Morphological and Molecular Species Identification of Termites Attacking Ironwood Trees, Casuarina equisetifolia (Fagales: Casuarinaceae), in Guam.

Ironwood trees (Casuarina equisetifolia subsp. equisetifolia L.) are ecologically and economically important trees in tropical and subtropical regions of the Indo-Pacific. Ironwood is one of the dominant tree species in Guam, but since 2002, this tree has been declining dramatically. A previous study showed that numerous sick or dead trees were under termite attack. However, the species of termites were not identified. As a first step to investigate causal relationships between termites and ironwood tree death, we assigned termites collected from ironwood trees to species using a combination of morphological characters and DNA barcoding of the 12S, 16S, COI, COII, and ITS2 regions. Based on morphology and comparisons to reference sequences in NCBI GenBank, the most likely species assignments were Nasutitermes takasagoensis (Nawa) (Blattodea: Termitidae) found to infest 45 trees, followed by Coptotermes gestroi (Wasmann) (Blattodea: Rhinotermitidae) (2 trees), Microcerotermes crassus Snyder (Blattodea: Termitidae) (2 trees), and an additional unidentified Microcerotermes species (1 tree) with no close sequence match to identified species in NCBI GenBank. However, taxonomic revisions and broader representation of DNA markers of well-curated specimen in public databases are clearly needed, especially for the N. takasagoensis species complex.

Recovery of horse fly populations in Louisiana marshes following the Deepwater Horizon oil spill.

The Deepwater Horizon oil spill in April 2010 had unprecedented impact on the Gulf of Mexico. We established the greenhead horse fly (Tabanus nigrovittatus Macquart) as a bioindicator of marsh health. This species is bound to coastal marshes, since its larvae develop as top invertebrate predators in the marsh soil. Immediately after the oil spill (2010-2011), populations of this horse fly declined in oiled areas of Louisiana marshes with significant impacts on genetic structure. In this follow-up study five years after the catastrophic event (2015-2016), we now report signs of recovery of populations in formerly oiled areas. Fly numbers increased compared to previous counts. Previously detected genetic bottlenecks in oiled populations have disappeared. Migration into oiled areas began to replenish formerly depleted horse fly populations in impacted regions with populations from non-oiled areas as an important source of migrants. Parameters of family structure that had been impacted by the oil spill (number of breeding parents, effective population size, number of family clusters) rebounded to levels similar to or exceeding those in non-oiled control areas.

Isolation and assessment of gut bacteria from the Formosan subterranean termite, Coptotermes formosanus (Isoptera: Rhinotermitidae), for paratransgenesis research and application.

Paratransgenesis targeting the gut protozoa is being developed as an alternative method for the control of the Formosan subterranean termite (FST). This method involves killing the cellulose-digesting gut protozoa using a previously developed antiprotozoal peptide consisting of a target specific ligand coupled to an antimicrobial peptide (Hecate). In the future, we intend to genetically engineer termite gut bacteria as "Trojan Horses" to express and spread ligand-Hecate in the termite colony. The aim of this study was to assess the usefulness of bacteria strains isolated from the gut of FST as "Trojan Horses." We isolated 135 bacteria from the guts of workers from 3 termite colonies. Sequencing of the 16S rRNA gene identified 20 species. We tested 5 bacteria species that were previously described as part of the termite gut community for their tolerance against Hecate and ligand-Hecate. Results showed that the minimum concentration required to inhibit bacteria growth was always higher than the concentration required to kill the gut protozoa. Out of the 5 bacteria tested, we engineered Trabulsiella odontotermitis, a termite specific bacterium, to express green fluorescent protein as a proof of concept that the bacteria can be engineered to express foreign proteins. Engineered T. odontotermitis was fed to FST to study if the bacteria are ingested. This feeding experiment confirmed that engineered T. odontotermitis is ingested by termites and can survive in the gut for at least 48 h. Here we report that T. odontotermitis is a suitable delivery and expression system for paratransgenesis in a termite species.

Impact of the 2010 Deepwater Horizon oil spill on population size and genetic structure of horse flies in Louisiana marshes.

