Repellent and toxic effects of plant extracts on subterranean termites (Isoptera: Rhinotermitidae).

The effects of four plant extract formulations on the orientation and survival of subterranean termites were tested. In choice experiments, extract-treated filter paper had a significantly repellent effect on groups of Reticulitermes santonensis De Feytaud, R. virginicus (Banks), Coptotermes formosanus Shiraki, and Schedorhinotermes intermedius Breinli. There was no species-specific difference in avoidance behavior toward the tested concentrations. No-choice experiments revealed toxic properties of all investigated extracts by contact or airborne compounds against R. santonensis. However, high termite mortality was only achieved by forced direct or forced indirect exposure to the plant material, Feeding deterrence or digestive toxicity could not be judged in these experiments. One of the extracts was efficiently used for soil treatments to protect a food substrate against R. santonenis infestation. Extract-treated barriers in the experiments did not affect mortality compared with control trials but prevented termites from penetrating treated soil.     

Composition of the essential oil of Lepidium meyenii (Walp)

The essential oil profile of maca (Lepidium meyenii) obtained from Lima, Peru, was examined. Steam distillates of the aerial parts of L. meyenii were continuously extracted with pentane and the pentane extracts analyzed by GC/MS. Retention indices and mass spectral data were used to identify 53 oil components. Phenyl acetonitrile (85.9%), benzaldehyde (3.1%), and 3-methoxyphenylacetonitrile (2.1%) were the major components of the steam distilled oil. The oil of L. meyenii was tested for phytotoxic, cyanobactericidal, and antitermite activity. The oil was selectively toxic towards the cyanobacterium Oscillatoria perornata compared to the green alga Selenastrum capricornutum, with complete growth inhibition at 100 microg/ml. Mortality of the Formosan subterranean termite, Coptotermes formosanus, was numerically, but not significantly, higher when held on filter paper treated with maca oil. At 1% (w/w), maca oil also appeared to act as a feeding deterrent to termites. Several minor components of the essential oil of maca including 3-methoxyphenylacetonitrile and benzylthiocyanate were significantly active against the Formosan termite. This is the first report on the essential oil composition of L. meyenii.     

Antiprotozoal and cytotoxic naphthalene derivatives from Diospyros assimilis

Chemical investigation of the roots of Diospyros assimilis had led to the isolation and characterization of six naphthalene derivatives, two 2-naphthaldehyes, namely 4-hydroxy-3,5-dimethoxy-2-naphthaldehyde 1, 4-hydroxy-5-methoxy-2-naphthaldehye 2, its related isomer 5-hydroxy-4-methoxy-2-naphthaldehyde 3 and three commonly occurring naphthoquinones, diospyrin 4, 8'-hydroxyisodiospyrin 5 and the simple monomer, plumbagin 6. Their chemical structures were established by detailed NMR investigations including 1H and 13C NMR, HSQC, HMBC and NOESY experiments. In addition, the naphthalene derivatives 1-5 were evaluated for their in vitro antiprotozoal activity against protozoan parasites belonging to the genera Trypanosoma, Leishmania and Plasmodium. Among the tested compounds, naphthaldehyde 1 showed moderate inhibition of the growth of the parasites, T. brucei, T. cruzi, L. donovani with IC50 values of 19.82, 12.28 and 38.78 microM and displayed cytotoxicity towards rat skeletal myoblasts (L-6 cells) with IC50 of 174.94 microM, while 2 and 3 were found to be comparatively less active to 1. The dimeric quinones 4 and 5 exhibited good activity against T. brucei and L. donovani with IC50 of 1.12 and 8.82 microM and 12.94 and 16.66 microM respectively.     

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.     

