered an undesirable trait. Both types of fibers are believed to be controlled by the same regulators; however, their mechanisms of actions are still obscure. Cotton fiber mutants provide an excellent system to study the genes that regulate fiber development. Here we described four uncharacterized and three previously reported cotton mutants with fuzzless seed phenotypes. To evaluate whether or not the genes previously associated with fuzzless seed phenotypes have mutations we sequenced whole genomic DNA of seven mutants and wild type varieties. We identified multiple polymorphic changes among the tested genes. Non-synonymous SNPs in the coding region of the MML3-A gene was common in the six mutant lines tested in this study, showing both dominant and recessive fuzzless phenotypes. We have mapped the locus of the causative mutation for one of the uncharacterized fuzzless lines using an F2 population that originated from a cross between the dominant fuzzless mutant and a wild type. Further, we have clarified the current knowledge about the causative n2 mutations by analyzing the sequence data and previously reported mapping data. The key genes and possible mechanisms of fiber differentiation are discussed in this study.

An EMS-induced mutation in a tetratricopeptide repeat-like superfamily protein gene (Ghir_A12G008870) on chromosome A12 is responsible for the liy short fiber phenotype in cotton.

KEY MESSAGE: The EMS-induced threonine/isoleucine substitution in a tetratricopeptide repeat-like superfamily protein encoded by gene Ghir_A12G008870 is responsible for the Ligon-lintless-y (liy) short fiber phenotype in cotton. A short fiber mutant Ligon-lintless-y was created through treating the seeds of the cotton line MD15 with ethyl methanesulfonate. Genetic analysis indicated that the short fiber phenotype is controlled by a single recessive locus designated liy. From F2 populations derived from crosses between the mutant and its wild type (WT), we selected 132 short fiber progeny (liy/liy) and made two DNA bulks. We sequenced these DNA bulks along with the two parents of the population. The liy locus was located on chromosome A12. Using multiple F2 populations and F3 progeny plants, we mapped the liy locus within a genomic region of 1.18 Mb. In this region, there is only one gene, i.e., Ghir_A12G008870 encoding a tetratricopeptide repeat-like superfamily protein that has a non-synonymous mutation between the liy mutant and its WT. Analysis of a SNP marker representing this gene in the F2 and F3 progeny plants demonstrated its complete linkage with the liy short fiber phenotype. We further analyzed this SNP marker in a panel of 384 cotton varieties. The mutant allele is absent in all varieties analyzed. RNAseq and RT-qPCR analysis of the gene Ghir_A12G008870 during fiber development showed a significant expression difference between the liy mutant and its WT in developing fiber cells beginning at 12 days post-anthesis. Virus-induced gene silencing of the gene Ghir_A12G008870 significantly reduced the fiber length of the WT cotton line MD15. Taken together, our results suggest that the gene Ghir_A12G008870 is involved in the cotton fiber cell elongation process and is a promising candidate gene responsible for the liy short fiber phenotype.

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

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.

The P450 gene CYP749A16 is required for tolerance to the sulfonylurea herbicide trifloxysulfuron sodium in cotton (Gossypium hirsutum L.).

