The Formosan subterranean termite (FST), Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), is an invasive pest species that causes billions of dollars in economical damage in the southeastern U.S., especially in Louisiana. The results of this study will facilitate a genomic approach to termite control, likely reducing the need for chemical insecticides.
The principal goals of the proposed study will be to identify genes vital to immune defense and detoxification in the FST and to test if there is differential gene expression of immune related and detoxification genes among workers and soldiers and among heterozygous and homozygous male and female alates. To achieve these goals, I propose the following specific aims:
Aim 1: Identify genes associated with immune response and detoxification in termites and develop primers for them (completed). To achieve this objective, I have screened the recently completed expressed sequence tag (EST) library of FST and public databases for putative genes associated with immunity and detoxification. I then designed and tested primers for target genes and housekeeping genes and optimized them for quantitative reverse transcriptase polymerase chain reaction (qRT PCR).
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Aim 2: Test for differences in the constitutive and inducible expression of immune response and detoxification genes in workers and soldiers (in progress). I have determined sub-lethal doses and times for toxic and bacterial challenges of FST workers and soldiers. I will next determine the levels of constitutive expression (untreated controls) vs. induced expression (challenged with bacteria or xenobiotics) in workers and soldiers via qRT PCR.
Aim 3: Test whether constitutive and induced expression levels of immune response and detoxification genes differ between male and female alates and/or between heterozygous and homozygous alates. I first determined sub-lethal doses and times for bacterial and toxin challenge of FST alates. I have determined the sexes of the alates and will next determine the levels of heterozygosity (via microsatellite genotyping) and the levels of constitutive expression (untreated controls) vs. induced expression (challenged with bacteria or xenobiotics) in heterozygous and homozygous male and female alates via qRT PCR.
A.3. Expected Significance:
The proposed study will be used to extend the current research of termite molecular biology into the field of genomics. Data generated from this research into termite gene expression will produce several major publications in national and international journals, as well as presentations at scientific meetings. Results will be used as preliminary data to compete for sustainable federal funding for research and development with the long-term goal to apply termite genomics to devise novel biotechnologies for reducing the amount of pesticide used for termite control. Subterranean termites are an excellent model for investigating the effects of inbreeding on gene expression, since inbreeding is a natural part of subterranean termite life history. The basic and applied thrust of the genomics based research program will be attractive to several federal funding agencies (e.g. USDA-AFRI).
B. BACKGROUND and SIGNIFICANCE
The Formosan subterranean termite is one of the most aggressive and economically important agricultural, rural and urban pests in Louisiana. In addition to the destruction the FST causes to urban and rural structures, this termite damages at least 23 plant species in Louisiana. Trees are considered a major crop in Louisiana and the infestation rate is alarming . Tree damage caused by FST is severe because this species primarily feeds on the heartwood, which reduces structural integrity so that trees collapse during inclement weather. For example, after hurricane Andrew in 1992, surveys by the New Orleans Mosquito Control Board and LSU Agricultural Center revealed that 30-50% of the felled trees were termite infested . The economic losses due to the FST are estimated to be over $1.5 billion per year in the USA and $500 million per year in Louisiana alone.
B.1. Need for improved termite treatments:
Conventional termite treatment of structures and trees relies heavily on chemical insecticides. Humans and termites live in close contact in the urban landscape, and public health concerns about adverse affects of chemicals are increasing. Therefore, it is important to develop new termite control technologies that require less pesticides to reduce termite damage to urban structures, agriculture and forestry, reduce the spread invasive termites and lower the costs for termite control and damage repair.
Subterranean termite defense against pathogens and toxic chemicals are of high interest due to the significance for colony propagation, fitness and health of these economically important insects. Studies of gene expression are the first crucial steps to elucidate the genetic underpinnings of termite immunology and detoxification. Disruption of colony defense mechanisms against pathogens and pesticides by use of biotechnology-based treatments will be a novel and promising alternative to conventional chemical treatment in the future.