Behavioral changes in the termite, Coptotermes formosanus (Isoptera), inoculated with six fungal isolates

The studies of pathogen-prevention behaviors of termites have focused on hygiene behavior directed only against highly virulent pathogens. Therefore, we compared behavioral changes in the subterranean termite Coptotermes formosanus following contact with entomopathogenic fungi with different levels of virulence. The fungal virulence was inferred from the daily mortality and the LD₅₀ value in previous data. When untreated termites were allowed to contact their fungus-inoculated nestmates, mutual grooming was frequent during 30 min after inoculation. The inoculated termites were often attacked and eaten by their uninoculated nestmates, and then buried after death. Notably, there was no influence of fungal virulence on these pathogen-prevention behaviors. However, the fungal isolates and genera affected not only the frequency of the behaviors but also the horizontal transmission pattern, the number of dead individuals and the survival period before the first death following infection.     

Termite resistance of solid wood and plywood treated with quaternary ammonia compounds and common fire retardants

The ability of termites to attack solid wood and plywood treated with quaternary ammonia compounds and common fire retardants was evaluated. The plywood and solid-wood specimens treated with either monoammonium phosphate (MAP), diammonium phosphate (DAP), ammonium sulfate (AS), didecyl dimethyl ammonium chloride (DDAC), or didecyl dimethyl ammonium tetrafluoroborate (DBF) were subjected to termite resistance tests using the subterranean termites Coptotermes formosanus Shiraki under laboratory conditions. The lowest mass losses and the highest termite mortalities were obtained for the solid-wood and plywood specimens treated with DDAC and DBF. Higher termite mortalities were seen in the plywood specimens treated with the fire retardants when compared to the solid-wood specimens. The MAP, DAP, and AS treatments lowered the mass losses in both solid-wood and plywood specimens in comparison with control specimens; however, DBF and DDAC protected specimens well against termite attack at both concentration levels tested.     

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.     

Naphthoquinones and triterpenes from african Diospyros species

Investigation of the bark and/or wood of seventeen African species of Diospyros has revealed the presence of a number of triterpenes and naphthoquinones. The triterpenes lupeol, betulin and betulinic acid were detected in all samples but three other triterpenes were each found in only one species; betulinaldehyde in D. canaliculata, cerin in D. iturensis, and glutinol in D. zenkeri. Naphthoquinones were detected in fourteen of the seventeen species and the common dimeric compounds diospyrin, isodiospyrin and diosindigo-A were isolated. D. canaliculata differed from all other species in producing derivatives of plumbagin including its reduced form, isoshinanolone (1R,2R) and a novel stereoisomer which was given the trivial name epiisoshinanolone (1S,2R).     

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.     

Sporulation of Metarhizium anisopliae and Beauveria bassiana on Coptotermes formosanus and in vitro

The sporulation of 22 total isolates of Metarhizium anisopliae and Beauveria bassiana was quantified on cadavers of the Formosan subterranean termite, Coptotermes formosanus. Conidial production increased significantly over 11 days post-death. Effects of isolates of M. anisopliae and B. bassiana on in vivo sporulation were significant. Although the overall effects of fungal species on in vivo sporulation were not significant, the interactions between fungal species and certain times post-death were significant, indicating different sporulation patterns between the two fungal species. B. bassiana isolates could be categorized into a group with high total sporulation (day 11) and low quick sporulation (on days 2 and 3), while M. anisopliae isolates fell into another group with high quick sporulation and low total sporulation. This could give M. anisopliae an advantage over B. bassiana in termite microbial control due to termite defensive social behaviors. Conidial production was significantly higher in vitro than in vivo. In vitro and in vivo sporulation differed by as much as 89x and 232x among the selected isolates of B. bassiana and M. anisopliae, respectively. Correlation between in vivo and in vitro conidial production was positive and significant. This may allow preliminary in vitro screening of a large number of isolates for high in vivo sporulation.     