BACKGROUND: Weed management is critical to global crop production and is complicated by rapidly evolving herbicide resistance in weeds. New sources of herbicide resistance are needed for crop plants so that applied herbicides can be rotated or combined to thwart the evolution of resistant weeds. The diverse family of cytochrome P450 proteins has been suggested to be a source of detoxifying herbicide metabolism in both weed and crop plants, and greater understanding of these genes will offer avenues for crop improvement and novel weed management practices. RESULTS: Here, we report the identification of CYP749A16 (Gh_D10G1401) which is responsible for the natural tolerance exhibited by most cotton, Gossypium hirsutum L., cultivars to the herbicide trifloxysulfuron sodium (TFS, CGA 362622, commercial formulation Envoke). A 1-bp frameshift insertion in the third exon of CYP749A16 results in the loss of tolerance to TFS. The DNA marker designed from this insertion perfectly co-segregated with the phenotype in 2145 F2 progeny of a cross between the sensitive cultivar Paymaster HS26 and tolerant cultivar Stoneville 474, and in 550 recombinant inbred lines of a multi-parent advanced generation inter-cross population. Marker analysis of 382 additional cotton cultivars identified twelve cultivars containing the 1-bp frameshift insertion. The marker genotypes matched perfectly with phenotypes in 188 plants from the selected twelve cultivars. Virus-induced gene silencing of CYP749A16 generated sensitivity in the tolerant cotton cultivar Stoneville 474. CONCLUSIONS: CYP749A16 located on chromosome D10 is required for TFS herbicide tolerance in cotton. This finding should add to the repertoire of tools available to farmers and breeders for the advancement of agricultural productivity.

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.

Genome-wide analysis of gene expression of EMS-induced short fiber mutant Ligon lintless-y (liy) in cotton (Gossypium hirsutum L.).

In this work we describe a chemically-induced short fiber mutant cotton line, Ligon-lintless-y (liy), which is controlled by a single recessive locus and affects multiple traits, including height of the plant, and length and maturity of fiber. An RNAseq analysis was used to evaluate global transcriptional changes during cotton fiber development at 3, 8 and 16days post anthesis. We found that 613, 2629 and 3397 genes were significantly down-regulated, while 2700, 477 and 3260 were significantly up-regulated in liy at 3, 8 and 16 DPA. Gene set enrichment analysis revealed that many metabolic pathways, including carbohydrate, cell wall, hormone metabolism and transport were substantially altered in liy developing fibers. We discuss perturbed expression of genes involved in signal transduction and biosynthesis of phytohormones, such as auxin, abscisic acid, gibberellin and ethylene. The results of this study provide new insights into transcriptional regulation of cotton fiber development.

Role of xyloglucan in cotton (Gossypium hirsutum L.) fiber elongation of the short fiber mutant Ligon lintless-2 (Li2).

Xyloglucan is a matrix polysaccharide found in the cell walls of all land plants. In growing cells, xyloglucan is thought to connect cellulose microfibrils and regulate their separation during wall extension. Ligon lintless-2 (Li2) is a monogenic dominant cotton fiber mutation that causes extreme reduction in lint fiber length with no pleiotropic effects on vegetative growth. Li2 represents an excellent model system to study fiber elongation. To understand the role of xyloglucan in cotton fiber elongation we used the short fiber mutant Li2 and its near isogenic wild type for analysis of xyloglucan content and expression of xyloglucan-related genes in developing fibers. Accumulation of xyloglucan was significantly higher in Li2 developing fibers than in wild type. Genes encoding enzymes for nine family members of xyloglucan biosynthesis were identified in the draft Gossypium hirsutum genome. RNAseq analysis revealed that most differentially expressed xyloglucan-related genes were down-regulated in Li2 fiber cells. RT-qPCR analysis revealed that the peak of expression for the majority of xyloglucan-related genes in wild type developing fibers was 5-16days post anthesis (DPA) compared to 1-3 DPA in Li2 fibers. Thus, our results suggest that early activation of xyloglucan-related genes and down regulation of xyloglucan degradation genes during the elongation phase lead to elevated accumulation of xyloglucan that restricts elongation of fiber cells in Li2.

A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and F-actin organization resulting in dwarf, lintless cotton plants.

Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single-celled trichomes that comprise cotton lint fibers. Using mapping-by-sequencing, virus-induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li1 short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F-actin in Li1 plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F-actin did not uniformly decorate the nuclear envelope. While wild-type lint fiber trichome cells contained long longitudinal actin cables, the short Li1 fiber cells accumulated disoriented transverse cables. The polymerization-defective Gly65Val allele in Li1 plants likely disrupts processive elongation of F-actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li1 mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth.

