Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.     

Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae)

Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.     

Presence of nitrogen fixing Klebsiella pneumoniae in the gut of the Formosan subterranean termite (Coptotermes formosanus)

A gram-negative facultative anaerobic enteric bacterium, Klebsiella pneumoniae was isolated from the hindgut of the Formosan subterranean termite (FST). It was characterized using, fatty acid methyl ester (FAME) analysis, BIOLOG assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and biochemical studies. The role of this isolate seems to be nitrogen fixation because the termite's diet is nitrogen deficient and the isolate produced significant amounts of ammonia when it was grown on nitrogen deficient medium under anaerobic condition with nitrogen gas in the headspace.     

Resistance of insecticide-treated foam board insulation against the eastern subterranean termite and the Formosan subterranean termite (Isoptera: Rhinotermitidae)

Three foam board types, one untreated control, one containing 2,000 ppm disodium octaborate tetrahydrate (DOT), and one containing 1,000 ppm deltamethrin, were exposed to field populations of the eastern subterranean termite, Reticulitermes flavipes (Kollar), and the Formosan subterranean termite, Coptotermes formosanus Shiraki. There was no significant difference in termite damage between foam boards treated with 2,000 ppm DOT and the untreated control. Form boards containing 1,000 ppm deltamethrin were not damaged by R. flavipes, whereas only minor damage occurred after exposure to C. formosanus.     

Marked variations in patterns of cellulase activity against crystalline- vs. carboxymethyl-cellulose in the digestive systems of diverse, wood-feeding termites

Abstract.  Throughout the history of studies on cellulose digestion in termites, carboxymethyl-cellulose has been preferably used as a substrate for measuring cellulase activity in termites due to its high solubility. However, carboxymethyl-cellulose degradation is not directly related to digestibility of naturally occurring cellulose because many noncellulolytic organisms can also hydrolyse carboxymethyl-cellulose. To address this issue, a comparative study of microcrystalline cellulose digestion is performed in diverse xylophagous termites, using gut homogenates. For those termites harbouring gut flagellates , the majority of crystalline cellulose appears to be digested in the hindgut, both in the supernatant and the pellet. For Nasutitermes takasagoensis , a termite free of gut flagellates, crystalline cellulose is degraded primarily in the midgut supernatant, and partially in the pellet of the hindgut. The fungus-growing termite Odontotermes formosanus , which also does not possess intestinal flagellates, shows only a trace of crystalline cellulose hydrolysis throughout the gut. Comparison of levels of activity against crystalline cellulose with previously reported levels of activity against carboxymethyl-cellulose in the gut of each termite reveals significant differences between these activities. The results suggest that the hindgut flagellates produce commonly cellobiohydrolases in addition to endo-β-1,4-glucanases, which presumably act synergistically to digest cellulose. Preliminary evidence for the involvement of bacteria in the cellulose digestion of N. takasagoensis is also found.     

Time trends in mortality for thiamethoxam and fipronil against Formosan subterranean termites and eastern subterranean termites (Isoptera: Rhinotermitidae)

Time trends in mortality for the Formosan subterranean termite, Coptotermes formosanus Shiraki, and eastern subterranean termite, Reticulitermes flavipes (Kollar), were determined for thiamethoxam and fipronil. Filter paper treated with 50 ppm thiamethoxam led to >80% mortality in 2-4 d for R. flavipes, whereas 5 ppm thiamethoxam resulted in >80% mortality in 2-3 d for C. formosanus. Filter paper treated with 1 ppm fipronil resulted in >80% mortality in 5 d for R. flavipes and 9 d for C. formosanus, indicating that thiamethoxam is faster acting than fipronil. As concentration decreases for slow-acting termiticides, the time required for adverse effects to be fully expressed increases.     

台湾乳白蚁种群兵蚁量度特征变异及其分类学意义分析

台湾乳白蚁Coptotermes formosanus是我国南方木材及其建筑结构最主要的有害白蚁,也是世界重要的入侵性物种。台湾乳白蚁兵蚁品级的量度特征是其描述鉴定的重要依据,本文对台湾乳白蚁兵蚁主要量度特征数据进行了整合,并就台湾乳白蚁11个地理种群兵蚁品级的主要量度特征进行了比较,探讨了台湾乳白蚁相应量度特征的分类学意义。     

Tunneling and mortality of eastern and Formosan subterranean termites (Isoptera: Rhinotermitidae) in sand treated with thiamethoxam or fipronil

Thiamethoxam and fipronil were examined for their termiticidal properties against the Formosan subterranean termite, Coptotermes formosanus Shiraki, and the eastern subterranean termite, Reticulitermes flavipes (Kollar). Concentrations > or =8 ppm thiamethoxam and > or =1 ppm fipronil provided an effective barrier against C. formosanus and R. flavipes. Sand was penetrated to some degree at all concentrations of thiamethoxam (0-800 ppm for C. formosanus and 0-1000 ppm for R. flavipes) and fipronil (0-64 ppm for both C. formosanus and R. flavipes) tested, indicating that both termiticides are nonrepellent. Thiamethoxam was found to be more toxic against C. formosanus than R. flavipes whereas fipronil showed similar toxicity for both species. Higher mortality prevented termites from penetrating the entire 5-cm segment of treated sand.     