Physical contact between soldier and worker is essential in soldier self-regulation of Coptotermes formosanus (Isoptera, Rhinotermitidae)

In a termite society, the soldier proportion is regulated at a species-specific level with a seasonal fluctuation. A feedback mechanism in soldier proportion regulation is well recognized, while the proximate means by which the presence of soldiers inhibits the new soldier formation from workers remains mostly elusive. In the present study, some possible means for this inhibition were tested with the Formosan subterranean termite, Coptotermes formosanus Shiraki. The results showed that neither soldier-originated volatiles nor non-volatile chemicals without physical contact were responsible for the inhibition of new soldier formation. The essentiality of physical contact between workers and soldiers in soldier regulation was established. However, by physical contacts, as the soldier-replacement experiment showed, workers could not individually recognize or “count” soldiers to maintain a set soldier proportion. The possible feedback mechanisms for soldier regulation are discussed.     

Learning by embryos and the ghost of predation future

Most research on the effects of exposure to stressful stimuli during embryonic development has focused on post-embryonic behaviour that appears to be abnormal or maladaptive. Here, we tested whether exposure to some stressful stimuli (predatory cues) can lead to post-embryonic behaviour that is adaptive. When eggs of ringed salamanders (Ambystoma annulatum) were exposed to chemical cues from predators, post-hatching larvae showed reduced activity and greater shelter-seeking behaviour; larvae that had been exposed to control cues did not show these behaviours. In addition, wood frog (Rana sylvatica)tadpoles learned to respond to chemical cues from unfamiliar predators with danger based on embryonic conditioning. Therefore, if embryonic experience is a good predictor of future risk, learning associated with exposure to negative stimuli during development may be adaptive.     

A Novel Strain of Steinernema riobrave (Rhabditida: Steinernematidae) Possesses Superior Virulence to Subterranean Termites (Isoptera: Rhinotermitidae)

Subterranean termites are major global pests of wood structures and wood products. Among the most economically important subterranean termite species in the US are Heterotermes aureus, Reticulitermes flavipes, and Coptotermes formosanus. In prior studies, the entomopathogenic nematode, Steinernema riobrave strain 355, exhibited a high level of virulence to H. aureus compared with other nematode species. However, S. riobrave 355 was reported to be poorly or only moderately virulent to R. flavipes and C. formosanus, respectively. We hypothesized that other strains of S. riobrave may possess a high level of virulence to all three termite species. Under laboratory conditions we compared three novel strains of S. riobrave (3-8b, 7-12, and TP) with the 355 strain for virulence to H. aureus, R. flavipes, and C. formosanus workers. H. aureus was very susceptible to all the S. riobrave strains, and termites in all nematode treatments were dead after 4 d. The TP strain of S. riobrave caused greater mortality in R. flavipes and C. formosanus compared to the other nematode strains. Specifically, the TP strain caused 75% and 91% mortality in R. flavipes and C. formosanus, respectively, which was more than 300% and 70% higher than the mortality caused by other strains. Additional studies are warranted to determine the ability of S. riobrave (TP) to control the targeted termite species under field conditions.     

Exogenous quinones directly inhibit the respiratory NADH dehydrogenase in Escherichia coli.

The ability of naphthoquinones to generate reactive oxygen species has been widely exploited in studies of oxidative stress. However, excess superoxide dismutase and catalase failed to protect Escherichia coli in rich medium against growth inhibition by plumbagin, indicating that its toxic effect was not due to the production of partially reduced oxygen species. Respiration failed immediately upon the addition of growth-inhibitory levels of plumbagin. Studies in vitro showed that plumbagin and other redox-active quinones intercept electrons from NADH dehydrogenase, the primary respiratory dehydrogenase in glucose-containing media. An excess of oxidative substrate, such as plumbagin, inactivates this enzyme, which appears to be redox-regulated. The resultant respiratory arrest is a cautionary example of metabolic dysfunction from redox-cycling drugs that cannot be attributed to superoxide or hydrogen peroxide.     