RNA-Seq Analysis of Developing Pecan (Carya illinoinensis) Embryos Reveals Parallel Expression Patterns among Allergen and Lipid Metabolism Genes.

The pecan nut is a nutrient-rich part of a healthy diet full of beneficial fatty acids and antioxidants, but can also cause allergic reactions in people suffering from food allergy to the nuts. The transcriptome of a developing pecan nut was characterized to identify the gene expression occurring during the process of nut development and to highlight those genes involved in fatty acid metabolism and those that commonly act as food allergens. Pecan samples were collected at several time points during the embryo development process including the water, gel, dough, and mature nut stages. Library preparation and sequencing were performed using Illumina-based mRNA HiSeq with RNA from four time points during the growing season during August and September 2012. Sequence analysis with Trinotate software following the Trinity protocol identified 133,000 unigenes with 52,267 named transcripts and 45,882 annotated genes. A total of 27,312 genes were defined by GO annotation. Gene expression clustering analysis identified 12 different gene expression profiles, each containing a number of genes. Three pecan seed storage proteins that commonly act as allergens, Car i 1, Car i 2, and Car i 4, were significantly up-regulated during the time course. Up-regulated fatty acid metabolism genes that were identified included acyl-[ACP] desaturase and omega-6 desaturase genes involved in oleic and linoleic acid metabolism. Notably, a few of the up-regulated acyl-[ACP] desaturase and omega-6 desaturase genes that were identified have expression patterns similar to the allergen genes based upon gene expression clustering and qPCR analysis. These findings suggest the possibility of coordinated accumulation of lipids and allergens during pecan nut embryogenesis.

Small RNA sequencing and degradome analysis of developing fibers of short fiber mutants Ligon-lintles-1 (Li 1 ) and -2 (Li 2 ) revealed a role for miRNAs and their targets in cotton fiber elongation.

BACKGROUND: The length of cotton fiber is an important agronomic trait that directly affects the quality of yarn and fabric. Understanding the molecular basis of fiber elongation would provide a means for improvement of fiber length. Ligon-lintless-1 (Li 1 ) and -2 (Li 2 ) are monogenic and dominant mutations that result in an extreme reduction in the length of lint fiber on mature seeds. In a near-isogenic state with wild type cotton these two short fiber mutants provide an effective model system to study the mechanisms of fiber elongation. Plant miRNAs regulate many aspects of growth and development. However, the mechanism underlying the miRNA-mediated regulation of fiber development is largely unknown. RESULTS: Small RNA libraries constructed from developing fiber cells of the short fiber mutants Li 1 and Li 2 and their near-isogenic wild type lines were sequenced. We identified 24 conservative and 147 novel miRNA families with targets that were detected through degradome sequencing. The distribution of the target genes into functional categories revealed the largest set of genes were transcription factors. Expression profiles of 20 miRNAs were examined across a fiber developmental time course in wild type and short fiber mutations. We conducted correlation analysis between miRNA transcript abundance and the length of fiber for 11 diverse Upland cotton lines. The expression patterns of 4 miRNAs revealed significant negative correlation with fiber lengths of 11 cotton lines. CONCLUSIONS: Our results suggested that the mutations have changed the regulation of miRNAs expression during fiber development. Further investigations of differentially expressed miRNAs in the Li 1 and Li 2 mutants will contribute to better understanding of the regulatory mechanisms of cotton fiber development. Four miRNAs negatively correlated with fiber length are good candidates for further investigations of miRNA regulation of important genotype dependent fiber traits. Thus, our results will contribute to further studies on the role of miRNAs in cotton fiber development and will provide a tool for fiber improvement through molecular breeding.