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B. 2. Expressed sequence tag (EST) library:
The first comprehensive genomic resource (EST library) for FSTs has been developed using eggs, multiple larva stages, workers, presoldiers, soldiers, male and female nymphs (immature reproductives), neotenic queens, male and female alates, primary kings and physogastric queens from an FST population in New Orleans by Husseneder et al. (oral communication). This material was sent to Invitrogen (Carlsbad, CA) to create a normalized cDNA library from equal amounts of RNA from samples of each category. Ultimately, 7,662 expressed sequence tags (ESTs) were submitted to dbEST and GenBank (acc. # FK829415 to FK837077, http://www.ncbi.nlm.nih.gov/ Genbank/index.html). The ESTs were assembled into 4,726 contiguous sequences (contigs). After removal of sequences which originated from the same contig and annotation using BLAST2GO, there were 4,154 unique EST sequences without redundancy to other ESTs (unigenes). Forty-four percent of the unigenes had significant homology to known sequences (with the majority matching to insect genes), and 16% of the contigs could be assigned Gene Ontology terms. Among those were genes putatively involved in termite reproduction (e.g. vitellogenin), ovary maturation, juvenile hormone binding/storage proteins (e.g. hexamerins, [2-3]), spermatogenesis, protein translation and folding, fatty acid biosynthesis, sugar-, ion- and lipid-transport, apoptosis, longevity assurance, immune response (genes expressing antibacterial peptides, gram-negative binding protein, lectin-like genes for binding glucanases, lipopolysaccarides and chitin and serine proteases that possibly activate enzymes of immune response pathways) and detoxification (genes from the cytochrome P450 and Glutathion-S-Transferase families), among others.
B.3. Termite immune system:
The insect immune system consists of several pathways that are triggered after different antigens are recognized, e.g., lipopolysaccharides on the cell surface of gram- bacteria, peptidoglucans on the cell wall of gram+ and gram- bacteria, beta-1,3-glucans and mannans on fungal cell walls. Once pathogens have been recognized, appropriate responses are initiated including opsonization, phagocytosis, melanization, encapsulation, coagulation, the release of cytotoxic molecules, as well as the production of antimicrobial peptides, lysozymes and proteases . These responses are initiated via four main pathways of immune defense. The Toll-signaling pathway is mainly triggered by infection with fungi and gram+ bacteria and leads to the release of cytokines (signaling factors), phagocytosis and the production of antimicrobial peptides; the Immune-deficient mutation (Imd) pathway is triggered mainly by gram- bacteria and leads to the production of antimicrobial peptides; the lesser known JAK/STAT pathway possibly regulates cellular responses (opsonization, phagocytosis); the prophenoloxidase system is activated by serine proteases and leads to melanization and encapsulation of microbes and the release of cytotoxic molecules . All these pathways are regulated by a network of genes, which produce recognition molecules (e.g., Gram-negative binding proteins, which bind to lipopolysaccharides and glucans of bacteria surface, lectin- and ficolin-like proteins that "mark" (opsonize) microbes for phagocytosis, proteases that activate enzymes (e.g. serine proteinases), signaling factors and antimicrobial peptides (e.g. termicin, spinigerin, ), among other functions.
Termites are at increased risk for exposure to potentially pathogenic microorganisms because they nest and forage in high densities with genetically similar nestmates in an environment with high microbial density (decaying wood and soil) and a microclimate that is conducive to the growth of microorganisms . Therefore, termites are considered to be valuable model systems for studying insect immunity. Several studies have shown that termites (Zootermopsis angusticollis) can be immunized with killed bacteria or fungal spores leading to increased immune activity and the detection of inducible peptides in the hemolymph [7-8]. A number of genes and peptides related to innate immunity have been described in termites. Among them are a transferrin gene that was found to be upregulated in Mastotermes darwiniensis upon exposure to an entomopathogenic fungus , antimicrobial peptides (termicin, spinigerin from Pseudacanthotermes spiniger, ) and gram-negative binding proteins with are pattern recognition receptors and antimicrobial effectors in several termite species . However, these studies only captured a small part of the immune system. The only genome-wide resource in termites available to date (besides genomics of the termite gut, ) is the previously created EST library for FST, which contains many genes with putative immune-related functions similar to those mentioned above (Husseneder et al., oral communication).