Effects of nutrients on feeding activities of the pest termite Heterotermes tenuis (Isoptera: Rhinotermitidae)

Heterotermes tenuis is a native termite of large negative impact on the Brazilian economy. Many studies have focused on optimizing the baiting method for termite control by improving its use and its efficacy. In order to maximize termite bait consumption, this study aimed to select nutrient solutions which are able to enhance termite feeding. The effects of some sugars and urea on feeding preference of the subterranean termite H. tenuis were determined by double choice feeding tests, and the consumption was evaluated by variation in dry mass of filter paper after 28 days of termite feeding. Most of the solutions tested did not significantly influence feeding of H. tenuis . Termites fed preferentially on filter paper treated with 0.03 g/ml trehalose and 0.015 g/ml urea solutions. The data indicate that trehalose and urea solutions are phagostimulants to H. tenuis , and their future use in bait matrices may increase consumption of baits and improve this control technique.     

Musty odor of entomopathogens enhances disease-prevention behaviors in the termite Coptotermes formosanus

Termites often eliminate pathogens directly through mutual grooming, and are thereby prevent infections from entomopathogenic fungi. Our previous study confirmed that the antennae of Coptotermesformosanus sensitively responded to the musty odor of entomopathogenic fungi. However, it is unclear if this odor has any effect on termite behavior. The purpose of this study was to clarify the effects of fungal odor on termite behavior, especially on conidia removal. The musty odor was prepared as an aqueous solution by immersing conidia in distilled water. When untreated termites were mixed with fungal-odor-treated termites at a ratio of 4:1, mutual grooming and attack of treated termites were frequently observed. This indicated that the fungal odor triggered these behavioral responses. While some components of the fungal odor were found in all of the entomopathogenic fungi tested, the odor profiles differed among the isolates.     

Effect of orange oil extract on the formosan subterranean termite (Isoptera: Rhinotermitidae)

The Formosan subterranean termite, Coptotermesformosanus Shiraki (Isoptera: Rhinotermitidae), accidentally brought into the United States, has become a major urban pest, causing damage to structures and live trees. Because of increasing restrictions on the use of conventional termiticides, attention is focused on finding safer alternative methods for termite management. Oil from citrus peel, referred to here as orange oil extract (OOE), contains -92% d-limonene, and it is generally known to be toxic to insects. In laboratory experiments, 96 and 68% termites were killed in 5 d when OOE at 5 ppm (vol:vol) was dispensed from the top or bottom, respectively, with termites held at the opposite end of a tight-fitting plastic container. Apart from high mortality, workers exposed to vapor consumed significantly less filter paper than controls. However, when termites were exposed to OOE vapor, even at 10 ppm, in the void of a model wall, there was very little mortality. Termites did not tunnel through glass tubes filled with sand treated with 0.2 or 0.4% OOE. Sand treated with OOE was extracted each week for 8 wk to determine the remaining amount of d-limonene. Results indicated that there was a sharp decline in the quantity of d-limonene during the first 3 wk to a residual level that gradually decreased over the remaining period. With a suitable method of application and in combination with other control practices, OOE can be effectively used for the control of subterranean termites.     

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.     

Toxic effects of 2-deoxy-D-galactose on Coptotermes formosanus (Isoptera: Rhinotermitidae) and symbionts

In the interest of developing interventions to infestations by Formosan subterranean termites, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), several rare sugars were tested for effects on the termites and symbionts. Among these, the D-galactose analog, 2-deoxy-D-galactose (2deoxyGal) showed promise as a potential control chemical. At a test concentration of 2deoxyGal (320.4 microg/mm3) in water applied to 5-cm filter paper, in bioassays with 20 termite workers, we found that worker termite mortality was significantly affected over a 2-wk period. Subsequent dose-mortality feeding studies confirmed these findings. In addition, consumption of the sugar-treated filter paper by termites caused a significant decrease in hindgut protozoan populations. 2deoxyGal caused dose-dependent termite mortality, taking on average 1 wk to begin killing workers, indicating that it may have promise as a delayed action toxin, which, if added to baits, could allow time after bait discovery for an entire colony to be affected.     

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.