Improved mortality of the Formosan subterranean termite by fungi, when amended with cuticle-degrading enzymes or eicosanoid biosynthesis inhibitors.

Formosan subterranean termites (FST) were exposed to strains of Beauveria pseudobassiana (Bpb) and Isaria fumosorosea (Ifr) to determine virulence of the fungi. Once lethality was determined, sublethal doses of Bpb were combined with enzymes capable of degrading the insect cuticle to measure the potential to enhance fungal infection. Bpb applied to FST in combination with proteinases and a chitinase caused increased mortality over the fungus alone. Mortality was enhanced when Ifr was applied to FST in combination with a chitinase isolated from Serratia marcesans. A lipase isolated from Pseudomonas cepacia, when combined with Ifr, also resulted in greater mortality than all control treatments. FST were also exposed to the eicosanoid biosynthesis inhibitors (EBIs) dexamethasone (DEX), ibuprofen (IBU), and ibuprofen sodium salt (IBUNA), in combination with Ifr. Combining Ifr with IBUNA caused significantly increased mortality on days 6, 7, and 9. Cuticle-degrading enzymes and EBIs may have potential to enhance the pathogenic effect of a fungal control agent against the Formosan subterranean termite.

Combined effect of microbial and chemical control agents on subterranean termites.

Termite mortality was measured when fungi were combined with bacteria or a chemical termiticide to determine whether a synergistic effect occurred. The fungus Beauveria bassiana was combined with the non-repellant chemical termiticide imidacloprid. Of the three B. bassiana strains tested one, B. bassiana ATCC 90519, was sufficiently pathogenic on its own that the advantage of a supplementary chemical treatment was marginal. The mortality caused by another fungal strain, B. bassiana ATCC 26037, was improved in combination with imidacloprid at both of the tested chemical concentrations over the first 14 days. The remaining fungal strain, B. bassiana ATCC 90518, demonstrated an overall mortality rate in combination with imidacloprid of 82.5%, versus a rate of 65.0% for the fungus alone. The fungus Isaria fumosorosea (Ifr) was combined with the bacterium Bacillus thuringiensis (Bt). On day 5, Ifr, Bt, and the combined treatment at a 10(6) spores or cells/ml dosage caused 8.8%, 22.5%, and 15.0% mortality, respectively. The Bt and combined mortality rates are not significantly different. Control mortality on day 5 was 5.0%. On day 13 the combined 10(6) treatment mortality rate was 91.3%, which was significantly higher than all other treatments: control at 17.5%, Ifr at 36.3% and Bt at 35.0%. When Ifr and Bt were applied at a 10(9) spores or cells/ml dosage, Ifr alone caused a mortality rate of 97.5% as early as day 5. The combination with Bt could not significantly increase the effectiveness of this dosage. These data demonstrate the potential for synergistic effects of fungal and chemical treatment methods, thereby broadening the use of microbial control agents and reducing the quantity of chemical agents necessary to effect control.

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.

Functional analyses of the digestive ß-glucosidase of Formosan subterranean termites (Coptotermes formosanus).

The research was to elucidate the function of the ß-glucosidase of Formosan subterranean termites in vitro and in vivo. The gene transcript was detected predominantly in the salivary gland tissue, relative to the midgut and the hindgut of the foraging worker caste, indicating salivary glands were the major expression sites of the ß-glucosidase. Using recombinant ß-glucosidase produced in Escherichia coli, the enzyme showed higher affinity and activity toward cellobiose and cellotriose than other substrates tested. In assessing impacts of specific inhibitors, we found that the ß-glucosidase could be irreversibly inactivated by conduritol B epoxide (CBE) but not gluconolactone. Termite feeding assays showed that the CBE treatment reduced the glucose supply in the midgut and resulted in the body weight loss while no effect was observed for the gluconolactone treatment. These findings highlighted that the ß-glucosidase is one of the critical cellulases responsible for cellulose degradation and glucose production; inactivation of these digestive enzymes by specific inhibitors may starve the termite.

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.

Characterization of a new endogenous endo-ß-1,4-glucanase of Formosan subterranean termite (Coptotermes formosanus).