Effect of imidacloprid soil treatments on occurrence of Formosan subterranean termites (Isoptera: Rhinotermitidae) in independent monitors

Periodic sampling of 30 independent monitors, initially active with the Formosan subterranean termite, Coptotermes formosanus Shiraki, was conducted to evaluate the effects of soil treated with imidacloprid on nearby termite activity. Monitors were located adjacent (1-3 m) to the buildings. Soil around and under the buildings was treated with 0.05% imidacloprid. None of the termites collected showed latent mortality attributed to imidacloprid intoxication. Imidacloprid soil treatments did not measurably reduce C. formosanus populations adjacent to the treatments. Imidacloprid does not seem to fit the liquid-bait model.     

Hermetically sealed baits for subterranean termites (Isoptera: Rhinotermitidae)

Cellulose baits containing 0.5% hexaflumuron were hermetically sealed in a closed cell polyethylene sheet envelope and placed in soil to test their durability and efficacy against field colonies of the Formosan subterranean termite, Coptotermes formosanus Shiraki, or the eastern subterranean termite, Reticulitermes flavipes (Kollar). The closed cell polyethylene sheet was readily tunneled through by termites, yet it was impervious to water and protected the cellulose baits and hexaflumuron from the environment. Only in a few incidents did the polyethylene envelope become infiltrated by plant roots, resulting in water intrusion and apparent degradation of cellulose baits. After consuming one to three sealed baits, three colonies each of both termite species were eliminated. The sealed baits may be placed in soil for months or years without the need of monitoring, and they are readily penetrated and fed upon by termites when they are present. Application of baits hermetically sealed in a protective sheet may save labor costs by bypassing the monitoring phase, circumvent the station avoidance by some termite species, and enable the use of baiting technologies in large areas such as agricultural fields in which the manual monitoring is impractical.     

Use of a bait impact index to assess effects of bait application against populations of Formosan subterranean termite (Isoptera: Rhinotermitidae) in a large area

The effects of bait applications on the overall activity of the Formosan subterranean termite, Coptotermes formosanus Shiraki, in a town of 1.2 km2 were assessed between 2001 and 2003 by using a bait impact index. The index incorporated the amount of baits applied and the distance between bait application loci and the monitoring stations from which termite activity was measured. Even with the collaboration of only 35% of the residents and incomplete treatment records, the bait impact index demonstrated that more baits applied in proximity to monitoring loci significantly caused the decline of C. formosanus activity.     

DNA technology,interstate commerce,and the likely origin of formosan subterranean termite (Isoptera: Rhinotermitidae) infestation in Atlanta,Georgia

This study was the first to use DNA sequencing data to infer the origin of a Formosan subterranean termite, Coptotermes formosanus Shiraki, infestation. Cytochrome oxidase II gene sequence was used to define eight specific maternal lineages from 14 geographic locations across the southeastern United States, Hawaii, and China. One maternal lineage, A, was unique to all the Atlanta collections and one New Orleans collection, which indicated that termites at these sites shared a common maternal ancestor. The impact of interstate commerce in terms of Formosan subterranean termite introductions is addressed as is the industry response needed to arrest the spread of these termites via commercial traffic in used railroad cross ties.     

Areawide field study on effect of three chitin synthesis inhibitor baits on populations of Coptotermes formosanus and Reticulitermes flavipes (Isoptera: Rhinotermitidae)

Periodic sampling of 43 independent monitors, initially active with Formosan subterranean termite, Coptotermes formosanus Shiraki, or the eastern subterranean termite, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae), was conducted to evaluate the effects of cellulose baits containing one of three chitin synthesis inhibitors (CSIs)-diflubenzuron, hexaflumuron, or chlorfluazuron-on termite populations. Diflubenzuron at 0.1% active ingredient (AI, wt:wt) had no noticeable effect on termite populations. Chlorfluazuron (0.25% [AI]) significantly reduced termite populations in approximately 3 yr. Chlorfluazuron used after > 2-yr diflubenzuron treatment significantly reduced termite populations within months. This suggests diflubenzuron exposure increased the termite's sensitivity to chlorfluazuron accelerating population collapse. Hexaflumuron (0.5% [AI]) also reduced termite populations in approximately 2 yr. The process of removing most detectable termite populations from the approximately 160,000-m2 campus of the Southern Regional Research Center, New Orleans, LA, with CSIs baits required approximately 3 yr. Adjustments in the specific bait formulations and application procedures might reduce time to suppression. Establishment of new independent termite populations provides a mechanism to minimize the effects of baits. Remedial control measures around and under structures should be considered when implementing an area wide management strategy.     