Mapping-by-sequencing of Ligon-lintless-1 (Li 1 ) reveals a cluster of neighboring genes with correlated expression in developing fibers of Upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Mapping-by-sequencing and SNP marker analysis were used to fine map the Ligon-lintless-1 ( Li 1 ) short fiber mutation in tetraploid cotton to a 255-kb region that contains 16 annotated proteins. The Ligon-lintless-1 (Li 1 ) mutant of cotton (Gossypium hirsutum L.) has been studied as a model for cotton fiber development since its identification in 1929; however, the causative mutation has not been identified yet. Here we report the fine genetic mapping of the mutation to a 255-kb region that contains only 16 annotated genes in the reference Gossypium raimondii genome. We took advantage of the incompletely dominant dwarf vegetative phenotype to identify 100 mutants (Li 1 /Li 1 ) and 100 wild-type (li 1 /li 1 ) homozygotes from a mapping population of 2567 F2 plants, which we bulked and deep sequenced. Since only homozygotes were sequenced, we were able to use a high stringency in SNP calling to rapidly narrow down the region harboring the Li 1 locus, and designed subgenome-specific SNP markers to test the population. We characterized the expression of all sixteen genes in the region by RNA sequencing of elongating fibers and by RT-qPCR at seven time points spanning fiber development. One of the most highly expressed genes found in this interval in wild-type fiber cells is 40-fold under-expressed at the day of anthesis (DOA) in the mutant fiber cells.  This gene is a major facilitator superfamily protein, part of the large family of proteins that includes auxin and sugar transporters. Interestingly, nearly all genes in this region were most highly expressed at DOA and showed a high degree of co-expression. Further characterization is required to determine if transport of hormones or carbohydrates is involved in both the dwarf and lintless phenotypes of Li 1 plants.

Next generation genetic mapping of the Ligon-lintless-2 (Li2) locus in upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Mapping-by-sequencing and novel subgenome-specific SNP markers were used to fine map the Ligon-lintless 2 ( Li 2 ) short-fiber gene in tetraploid cotton. These methodologies will accelerate gene identification in polyploid species. Next generation sequencing offers new ways to identify the genetic mechanisms that underlie mutant phenotypes. The release of a reference diploid Gossypium raimondii (D5) genome and bioinformatics tools to sort tetraploid reads into subgenomes has brought cotton genetic mapping into the genomics era. We used multiple high-throughput sequencing approaches to identify the relevant region of reference sequence and identify single nucleotide polymorphisms (SNPs) near the short-fiber mutant Ligon-lintless 2 (Li 2) gene locus. First, we performed RNAseq on 8-day post-anthesis (DPA) fiber cells from the Li 2 mutant and its wild type near isogenic line (NIL) Gossypium hirsutum cv. DP5690. We aligned sequence reads to the D5 genome, sorted the reads into A and D subgenomes with PolyCat and called SNPs with InterSNP. We then identified SNPs that would result in non-synonymous substitutions to amino acid sequences of annotated genes. This step allowed us to identify a 1-Mb region with 24 non-synonymous SNPs, representing the introgressed region that differentiates Li 2 from its NIL. Next, we sequenced total DNA from pools of F2 plants, using a super bulked segregant analysis sequencing (sBSAseq) approach. The sBSAseq predicted 82 non-synonymous SNPs among 3,494 SNPs in a 3-Mb region that includes the region identified by RNAseq. We designed subgenome-specific SNP markers and tested them in an F2 population of 1,733 individuals to construct a genetic map. Our resulting genetic interval contains only one gene, an aquaporin, which is highly expressed in wild-type fibers and is significantly under-expressed in elongating Li 2 fiber cells.

The Li2 mutation results in reduced subgenome expression bias in elongating fibers of allotetraploid cotton (Gossypium hirsutum L.).