The greenhead horse fly, Tabanus nigrovittatus Macquart, is frequently found in coastal marshes of the Eastern United States. The greenhead horse fly larvae are top predators in the marsh and thus vulnerable to changes in the environment, and the adults potentially are attracted to polarized surfaces like oil. Therefore, horse fly populations could serve as bioindicators of marsh health and toxic effects of oil intrusion. In this study, we describe the impact of the April 2010 Deep Water Horizon oil spill in the Gulf of Mexico on tabanid population abundance and genetics as well as mating structure. Horse fly populations were sampled biweekly from oiled and unaffected locations immediately after the oil spill in June 2010 until October 2011. Horse fly abundance estimates showed severe crashes of tabanid populations in oiled areas. Microsatellite genotyping of six pristine and seven oiled populations at ten polymorphic loci detected genetic bottlenecks in six of the oiled populations in association with fewer breeding parents, reduced effective population size, lower number of family clusters and fewer migrants among populations. This is the first study assessing the impact of oil contamination at the level of a top arthropod predator of the invertebrate community in salt marshes.

Protozoacidal Trojan-Horse: use of a ligand-lytic peptide for selective destruction of symbiotic protozoa within termite guts.

For novel biotechnology-based termite control, we developed a cellulose bait containing freeze-dried genetically engineered yeast which expresses a protozoacidal lytic peptide attached to a protozoa-recognizing ligand. The yeast acts as a 'Trojan-Horse' that kills the cellulose-digesting protozoa in the termite gut, which leads to the death of termites, presumably due to inefficient cellulose digestion. The ligand targets the lytic peptide specifically to protozoa, thereby increasing its protozoacidal efficiency while protecting non-target organisms. After ingestion of the bait, the yeast propagates in the termite's gut and is spread throughout the termite colony via social interactions. This novel paratransgenesis-based strategy could be a good supplement for current termite control using fortified biological control agents in addition to chemical insecticides. Moreover, this ligand-lytic peptide system could be used for drug development to selectively target disease-causing protozoa in humans or other vertebrates.

Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and Cry1Ab-resistant strains of sugarcane borer, Diatraea saccharalis.

Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is responsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin-like proteinases were sequenced from Cry1Ab-SS and Cry1Ab-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, including signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab-SS and Cry1Ab-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab-SS and Cry1Ab-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab-SS and Cry1Ab-RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly associated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.

Transcriptome profiling of female alates and egg-laying queens of the Formosan subterranean termite.

Termites are known to have an extraordinary reproductive plasticity and capacity, but the underlying genetic patterns of termite reproductive biology are relatively understudied. The goal of this study was to identify genes for which expression levels differ between dealated precopulatory females (virgins) and egg-laying queens of the Formosan subterranean termite, Coptotermes formosanus Shiraki. We constructed a normalized polyphenic expressed sequence tag (EST) library that represents genomic material from most of the castes and life stages of the Formosan subterranean termite. Microarrays were designed using probes from this EST library and public genomic resources. Virgin females and queens were competitively hybridized to these microarrays and differentially expressed candidate genes were identified. Differential expression of eight genes was subsequently confirmed via reverse transcriptase quantitative PCR (RT-QPCR). When compared to virgins, queens had higher expression of genes coding for proteins related to immunity (gram negative binding protein), nutrition (e.g., termite-derived endo-beta-1,4-glucanase), protein storage, regulation of caste differentiation and reproduction (hexamerin, juvenile hormone binding protein). Queens also had higher transcript levels for genes involved in metabolism of xenobiotics, fat, and juvenile hormone (glutathione-S-transferase-like proteins, and cytochrome P450), among others. In particular, hexamerin, juvenile hormone binding protein, and a cytochrome P450 from the 4C subfamily are likely to be involved in initiating the inactive period during the reproductive cycle of the queen. Vice versa, virgins had higher expression than queens of genes related to respiration, probably due to recent flight activity, and several genes of unknown function.

Testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut).

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004). In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni,Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks. In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.