Nitrogen-fixing Enterobacter agglomerans isolated from guts of wood-eating termites.

Two strains of facultatively anaerobic, N2-fixing bacteria were isolated from guts of Coptotermes formosanus and identified as Enterobacter agglomerans. The deoxyribonucleic acid base composition of isolates was 52.6 and 53.1 mol% guanine plus cytosine. Both isolates and a known strain of E. agglomerans carried out a mixed acid type of glucose fermentation. N2 fixation by E. agglomerans was inhibited by O2; consequently, N2 served as an N source only for cells growing anaerobically in media lacking a major source of combined N. However, peptone, NH4Cl, or KNO3 served as an N source under either aerobic or anaerobic conditions. It was estimated that 2 x 10(2) cells of E. agglomerans were present per termite gut. This value was 100-fold lower than expected, based on N2 fixation, low recoveries of E. agglomerans may be related to the marked decrease in N2 fixation rates observed when intact termites or their extracted guts were manipulated for the isolation of bacteria. It was concluded that the N2-fixing activity of E. agglomerans may be important to the N economy of C. formosanus.     

Lignocellulose degradation by microorganisms from termite hills and termite guts: A survey on the present state of art

Abstract: In several aspects termites are a fascinating group of insects having attracted the interest of many researchers. They exhibit a complex social behavior and caste differentiation occurring elsewhere only among the hymenoptera. In an enlarged part of the hindgut, the paunch, termites have established a unique symbiotic association with prokaryotic and eukaryotic microorganisms. A similar flora is also found in wood-eating roaches of the genus Cryptocercus . The study of symbiosis between termites and their intestinal microbes is of general interest, because due to this symbiotic interaction termites can feed on complex biopolymers such as wood. Flagellates and bacteria occur in the gut of lower termites, while higher termites possess only bacteria. In particular spirochetes are abundant in the termite gut. Apart from spirochetes and other more common bacteria, actinomycetes, yeasts and fungi have also been isolated from different species of termites. This review summarizes the distinct role of the intestinal flora in degradation of wood components such as cellulose, hemicellulose and lignin.     

Effect of imidacloprid tree treatments on the occurrence of Formosan subterranean termites,Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), in independent monitors

Periodic sampling of 87 independent monitors, initially active with the Formosan subterranean termite, Coptotermes formosanus Shiraki, was conducted. Monitors, located in eight sectors adjacent to seven buildings, were various distances (1-46 m) from 57 trees treated with 0.1% imidacloprid foam. Termites collected from six of the eight sectors showed latent mortality attributed to imidacloprid intoxication at all monitor-tree distances. Approximately 6 mo after treatment, termite populations had recovered in these sectors. Another sector showed termite population suppression for approximately 15 mo, followed by recovery. Imidacloprid tree treatments did not control C. formosanus populations in independent monitors adjacent to the treatments.     

Prospects for the biological control of subterranean termites (Isoptera: rhinotermitidae), with special reference to Coptotermes formosanus

Costs associated with subterranean termite damage and control are estimated to approach $2 billion annually in the United States alone. The Formosan subterranean termite, Coptotermes formosanus Shiraki, is one of the more economically important subterranean species. In recent years, the shortcomings associated with conventional chemical control methods have prompted policymakers and scientists to evaluate the potential for biological control of subterranean termites (C. formosanus in particular), that is, to determine the potential for natural enemies - predators, parasitoids and pathogens - to suppress termite populations. Ants are the greatest predators of termites, and may have a considerable local impact on termite populations in some areas of the world. A few parasitoids of termites are known, but their potential for regulating termite populations seems negligible. Characteristics of the colony, such as a protected, underground location (and, for the C. formosanus nest, its modular and dispersed nature), are likely to limit the impact predators and parasitoids have on subterranean termites. Thus, there seems little potential for use of these agents for subterranean termite control. For various reasons, pathogenic organisms, such as viruses, bacteria, Protozoa, nematodes and most fungi, have shown little promise for use in biological termite control. However, research suggests that strains of two well-studied, endoparasitic fungi, Beauveria bassiana and Metarhizium anisopliae, when employed in baiting schemes, may offer the potential for at least some measure of subterranean termite control, although their successful use is compromised by a number of inherent biological limitations and logistical problems that have yet to be solved. Although not strictly in the realm of classical biological control, recent studies suggest that natural products, such as ant semiochemicals and fungal metabolites (siderophores), or their synthetic analogues, eventually might find a use in termite control programmes as repellents or insecticides in wood treatments or soil applications if stable formulations can be developed.     