B.4. Termite detoxification system:
The three most important inducible detoxification systems in insects are the microsomal monooxidases (cytochrome P450, CYP450), the glutathione-S-transferases (GSTs) and the carboxyesterases . Multiple genes coding for isoforms of these enzymes are known to be transcriptionally regulated by xenobiotic inducers. Phenobarbital (PB), pentamethylbenzene (PMB) and atrazine are known as prototypical inducers for a broad variety of detoxification genes [13-14]. A number of genes whose transcription is regulated by these inducers have been reported to be associated with insecticide resistance [13-14]. Therefore, inducibility has been suggested as a risk factor for resistance development .
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Susceptibility to most of the commonly used termiticides varies among colonies of FST [15-17]. Similarly, activity of detoxification enzymes varies among colonies . Selection for more tolerant colonies and the possibility of tolerant individuals becoming secondary reproductives theoretically might increase resistance in the population, which would lead to treatment failures . Challenges with a wide variety of pesticides [15-16] suggest that workers are more resistant to insecticides than soldiers presumably due to higher levels of enzymes that metabolize/detoxify pesticide components. Soldiers show only a fraction of detoxification enzyme activity of workers .
C. RESEARCH PLAN: DESIGN and METHODS
C.1. Gene Identification and primer development:
The work for this objective is completed. I have screened, designed and tested primers for each of five putative immunity related genes, five toxicity related genes and one housekeeping gene (elongation factor 1 alpha). I screened and selected putative genes from the EST library for FST that are most likely involved in immune response (i.e. genes expressing antibacterial peptides, gram-negative binding proteins, lectin-like genes for binding glucanases, lipopolysaccarides chitins and serine proteases) and detoxification (i.e. genes from the Cytochrome P450 and Glutathione-S-Transferase families). I designed three primer pairs each for a total of 11 genes using PRIMER-BLAST software suite located on the NCBI website. Primers were between 17-28 bases, with 50%-60% GC content. I tested the efficiency, reliability and specificity of these primers using quantitative reverse transcriptase (qRT) PCR to ensure their consistency, target amplicons were at 50-100 bp, melting curve had only one peak and that the efficiency is >1.8. For each gene, the primer pair with the best results according to these criteria was chosen.
C.2.1. Immunity Challenge with Non-pathogenic Bacteria (workers and soldiers):
I have conducted a pilot study to modify previous methods  for injecting FST with bacteria and to determine the appropriate non-lethal exposure time for the immune challenge. Non-pathogenic gram-negative Escherichia coli and gram-positive Pilibacter termitis, which occur naturally in the termite gut , were grown in BHI broth for one to three days, respectively. Sterile 28 gauge needles were each soaked with an equal mixture of cultured E. coli and P. termitis for approximately 60 seconds. Termites were cold immobilized and placed with their ventral surface exposed on a dissecting microscope stage. The abdomen was swabbed with 70% ethanol before being pierced through the intersegmental membrane of the fourth and fifth segments with the septic needles. This was done for 20 workers and 20 soldiers in total. Termites were grouped by caste and placed into arenas with moist paper towels for 48 hours. Mortality was assessed at 12, 24 and 48 hours. Ten control individuals (not injected with bacteria) per group were held in separate arenas. All workers and soldiers survived the septic challenge at both 12 and 24 hours. The highest mortality (over 70%) was found in soldiers at 48 hours. All control individuals survived over the 48 hour period. I determined that the 24 hour exposure treatment would be used for further analysis, based on these preliminary data, which are consistent with the rapid induction time of insect immune systems (i.e., peptide synthesis in Drosophila  and in P. spiniger ).
Next, I will inject 20 workers and 20 soldiers, collected from three separate FST colonies, with bacteria. Twenty workers and 20 soldiers per colony will be left unchallenged (controls). Termites will be held for 24 hours and then flash-frozen (-80C). For each colony, 5 challenged workers, 5 challenged soldiers, 5 control workers and 5 control soldiers will be randomly selected for measuring gene expression levels via qRT PCR.