Next generation sequencing (RNA-seq) technology was used to evaluate the effects of the Ligon lintless-2 (Li2) short fiber mutation on transcriptomes of both subgenomes of allotetraploid cotton (Gossypium hirsutum L.) as compared to its near-isogenic wild type. Sequencing was performed on 4 libraries from developing fibers of Li2 mutant and wild type near-isogenic lines at the peak of elongation followed by mapping and PolyCat categorization of RNA-seq data to the reference D5 genome (G. raimondii) for homeologous gene expression analysis. The majority of homeologous genes, 83.6% according to the reference genome, were expressed during fiber elongation. Our results revealed: 1) approximately two times more genes were induced in the AT subgenome comparing to the DT subgenome in wild type and mutant fiber; 2) the subgenome expression bias was significantly reduced in the Li2 fiber transcriptome; 3) Li2 had a significantly greater effect on the DT than on the AT subgenome. Transcriptional regulators and cell wall homeologous genes significantly affected by the Li2 mutation were reviewed in detail. This is the first report to explore the effects of a single mutation on homeologous gene expression in allotetraploid cotton. These results provide deeper insights into the evolution of allotetraploid cotton gene expression and cotton fiber development.

Methoprene and temperature effects on caste differentiation and protein composition in the Formosan Subterranean termite, Coptotermes formosanus.

The utilization of multiple castes is a shared feature of social insects. In termites, multiple extrinsic factors have been shown to impact caste differentiation; for example, increased temperature has been shown to increase soldier production. Also, application of exogenous methoprene has also been demonstrated to increase soldier production. The objective of this investigation was to examine and correlate the effects of temperature variation and methoprene treatments on termite caste differentiation, and identify the resulting changes in protein levels. Our results indicate that worker-to-soldier differentiation is modulated by temperature, where a greater number of soldiers developed at a higher rate at higher temperatures compared to lower temperatures. We analyzed total protein by sodium dodecyl sulfate Polyacrylamide gel electrophoresis and N-terminal sequencing and found several changes. Specifically, four proteins affected by temperature change were identified: Hexamerin-1, Hexamerin-2, Endo-beta 1,4 glucanase, and myosin. These proteins were further examined for their response to temperature, assay length (time), and exposure to the juvenile hormone analog methoprene. Hexamerin-1 protein showed a temperature-and assay length-dependent effect, while Hexamerin-2, Endo-beta 1, 4 glucanase, and myosin protein levels were all affected by temperature, assay length, and exposure to methoprene. Our analysis allows the correlation of temperature, assay length, and presence of methoprene with specific changes in protein levels that occur during caste differentiation. These results can be directly applied to better understand the complex developmental factors that control termite differentiation and guide the use of juvenile hormone analogs to maximize efficiency of termite eradication in the field.

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.

Diet-mediated inter-colonial aggression in the formosan subterranean termite Coptotermes formosanus.

In most social insects, intercolonial and interspecific aggression are expressions of territoriality. In termites, cuticular hydrocarbons (CHCs) have been extensively studied for their role in nestmate recognition and aggressive discrimination of nonnest-mates. More recently, molecular genetic techniques have made it possible to determine relatedness between colonies and to investigate the influence of genetics on aggression. In the Formosan subterranean termite, Coptotermes formosanus, however, the role of CHCs and genetic relatedness in inter-colony aggression has been ambiguous, suggesting the involvement of additional factors in nest-mate recognition. In this study we assess the range of aggression in this termite species and characterize the influence of genetic relatedness, CHC profiles and diet on aggression levels. We collected four colonies of C. formosanus, feeding either on bald cypress or birch, from three locations in Louisiana. Inter-colony aggression ranged from low to high. Differences in CHC profiles, as well as genetic distances between colonies determined by using microsatellite DNA markers, showed no significant correlation with aggression. However, termite diet (host tree) played a significant role in determining the level of aggression. Thus, two distantly related colonies, each feeding on different diets, showed high aggression that significantly diminished if they were fed on the same wood in the laboratory (spruce). Using headspace solid phase microextraction, we found three compounds from workers fed on birch that were absent in workers fed on spruce. Such diet-derived chemicals may be involved in the complex determination of nest-mate recognition in C. formosanus.