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.

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.

Transcriptomic and functional resources for the small hive beetle Aethina tumida, a worldwide parasite of honey bees.

The small hive beetle (SHB), Aethina tumida, is a major pest of managed honey bee (Apis mellifera) colonies in the United States and Australia, and an emergent threat in Europe. While strong honey bee colonies generally keep SHB populations in check, weak or stressed colonies can succumb to infestations. This parasite has spread from a sub-Saharan Africa to three continents, leading to immense management and regulatory costs. We performed a transcriptomic analysis involving deep sequencing of multiple life stages and both sexes of this species. The assembled transcriptome appears to be nearly complete, as judged by conserved insect orthologs and the ability to find plausible homologs for 11,952 proteins described from the genome of the red flour beetle. Expressed genes include each of the major metabolic, developmental and sensory groups, along with genes for proteins involved with immune defenses and insecticide resistance. We also present a total of 23,085 high-quality SNP's for the assembled contigs. We highlight potential differences between this beetle and its honey bee hosts, and suggest mechanisms of future research into the biology and control of this species. SNP resources will allow functional genetic analyses and analyses of dispersal for this invasive pest. All resources are posted as Supplemental Tables at https://data.nal.usda.gov/dataset/data-transcriptomic-and-functional-resources-small-hive-beetle-aethina-tumida-worldwide, and at NCBI under Bioproject PRJNA256171.

Genetics, Synergists, and Age Affect Insecticide Sensitivity of the Honey Bee, Apis mellifera.

The number of honey bee colonies in the United States has declined to half of its peak level in the 1940s, and colonies lost over the winter have reached levels that are becoming economically unstable. While the causes of these losses are numerous and the interaction between them is very complex, the role of insecticides has garnered much attention. As a result, there is a need to better understand the risk of insecticides to bees, leading to more studies on both toxicity and exposure. While much research has been conducted on insecticides and bees, there have been very limited studies to elucidate the role that bee genotype and age has on the toxicity of these insecticides. The goal of this study was to determine if there are differences in insecticide sensitivity between honey bees of different genetic backgrounds (Carniolan, Italian, and Russian stocks) and assess if insecticide sensitivity varies with age. We found that Italian bees were the most sensitive of these stocks to insecticides, but variation was largely dependent on the class of insecticide tested. There were almost no differences in organophosphate bioassays between honey bee stocks (<1-fold), moderate differences in pyrethroid bioassays (1.5 to 3-fold), and dramatic differences in neonicotinoid bioassays (3.4 to 33.3-fold). Synergism bioassays with piperonyl butoxide, amitraz, and coumaphos showed increased phenothrin sensitivity in all stocks and also demonstrated further physiological differences between stocks. In addition, as bees aged, the sensitivity to phenothrin significantly decreased, but the sensitivity to naled significantly increased. These results demonstrate the variation arising from the genetic background and physiological transitions in honey bees as they age. This information can be used to determine risk assessment, as well as establishing baseline data for future comparisons to explain the variation in toxicity differences for honey bees reported in the literature.

Differential viral levels and immune gene expression in three stocks of Apis mellifera induced by different numbers of Varroa destructor.

The viral levels and immune responses of Italian honey bees (IHB), Russian honey bees (RHB) and an outcross of Varroa Sensitive Hygienic bees (POL) deliberately infested with one or two foundress Varroa were compared. We found that the Deformed wing virus (DWV) level in IHB inoculated with one or two foundress Varroa increased to about 10(3) or 10(5) fold the levels of their uninfested brood. In contrast, POL (10(2) or 10(4) fold) and RHB (10(2) or l0(4) fold) supported a lower increase in DWV levels. The feeding of different stages of Varroa nymphs did not increase DWV levels of their pupal hosts. Analyses of their corresponding Varroa mites showed the same trends: two foundress Varroa yielded higher DWV levels than one foundress, and the addition of nymphs did not increase viral levels. Using the same pupae examined for the presence of viruses, 16 out of 24 genes evaluated showed significant differential mRNA expression levels among the three honey bee stocks. However, only four genes (Defensin, Dscam, PPOact and spaetzle), which were expressed at similar levels in uninfested pupae, were altered by the number of feeding foundress Varroa and levels of DWV regardless of stocks. This research provides the first evidence that immune response profiles of different honey bee stocks are induced by Varroa parasitism.

An evaluation of the associations of parameters related to the fall of Varroa destructor (Acari: Varroidae) from commercial honey bee (Hymenoptera: Apidae) colonies as tools for selective breeding for mite resistance.

