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.

Acquisition, persistence, and species susceptibility of the Hz-2V virus.

Hz-2V, formerly called gonad-specific virus, is known to infect the reproductive organs of both males and females of the corn earworm Helicoverpa zea, rendering them agonadal or sterile. The primary mode of transmission is through mating by asymptomatic carrier moths. In this report we show that Hz-2V can be acquired by first instar larvae, through feeding on virus laced diet, although the incidence of agonadal condition was significantly lower. In a laboratory study, the virus appeared to persist for no more than three generations, with the incidence of agonadal progeny decreasing with each generation. Although, Hz-2V has been reported only from H. zea, in our tests when nine species of insects were artificially infected, four of the Noctuid species showed some signs of agonadal condition. Out of the remaining five species, the diamondback moth Plutella xylostella and the German cockroach Blatella germanica, showed no evidence of the virus in progeny of adults that were injected with Hz-2V, even after using the very sensitive PCR based assay.

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.

Regulation of juvenile hormone titers by soldiers in the Formosan subterranean termite, Coptotermes formosanus.

In field collections of the Formosan subterranean termite Coptotermes formosanus, soldiers averaged less than 10%. The proportion of soldiers increased to about 25% or higher when termites were kept in the laboratory, as did the juvenile hormone III (JH III) titers for both workers and soldiers. In laboratory experiments with a proportion of soldiers 25% or higher, very few new pre-soldiers were formed and the JH titer in existing soldiers remained constant. On the contrary, workers from groups containing less than 25% soldiers formed more pre-soldiers. In such cases, both workers and soldiers showed higher JH titers. Newly formed soldiers also had higher JH titers than older soldiers. It is speculated that populations containing higher soldier proportions inhibit further soldier differentiation, eventually holding the JH titers under the threshold or suppressing the activity of corpora allata resulting in lower JH titers in workers.

Hydroquinone is not a phagostimulant for the Formosan subterranean termite.

It has been suggested that hydroquinone found in the labial glands of a number of termite species acts as a primary phagostimulating factor. We tested hydroquinone as a phagostimulant using workers from three colonies of the Formosan subterranean termite, Coptotermes formosanus, under both laboratory and field conditions. Hydroquinone at concentrations ranging from ca. 0.002-20.0 ng/cm2 did not increase visitation by C. formosanus workers to treated over control filter papers, and was actually repellent at a 20 ng/cm2 dose. No phagostimulant response to hydroquinone was observed in two colonies. In the third, there was a significant increase in feeding on filter paper treated with a 2 ng/cm2 dose, but was significantly lower at a 20 ng/cm2 dose. Furthermore, sand treated with a gradient of hydroquinone, did not evoke increased tunneling activity compared with controls. GC-MS analysis of C. formosanus workers indicated that hydroquinone was present at an average of 41 pg/worker. It was also determined that within one week about 11% hydroquinone in aqueous solution oxidized to 1,4-benzoquinone. Our findings indicate that hydroquinone alone does not act as a phagostimulant but instead may act as a repellent at higher concentrations. The attractant/arrestant of the Formosan termite may have multiple components of which hydroquinone, at low doses, could be one.

Trilinolein identified as a sex-specific component of tergal glands in alates of Coptotermes formosanus.

Hexane extracts of the tergal glands from female alates of the Formosan subterranean termite Coptotermes formosanus were analyzed by high performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry with collision-induced dissociation. Double bond configuration was determined by chemical modifications with gas chromatography-mass spectrometry. A single component, identified as the triacylglycerol, trilinolein, was unique to the female tergal glands. This compound was not found in other areas of the female alate abdomen or in the corresponding area of male alates. Neither gland extract nor trilinolein caused a behavioral response from male alates. However, significant differences were found between males and females for responses from neurons within sensilla of the maxillary palps.

Factors affecting post-flight behavior in primary reproductives of the Formosan subterranean termite, Coptotermes formosanus (Isoptera: Rhinotermitidae).

After swarming, reproductive dealates of the Formosan subterranean termite, Coptotermes formosanus, run together in tandem. The tandem running is an age-related behavioral activity in C. formosanus. This behavior was not evoked in pre-flight alates less than 6-d-old that were artificially dealated. Female age was more important than male age for evoking tandem behavior. Females and males, older than 35 days, did not exhibit this behavior. Mating status was not important for female and male dealates to form the tandem pairs. The titers of the major tergal gland component, trilinolein, did not decline significantly and remained high, not only in virgin females, but also in mated females for a period of time after swarming. On the other hand, increasing amounts of the female-specific compound trilinolein were detected in male dealates 7, 14, and 42 days after pairing. This suggests that trilinolein in the females might be transferred to the males as a nuptial gift in C. formosanus. In addition, females have structurally different lateral setae that may constitute a morphological factor involved in the tandem behavior in this species. Covering the setae with dimethyl sulfoxide prevented the tandem behavior.