C.2.2. Toxic Challenge at Sub-lethal Dosage (workers and soldiers):
Although several detoxification genes and isoforms of CYP450  and GSTs have been described in termites , no genome-wide screening for constitutively expressed and induced detoxification genes in termites has been performed. As the first step, I screened the EST library of FST for potential detoxification genes (see above) and conducted a pilot study to establish the sublethal dose and time for PB exposure of workers and soldiers. Phenobarbital was serially diluted with water (1%, 0.5%, 0.25% and 0.125%); each concentration was fed to 20 workers and 20 soldiers in 2 Î¼l droplets. Twenty individuals of each caste received water droplets (controls). Preliminary data showed no mortality in the controls. All workers and soldiers fed with 1% PB were dead at 48 h and 70% were dead at 24 h. All individuals fed with 0.5% PB survived at 24 h, but approximately 50% were dead at 48 h. Therefore, 0.5% was chosen as the sublethal dose at 24h for workers and soldiers. This value lies within the published time frame recommended for measuring effects of induction of detoxification systems in insects ranging from 4 hrs in Drosophila  to 5 days in cockroaches .
As with the bacterial challenge, I will collect samples from three separate FST colonies and feed 20 workers and 20 soldiers from each colony 0.5% PB for 24 hours; 20 workers and 20 soldiers will be given only water (controls). The workers and soldiers will then be flash frozen (-80 C). For each colony, 5 challenged workers, 5 challenged soldiers, 5 control workers and 5 control soldiers will be randomly selected for measuring gene expression via qRT PCR.
C.2.3. RNA extraction, normalization, cDNA synthesis and qRT PCR (workers and soldiers):
Total RNA will be extracted from whole bodies according to manufacturer protocols (Qiagen RNA Purification Microkit). I will normalize RNA to 10 ng per ïl and then synthesize double- stranded cDNA using the BioRad IScript Kit. Next, qRT PCR reactions will be run using the Biorad IQ5 iCycler machine. Template will be amplified in reactions each containing 10 ng of cDNA, 12.5 ïl of SYBR-green master mix (BioRad), 150 nm of forward and reverse primer and water for a total volume of 25 ïl.
I will be using LinReg PCR software to determine cT and reaction efficiency for each reaction. This is determined using RFUs (relative fluorescence units) for reactions as given by the iCycler qRT PCR software. I will calculate relative concentration of target sequences using the formula [relative concentration = (ErcT(ref)/ErcT(target))], where Er is the reaction specific amplification efficiency estimated using LinRegPCR software, cT is the fractional cycle at which amplification reaches a detection threshold, ref is the endogenous control gene and, target is the amplicon under consideration. Differential expression is based on calculated baseline fluorescence using standard optimization methods (i.e. melt-curve analysis to ensure amplification of a single product). The controls will be used as the baseline, and normalization and replicates will be used as needed to reduce variability between individual reactions.
Gene expression levels will be compared between (1) workers and soldiers injected with bacteria vs. unchallenged workers and soldiers (2) PB challenged workers and soldiers vs. unchallenged workers and soldiers for each of the 3 colonies using a mixed model analysis of variance with the SAS system software (PROC MIXED, SAS/STAT Software, SAS Institute, Inc., 1999). Differences in expression will be considered significant when P < 0.05.
C.3.1. Immunity Challenge and Toxic Challenge (alates):
I first conducted pilot studies similar to those performed on workers and soldiers for determining the appropriate exposure times and sub-lethal doses for bacterial and PB challenges in alates. Similar to workers and soldiers, the 24 hour bacterial exposure treatment was found to be most appropriate. However, the 1% PB concentration will be used for the alates, rather than the 0.5% PB concentration used for workers and soldiers (see Table 1).
I have performed bacterial and 1% PB challenges with a total of 300 alates (100 challenged and 50 controls each for septic exposure and PB exposure) sampled from a single swarming event in the French Quarter, New Orleans (May 16, 2009). After 24 hours, alates were cold immobilized and sexed. For my objective, I must determine the level of heterozygosity of alates before measuring their differential gene expressions; therefore, the heads of the alates were removed and stored in 95% ethanol for future DNA analysis.
Table 1. Phenobarbital concentrations, exposure times, caste and mortality for toxic challenge.