Varroa destructor (Anderson and Trueman) trapped on bottom boards were assessed as indirect measurements of colony mite population differences and potential indicators of mite resistance in commercial colonies of Russian and Italian honey bees (Apis mellifera L.) by using 35 candidate measurements. Measurements included numbers of damaged and nondamaged younger mites, nymphs, damaged and nondamaged older mites, fresh mites, and all mites, each as a proportion of total mites in the colonies and as a proportion of all trapped mites or all trapped fresh mites. Several measurements differed strongly between the stocks, suggesting that the detailed characteristics of trapped mites may reflect the operation of resistance mechanisms in the Russian honey bees. Regression analyses were used to determine the relationships of these candidate measurements with the number of mites in the colonies. The largest positive regressions differed for the two stocks (Italian honey bees: trapped mites and trapped younger mites; Russian honey bees: trapped younger mites and trapped fresh mites). Also, the regressions for Italian honey bees were substantially stronger. The largest negative regressions with colony mites for both stocks were for the proportion of older mites out of all trapped mites. Although these regressions were statistically significant and consistent with those previously reported, they were weaker than those previously reported. The numbers of mites in the colonies were low, especially in the Russian honey bee colonies, which may have negatively influenced the precision of the regressions.

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.

Cyp15F1: a novel cytochrome P450 gene linked to juvenile hormone-dependent caste differention in the termite Reticulitermes flavipes.

Termites are eusocial insects that jointly utilize juvenile hormone (JH), pheromones, and other semiochemicals to regulate caste differentiation and achieve caste homeostasis. Prior EST sequencing from the symbiont-free gut transcriptome of Reticulitermes flavipes unexpectedly revealed a number of unique cytochrome P450 (Cyp) transcripts, including fragments of a Cyp15 family gene (Cyp15F1) with homology to other insect Cyp15s that participate in JH biosynthesis. The present study investigated the role of Cyp15F1 in termite caste polyphenism and specifically tested the hypothesis that it plays a role in JH-dependent caste differentiation. After assembling the full-length Cyp15F1 cDNA sequence, we (i) determined its mRNA tissue expression profile, (ii) investigated mRNA expression changes in response to JH and the caste-regulatory primer pheromones γ-cadinene (CAD) and γ-cadinenal (ALD), and (iii) used RNA interference (RNAi) in combination with caste differentiation bioassays to investigate gene function at the phenotype level. Cyp15F1 has ubiquitous whole-body expression (including gut tissue); is rapidly and sustainably induced from 3 h to 48 h by JH, CAD, and ALD; and functions at least in part by facilitating JH-dependent soldier caste differentiation. These findings provide the second example of a termite caste regulatory gene identified through the use of RNAi, and significantly build upon our understanding of termite caste homeostatic mechanisms. These results also reinforce the concept of environmental caste determination in termites by revealing how primer pheromones, as socioenvironmental factors, can directly influence Cyp15 expression and caste differentiation.

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.

Soldier caste influences on candidate primer pheromone levels and juvenile hormone-dependent caste differentiation in workers of the termite Reticulitermes flavipes.

Caste systems and the division of labor they make possible are common underlying features of all social insects. Multiple extrinsic factors have been shown to impact caste composition in social insect colonies. Primer pheromones are one type of extrinsic caste-regulatory factor; they are chemical signaling molecules produced by certain colony members to impact developmental physiology of recipient nestmates. However, only limited evidence exists regarding primer pheromones and their actions in eusocial termites. In previous research we identified two soldier-produced terpenes, γ-cadinene (CAD) and γ-cadinenal (ALD), as candidate primer pheromones of the lower termite Reticulitermes flavipes. In the present study we tested hypotheses related to CAD and ALD action in recipient individuals. We examined the influences of terminally developed soldier termites on (1) CAD and ALD levels and (2) caste differentiation in developmentally totipotent workers. Our findings show CAD and ALD (respectively) are caste stimulatory and inhibitory components of chemical blends present in soldier heads, ALD levels increase significantly (10.9×) in workers only in the presence of soldiers, and soldiers can reduce developmental-hormone response thresholds of workers, presumably via ALD action. These findings provide novel evidence supporting that CAD and ALD are authentic caste-regulatory primer pheromones in Reticulitermes.

Socio-environmental and endocrine influences on developmental and caste-regulatory gene expression in the eusocial termite Reticulitermes flavipes.

BACKGROUND: Strict regulation of caste differentiation, at the molecular level, is thought to be important to maintain social structure in insect societies. Previously, a number of extrinsic and intrinsic factors have been shown to influence caste composition in termite colonies. One important factor is the influence of nestmates; in particular, soldier termites are known to inhibit hormone-dependent worker-to-soldier differentiation. However, soldier influences on nestmates at the molecular level are virtually unknown. Here, to test the hypothesis that soldiers can influence nestmate gene expression, we investigated the impact of four treatments on whole-body gene expression in totipotent Reticulitermes flavipes workers: (i) juvenile hormone III (JHIII; a morphogenetic hormone), (ii) soldier head extracts (SHE), (iii) JHIII+SHE, and (iv) live soldiers. RESULTS: Using quantitative-real-time PCR we determined the expression patterns of 49 previously identified candidate genes in response to the four treatments at assay days 1, 5, and 10. Thirty-eight total genes from three categories (chemical production/degradation, hemolymph protein, and developmental) showed significant differential expression among treatments. Most importantly, SHE and live soldier treatments had a significant impact on a number of genes from families known to play roles in insect development, supporting previous findings and hypotheses that soldiers regulate nestmate caste differentiation via terpene primer pheromones contained in their heads. CONCLUSIONS: This research provides new insights into the impacts that socio-environmental factors (JH, soldiers, primer pheromones) can have on termite gene expression and caste differentiation, and reveals a number of socially-relevant genes for investigation in subsequent caste differentiation research.

Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes.

Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when alpha-naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub-cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)-derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional-genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut.

Effects of soldier-derived terpenes on soldier caste differentiation in the termite Reticulitermes flavipes.

Primer pheromones play key roles in regulating division of labor, which is a fundamental and defining aspect of insect sociality. Primer pheromones are chemical messengers that transmit hormone-like messages among colony members; in recipients, these messages can either induce or suppress phenotypic caste differentiation. Here, we investigated soldier caste-derived chemicals as possible primer pheromones in the lower termite Reticulitermes flavipes, a species for which no primer pheromones have yet been identified. We determined that soldier head extracts (SHE), when provided to totipotent workers along with the insect morphogenetic juvenile hormone (JH), significantly enhanced soldier caste differentiation. When applied alone, however, SHE had no impacts on caste differentiation, survivorship, or any other aspect of worker biology. These findings support a function of soldier chemicals as primer pheromones that enhance the action of the endogenous JH. In accord with previous studies, gamma-cadinene and the corresponding aldehyde, gamma-cadinenal, were identified by gas chromatography-mass spectrometry and nuclear magnetic resonance analyses as the two most abundant components of R. flavipes SHE. Validative bioassays with commercially available cadinene confirmed activity. Several other terpenes, previously identified in R. flavipes soldiers, also were found to be active. These findings reveal a novel primer pheromone-like function for soldier-derived terpenes in termites and further suggest convergent evolution of terpene functions in enhancing JH-dependent soldier caste differentiation.

Pyramiding of insecticidal compounds for control of the cowpea bruchid (Callosobruchus maculatus F.).

The cowpea bruchid (Callosobruchus maculatus F.) (Chrysomelidae: Bruchini) is a major pest of stored cowpea grain. With limited available technologies for controlling the bruchid, transgenic cowpeas with bruchid resistance genes engineered into them could become the next management tools. An investigation was made of two different sets of potential transgenic insecticidal compounds using an artificial seed system: (i) CIP-PH-BT-J and recombinant egg white avidin, and (ii) avidin and wheat alpha-amylase inhibitor. CIP-PH-BT-J (0.1%; 1000 mg kg(-1)) and recombinant egg white avidin (0.006%; 60 mg kg(-1)) incorporated separately into artificial seeds caused 98.2 and 99% larval mortality rates respectively. Combining CIP-PH-BT-J and avidin in the same artificial seed provided additional mortality compared with each factor incorporated singly; no insects survived in seeds with the combined toxins. Similarly, when avidin and wheat alpha-amylase inhibitor (alphaAI) (1%; 10 g kg(-1)) were incorporated separately into artificial seeds, this caused 99.8 and 98% mortality respectively. However, in combination, avidin and alphaAI did not increase mortality, but they did cause a significant increase in developmental time of the cowpea bruchids. These results emphasize that the joint action of potential insecticidal compounds cannot be predicted from results obtained separately for each compound, and they suggest potential transgenes for further consideration.

Hexamerin-based regulation of juvenile hormone-dependent gene expression underlies phenotypic plasticity in a social insect.

Worker termites of the genus Reticulitermes are temporally-arrested juvenile forms that can terminally differentiate into adultsoldier- or reproductive-caste phenotypes. Soldier-caste differentiation is a developmental transition that is induced by high juvenile hormone (JH) titers. Recently, a status quo hexamerin mechanism was identified, which reduces JH efficacy and maximizes colony fitness via the maintenance of high worker-caste proportions. Our goal in these studies was to investigate more thoroughly the influences of the hexamerins on JH-dependent gene expression in termite workers. Our approach involved RNA interference (RNAi), bioassays and quantification of gene expression. We first investigated the expression of 17 morphogenesis-associated genes in response to RNAi-based hexamerin silencing. Hexamerin silencing resulted in significant downstream impacts on 15 out of the 17 genes, suggesting that these genes are members of a JH-responsive genomic network. Next, we compared gene-expression profiles in workers after RNAi-based hexamerin silencing to that of (i) untreated workers that were held away from the colony; and (ii) workers that were also held away from the colony, but with ectopic JH. Here, although there was no correlation between hexamerin silencing and colony-release effects, we observed a significant correlation between hexamerin silencing and JH-treatment effects. These findings provide further evidence supporting the hypothesis that the hexamerins modulate JH availability, thus limiting the impacts of JH on termite caste polyphenism. Results are discussed in a context relative to outstanding questions on termite developmental biology, particularly on regulatory gene networks that respond to JH-, colony- and environmental-cues.