The present work characterized a new endogenous cellulase (endo-ß-1,4-glucanase) gene, CfEG5, uncovered in the transcriptome of Formosan subterranean termite (Coptotermes formosanus). The full-length gene was cloned and sequenced. It is similar to the CfEG3a described earlier (Zhang et al., 2009) but not likely an allelic variant. GenomeWalker™ DNA walking analysis indicated that there may be one copy of CfEG5 and two copies of CfEG3a in the termite genome. As with CfEG3a, the transcript of CfEG5 was detected predominantly in the salivary gland based on quantitative RT-PCR. Phylogenetic analysis of translated amino acid sequence showed that the CfEG5 is more related to CaEG, derived from an Australian subterranean termite (Coptotermes acinaciformis), than CfEG3a and other cellulases from Coptotermes formosanus, Reticulitermes speratus, or Reticulitermes flavipes. Recombinant CfEG5, produced in Escherichia coli, was active against filter-paper cellulose, resulting in mostly cellobiose and cellotriose, similar to the enzymatic and biochemical properties of CfEG3a. These findings would lead to further investigation of both the evolutionary origin of eukaryotic cellulase genes and the evolutionary relationship of termite species. The cellulose-degrading enzyme is applicable for bioconversion of wood to simple sugars and production of biofuels. The recombinant cellulase should also be useful for designing and screening of inhibitors for the development of target-specific and environment-friendly bio-termicides.

Efficacy of bait supplements for improving the rate of discovery of bait stations in the field by formosan subterranean termites (Isoptera: Rhinotermitidae).

Field tests of four different bait supplements were conducted in City Park, New Orleans, LA. The four bait supplements tested included two different formulations of decayed material, a sports drink, and the combination of an application of an aqueous solution of Summon Preferred Food Source disks with the disk itself. Although all the bait supplements in this study resulted in a slightly greater number of treated stations discovered compared with control stations, only the application of the aqueous solution combined with the disk caused a significant increase in the number of stations discovered by termites. This treatment resulted in a significantly greater rate of discovery of treated stations versus control stations after only 14 d in the field. Termites were able to discover six times as many treated as control stations after 14 d, 9 times as many after 28 d, and 12 times as many after 42 d. These findings provide evidence that the diffusion of an aqueous solution into the soil underneath monitoring stations significantly decreased the length of time required for termites to infest stations.

Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from Coptotermes formosanus and expressed in E. coli.

An endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in Escherichia coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrates. The nCfEG was more active and stable than tCfEG even though the latter could be purified to near homogeneity with a simple procedure. The differential activities of nCfEG and tCfEG were also evidenced by hydrolytic products they produced on different substrates. On CMC, both acted as an endoglucanase, randomly hydrolyzing internal beta-1,4-glycosidic bonds and resulting in a smear of polymers with different lengths, although cellobiose, cellotriose, and cellotetraose equivalents were noticeable. The hydrolytic products of tCfEG were one unit sugar less than those produced by nCfEG. Using filter paper as substrate, however, the major hydrolytic products of nCfEG were cellobiose, cellotriose and trace of glucose; those of tCfEG were cellobiose, cellotriose and trace of cellotetraose, indicating a property similar to that of cellobiohydrolase, an exoglucanase. The results presented in this report uncovered the biochemical properties of the recombinant cellulase derived from the intact gene of Formosan subterranean termites. The recombinant cellulase would be useful in designing cellulase-inhibiting termiticides and incorporating into a sugar-based biofuel production program.

Effects of flooding on field populations of Formosan subterranean termites (Isoptera: Rhinotermitidae) in New Orleans, Louisiana.

Hurricane Katrina (2005) resulted in extensive flooding in the city of New Orleans, LA. Periodic sampling of monitors before the flood, and of different monitors in the same areas after the flood, was used to evaluate the effects of long-term flooding on populations of Formosan subterranean termites, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Monitors were located adjacent to buildings and in urban forests. Significant population reductions occurred in areas that flooded 2-3 wk with brackish water, with termite populations associated with pine (Pinus spp.) trees and buildings slower to recover than populations associated with oak trees. Alate production in flooded areas showed no reduction from previous years.

Activity of 1,4-benzoquinones against formosan subterranean termites (Coptotermes formosanus).