Behavioral responses of two subterranean termite species (Isoptera: Rhinotermitidae) to instant freezing or chilling temperatures

The behavioral responses to instant freezing or chilling temperatures and survivorship of the Formosan subterranean termite, Coptotermes formosanus Shiraki, and the Eastern subterranean termite, Reticulitermes flavipes (Kollar), were studied using a novel experimental design that closely simulated subterranean termites' natural in-ground environment. Both termite species responded to changes in temperature by exhibiting a downward mass movement from the cold to warmer area of constant temperature. However, the degrees of response were specific to the species and temperature regimen. Approximately 88 and 96% of R. flavipes escaped from instant 0 degrees C and chilling regimens (from 24 to 0 degrees C at a rate of 1 degrees C/h or 1 degrees C/12 h), respectively, compared with approximately 77 and 91% of C. formosanus. No significant difference was detected between the two cooling regimens in either termite species. Controls resulted in a relatively even distribution within test tubes in both termite species. The small portion of the termites that did not escape endured a cold coma at a 24-h 0 degrees C and had low mortality of 2.2 and <1% in R. flavipes and <5.2 and <3% in C. formosanus at instant and chilling regimens, respectively. This result may have implications for understanding group intelligence and decision making evolved by subterranean termites to survive temporary freezing cold.     

Molecular phylogenetic identification of the intestinal anaerobic microbial community in the hindgut of the termite, Reticulitermes speratus, without cultivation

A termite maintains an anaerobic microbial community in its hindgut, which seems to be the minimum size of an anaerobic habitat. This microbial community consists of bacteria and various anaerobic flagellates, and it is established that termites are totally dependent on the microbes for the utilization of their food. The molecular phylogene-tic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy that does not rely on cultivation of the resident microorganisms. Small subunit ribosomal RNA (ssrRNA) genes were directly amplified from the mixed-population DNA of the termite gut by polymerase chain reaction (PCR) and clonally isolated. Most sequenced clones were phylogenetically affiliated with the four major groups of the domain Bacteria: the Proteobacteria group, the Spirochete group, the Bacteroides group, and the Low G + C gram-positive bacteria. The 16S rRNA sequence data show that the majority of the intestinal microflora of the termite consists of new species that are yet to be cultured. The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite (R. speratus) was analyzed without cultivation. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order. The diversity of nitrogen-fixing organ-isms was also investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the mixed-population DNA of the termite gut and were clonally isolated. The phylogenetic analysis of the nifH amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. The molecular phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of R. speratus was also examined by the same strategy. The whole-cell hybridization experiments indicated that the sequence originated from a large hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that the hypermastigote and trichomonads shared a recent common ancestry. Received: January 22, 1998 / Accepted: February 16, 1998     

Valuation of efficacy and nonrepellency of indoxacarb and fipronil-treated soil at various concentrations and thicknesses against two subterranean termites (Isoptera: Rhinotermitidae)

The efficacy and nonrepellency of indoxacarb (150 SC, 150 g [AI]/liter) and fipronil (Termidor SC, 9.1% [Al]) against field-collected eastern subterranean termite, Reticulitermes flavipes (Kollar), and the Formosan subterranean termite, Coptotermes formosanus Shiraki, were evaluated for mortality and penetration into treated soil in laboratory glass tube bioassays. Both insecticides were tested at five concentrations (0, 1, 10, 50, and 100 ppm) and two thicknesses (20 and 50 mm) of treated soil. Indoxacarb caused significantly greater mortality than controls at all treatment thicknesses of > or = 10 ppm, but not at 1 ppm. Concentration and treatment thickness of indoxacarb significantly affected termite mortality. Eastern subterranean termites were significantly more susceptible to indoxacarb than Formosan subterranean termites, but there were no intercolony differences in either species. Termites completely penetrated through all treatment thickness of indoxacarb-treated soil at all concentrations, except one of the six Formosan subterranean termite replicates of 50 mm at 50 ppm, when all termites were killed before tunneling through the treated soil. Fipronil resulted in significantly faster and greater termite mortality than indoxacarb at corresponding concentrations. Concentration and treatment thickness of fipronil also significantly affected termite mortality. There was no intercolony difference in susceptibility to either insecticide in either termite. Both termite species completely penetrated 20-mm treatments of all tested fipronil concentrations, as well as 50-mm soil treated with fipronil at < or = 10 ppm. At 50 and 100 ppm fipronil, termites tunneled only a mean of 87 +/- 0.21 and 47 +/- 0.18% deep into 50-mm treated soil, respectively, before death. Both insecticides demonstrated a delayed mode of activity and nonrepellency against the two termite species.