C.3.2. DNA extraction and Genotyping, Significance testing (alates):
To estimate the levels of heterozygosity, the heads of 20 males and 20 females will be randomly selected from each from group (alates challenged with bacteria, alates challenged with PB and the respective controls) for a total of 160 alates. These alates will be genotyped at five polymorphic microsatellite loci developed for FST . The microsatellites will be amplified, run on a LICOR 4300 automated sequencer and scored according to established methods . Individual heterozygosity for each alate will be determined as the proportion of loci for which each alate is heterozygous . In addition, I will measure individual heterozygosity using the microsatellite-specific parameter d2  which uses the stepwise mutation model and is based on the measure of population differentiation . The methods of calculating individual heterozygosity are further detailed in a previous study .
Once individual heterozygosity is known for the alates, I will select the five most heterozygous and the five least heterozygous individuals from each of the following groups: bacterial challenged males, bacterial challenged females, PB challenged males, and PB challenged females and each of the respective controls. This will result in a total of 80 alates which will be used for further analysis.
C.3.3. RNA extraction, normalization, cDNA synthesis and qRT PCR (alates):
Total RNA will be extracted and subsequently normalized in the same way as will be done for the workers and soldiers (see above). Also described above are the methods of cDNA synthesis, qRT PCR analyses and determination of variance between constitutive and induced expression. The exception is that instead of investigation immune or detoxification response in workers and soldiers, I will determine whether there are differences in gene expression between heterozygous or homozygous, male or female alates exposed to immunity or toxic challenge.
D. CATALYZING FURTHER GENOMICS RESEARCH:
Insect immunology and detoxification mechanisms are important to applied entomology, since these factors directly influence the success of control methods . The goals of this study are to (1) identify genes vital to immune defense and detoxification in the FST, (2) determine if the gene expression level is different in unchallenged vs. challenged individuals of different castes subjected to immunity or toxic challenges and (3) determine if sex and/or degree of inbreeding influence the level of constitutive and/or inducible gene expression. Results from this study will provide information that may lead to more effective and environmentally friendly control of this invasive pest species. This genomic approach is expected to be utilized to reveal novel genes and targets for pest control by interfering with components of the immune system  and the detoxification system of FST. For example, future work using RNAi to disrupt genes involved in immune response and detoxification may be used to decrease fitness or increase susceptibility in the termites and thus increase efficiency and reduce the amount of insecticides needed for control.
Treatment with induction inhibitors could increase insecticide susceptibility thereby lowering the effective concentration of pesticides and prolong the time that pesticides are effective in the environment. Inhibition of induction of detoxification systems can be achieved by RNAi, which is a mechanism regulating gene expression in eukaryotes and can be used to silence candidate genes . This technique involves the homology-dependent breakdown of specific mRNAs in response to double-stranded RNA (dsRNA) molecules specific to the target gene's transcript. Small interfering RNAs (siRNAs) are small segments (20-25 nucleotides) of dsRNA which provide the same silencing of gene transcripts as long dsRNA, but with greater specificity . Introducing dsRNA or siRNA into termites by microinjection or feeding has been successful at initiating RNAi [34-36]. A dosage of 0.5 ng (15 pg/nl) siRNA injected in the lateral thorax silenced hexamerin and cellulase genes in Eastern subterranean termite workers, Reticulitermes flavipes [21, 35], and 36 ng siRNA silence the expression of a gene involved in reproductive suppression (neofem2) in Cryptotermes secundus . Maximum gene silencing in termites measured at the transcript (mRNA) level was observed at 24 h with a decline after 48 h [34-35]. In other insects the maximum silencing occurred within up to 7 days . The effect of gene knockdown measured at the protein level reaches its maximum at 48 h and decreased after day 4 . RNAi based pesticides that target genes vital in termite biology have been suggested, specifically, for termite control [35-36]. Here, I propose to identify both immunity and detoxification genes that can be used to facilitate future control of FSTs by reducing their ability to defend against pathogens and/or insecticides. Disrupting the expression of immunity and detoxification genes via RNAi will be the major goals of future genomics research in this species, utilizing and expanding upon the results of the present study.