A large number of naturally occurring and synthetic benzoquinones were evaluated for activity against the Formosan subterranean termite, Coptotermes formosanus, with potential use in termite control. Among these bioactive naturally occurring benzoquinones are 2-methyl-5-isopropyl-1,4-benzoquinone, 2-methoxy-6-pentyl-1,4-benzoquinone, 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone, 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone, and 2,3-dichloro-5,6-dimethyl-1,4-benzoquinone. All five of these compounds demonstrated 100% mortality against C. formosanus by day 11 at a concentration of 1% (wt/wt) or less. In general, benzoquinones with one or two hydrophobic groups on the 5 and/or 6 positions of the quinone ring along with one or two group(s) on the opposite side of the ring, at the 2 and/or 3 position, led to high rates of mortality against C. formosanus. Quantitative structure-activity relationship (QSAR) studies showed no correlation between lipophilicity (calculated log P) and mortality for the entire group of nonhalogenated benzoquinones. A correlation was observed between C-6 chain length and day 3 percent mortality for 2,3-dimethoxy-5-methyl-6-substituted aliphatic benzoquinones where short chain lengths resulted in higher mortality.

Antifeedant and toxicity effects of thiophenes from four Echinops species against the Formosan subterranean termite, Coptotermes formosanus.

Over 220 crude extracts from repositories generated from plants native to Greece and Kazakhstan were evaluated for termiticidal activity against the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Emerging from this screening effort were bioactive extracts from two Greek species (Echinops ritro L. and Echinops spinosissimus Turra subsp. spinosissimus) and extracts from two Kazakhstan species (Echinops albicaulis Kar. & Kir. and Echinops transiliensis Golosh.). Fractionation and isolation of constituents from the most active extracts from each of the four species has been completed, resulting in the isolation of eight thiophenes possessing varying degrees of termiticidal activity. 2,2':5',2"-Terthiophene and 5'-(3-buten-1-ynyl)-2,2'-bithiophene demonstrated 100% mortality against C. formosanus within 9 days at 1 and 2 wt% concentrations respectively. In addition, all but two of the eight compounds tested were significantly different from the solvent controls in the filter paper consumption bioassay.

A strain of the fungus Metarhizium anisopliae for controlling subterranean termites.

Alates of the Formosan subterranean termite, Coptotermes formosanus Shiraki, collected after swarming in 2002 died within 48 h, and the cadavers were visibly infected with a fungus. Fungi were picked from the cadavers, transferred to media, and ultimately isolated to purity. The individual fungal cultures were then used to infect Formosan subterranean termite workers. A single fungal isolate, C4-B, taxonomically identified as Metarhizium anisopliae (Metschnikoff), was found to cause rapid mortality of Formosan subterranean termite alates. This is the first report of a biological control agent for termite alates. In initial experiments, C4-B was more lethal to both alates and workers compared with M. anisopliae strain ESC 1, previously marketed as the termite biocontrol agent BioBlast. Dose-response assays in which Formosan subterranean termite alates were exposed to a known concentration of C4-B spores revealed that 10(6) spores/microl killed 100% of the alates in 3 d, both 10(5) and 10(4) spores/microl in 6 d, 10(3) spores/microl in 9 d, and 10(0) spores/microl in 12 d. Assays with workers demonstrated that 10(6) and 10(5) spores/microl killed 100% of the workers in 6 d. In an experiment to test the transfer of inoculum from infected workers to uninfected nestmates, 62.8% of the workers died in 21 d when only 20% of the workers had been inoculated. Mortality of alates caused by C4-B was tested at two field sites by dispersing fungal spores on grassy lawns and collecting alates from the treated areas. Alates thus infected showed 100% mortality by day 5, whereas only 64.8% of untreated control alates from the same collection area were dead on that day.

Effect of Summon Preferred Food Source on feeding, tunneling, and bait station discovery by the formosan subterranean termite (Isoptera: Rhinotermitidae).

This study evaluated the effect of Summon Preferred Food Source on feeding, tunneling, and bait station discovery by the Formosan subterranean termite, Coptotermes formosanus Shiraki. Bioassays were conducted to determine whether Summon disks affected the aggregation and feeding behavior of termites and to determine whether the presence of Summon disks caused increased recruitment of termites to wood blocks. When termites encountered the disk, they immediately clustered on top of the disk. Termites were observed aggregating on top of the disk throughout the experiment. Consumption of Summon disks was significantly greater than consumption of cardboard disks in paired choice tests. The presence of a Summon disk on top of a wood block caused a significant increase in consumption of the wood block. Bioassays also were conducted to determine whether water extracts of Summon disks affected termite behavior. Consumption of filter paper disks treated with a water extract of Summon disks was significantly greater than consumption of control filter paper disks. Termites tunneled through sand treated with a water extract of Summon disks faster than they tunneled through untreated sand. In a field test, the rate of infestation of monitoring stations with a Summon disk was 3 times greater than the rate of infestations of stations without a disk.

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.

Effect of a lignin-degrading fungus on feeding preferences of Formosan subterranean termite (Isoptera: Rhinotermitidae) for different commercial lumber.

The feeding preferences of the Formosan subterranean termite, Coptotermes formosanus Shiraki, for commercial lumber Alaska yellow cedar, Chamaecyparis nootkatensis (D. Don) Spach; yellow birch, Betula alleghaniensis Britton; northern red oak, Quercus rubra L.; redwood, Sequoia sempervirers (D. Don) Endl; and spruce (Picea spp.) were examined to determine whether the presence of the lignin-degrading basidiomycete Marasmiellus troyanus (Murrill) Singer could alter the relative preference of termites for these wood species. In paired choice tests with fungus-inoculated sawdust versus control sawdust, termites showed a strong preference for the fungus-inoculated sawdust for all wood species tested, except for Alaska yellow cedar. In a multiple-choice test using sawdust without fungus, termites showed a very strong preference for red oak sawdust over the other three species. In a paired choice test using fungus-inoculated sawdust, termites showed a preference for redwood over red oak sawdust. In a feeding test using autoclaved wood blocks without fungal decay, there was no difference in termite consumption of birch, red oak, or redwood. The relative preference of termites for redwood increased when blocks were decayed by M. troyanus for 3 and 8 wk. These results indicate that chemical modifications due to fungal decay affected the feeding preference of termites for different commercial lumber.

United States Department of Agriculture-Agriculture Research Service research on targeted management of the Formosan subterranean termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae).

The Formosan subterranean termite, Coptotermes formosanus Shiraki is currently one of the most destructive pests in the USA. It is estimated to cost consumers over US dollars 1 billion annually for preventative and remedial treatment and to repair damage caused by this insect. The mission of the Formosan Subterranean Termite Research Unit of the Agricultural Research Service is to demonstrate the most effective existing termite management technologies, integrate them into effective management systems, and provide fundamental problem-solving research for long-term, safe, effective and environmentally friendly new technologies. This article describes the epidemiology of the pest and highlights the research accomplished by the Agricultural Research Service on area-wide management of the termite and fundamental research on its biology that might provide the basis for future management technologies. Fundamental areas that are receiving attention are termite detection, termite colony development, nutrition and foraging, and the search for biological control agents. Other fertile areas include understanding termite symbionts that may provide an additional target for control. Area-wide management of the termite by using population suppression rather than protection of individual structures has been successful; however, much remains to be done to provide long-term sustainable population control. An educational component of the program has provided reliable information to homeowners and pest-control operators that should help slow the spread of this organism and allow rapid intervention in those areas which it infests.

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.

Effect of tolerance to insecticides on substrate penetration by Formosan subterranean termites (Isoptera: Rhinotermitidae).

Seven selected insecticides were applied to three substrates and bioassayed for barrier efficacy and toxicity against the Formosan subterranean termite, Coptotermes formosanus Shiraki, originating from colonies that differed in their tolerance to the insecticides. A profound substrate effect was seen with all insecticides tested. Sand was the most difficult substrate to penetrate and caused faster and greater mortality of C. formosanus. Increased tolerance in C. formosanus was accompanied by a decrease in penetration of treated substrata with chlordane, methoxychlor, chlorpyrifos, and deltamethrin. The opposite was true in the case of permethrin and bendiocarb. More tolerance C. formosanus displayed decreased mortality in the presence of chlorpyrifos, deltamethrin, bendiocarb, and fipronil. The opposite was true for permethrin.