Juvenile hormone III titers and regulation of soldier caste in coptotermes formosanus (Isoptera: Rhinotermitidae).

Juvenile hormone (JH) is an important growth hormone in insects that has also been implicated in caste determination in termites. Gas chromatography-mass spectrometry was used to establish that the JH in the Formosan subterranean termite, Coptotermes formosanus Shiraki, is JH III. JH III titers were measured in workers, pre-soldiers, and soldiers from samples collected from the field. The average titers of JH III in workers and soldiers were about 13 and 25 pg mg(-1), respectively. However, pre-soldiers contained a significantly higher amount, 596 pg mg(-1). As expected, treatment of workers with a JH-analogue, methoprene, triggered rapid formation of pre-soldiers. However, these pre-soldiers had a very low JH III titer (62 pg mg(-1)). It appears that the application of JHA, while inducing pre-soldier formation, does not increase the endogenous JH III titer. The titer, however, increased as the pre-soldiers aged and before transforming into soldiers.

Diet-mediated inter-colonial aggression in the formosan subterranean termite Coptotermes formosanus.

In most social insects, intercolonial and interspecific aggression are expressions of territoriality. In termites, cuticular hydrocarbons (CHCs) have been extensively studied for their role in nestmate recognition and aggressive discrimination of nonnest-mates. More recently, molecular genetic techniques have made it possible to determine relatedness between colonies and to investigate the influence of genetics on aggression. In the Formosan subterranean termite, Coptotermes formosanus, however, the role of CHCs and genetic relatedness in inter-colony aggression has been ambiguous, suggesting the involvement of additional factors in nest-mate recognition. In this study we assess the range of aggression in this termite species and characterize the influence of genetic relatedness, CHC profiles and diet on aggression levels. We collected four colonies of C. formosanus, feeding either on bald cypress or birch, from three locations in Louisiana. Inter-colony aggression ranged from low to high. Differences in CHC profiles, as well as genetic distances between colonies determined by using microsatellite DNA markers, showed no significant correlation with aggression. However, termite diet (host tree) played a significant role in determining the level of aggression. Thus, two distantly related colonies, each feeding on different diets, showed high aggression that significantly diminished if they were fed on the same wood in the laboratory (spruce). Using headspace solid phase microextraction, we found three compounds from workers fed on birch that were absent in workers fed on spruce. Such diet-derived chemicals may be involved in the complex determination of nest-mate recognition in C. formosanus.

Effects of plant flavonoids on fecundity, survival, and feeding of the Formosan subterranean termite.

Fecundity, mortality, and food consumption of the Formosan subterranean termite, Coptotermes formosanus Shiraki, were evaluated in response to five plant flavonoids (genistein, biochanin A, apigenin, quercetin, and glyceollin). Apigenin fed at 50 microg/primary reproductive pair proved to be the most toxic flavonoid. Biochanin A was most effective in reducing fecundity. Subsequently, these two flavonoids were tested through oral feeding and topical application at 100-microg dose. Significant reduction in the numbers of progeny was evident for biochanin A in both treatment methods. Choice feeding tests with termite workers showed that initially termites were attracted to filter paper treated with biochanin A, but over a period of 72 hr, consumed significantly less material when compared to controls. Biochanin A is a promising phytochemical with ability to reduce fecundity in primary reproductives of the Formosan subterranean termite, but it does not elicit phagostimulant activity.

A pheromonotropic peptide of Helicoverpa zea, with melanizing activity, interaction with PBAN, and distribution of immunoreactivity.

The sequence of an 18-amino acid residue peptide was deduced from the gene encoding PBAN and other peptides with common C-termini in Helicoverpa zea. The peptide caused melanization in larvae and pheromone production in females of H. zea, and was designated pheromonotropic melanizing peptide (Hez-PMP). The peptide has a 83% sequence homology with a pheromonotropic peptide isolated from Pseudaletia separata. PMP caused melanization and mortality when injected into larvae just before molting. Whereas intense melanization was caused with a dose of 1,000 pmol, peak mortality occurred at 100 pmol, with 50% of larvae dying within 48 h after injection. Pheromonotropic activity of PMP was dose dependent. Co-injection of Hez-PMP and Hez-PBAN into a female resulted in suppression of the pheromonotropic effect of PBAN. Whole-mount immunocytochemical studies revealed PMP-like immunoreactivity in frontal ganglion, subesophageal, thoracic, and abdominal ganglia as well as the esophageal nerve.

Ecdysone agonist halofenozide affects corpora allata and reproductive physiology of the Formosan subterranean termite, Coptotermes formosanus.

Following a short swarming flight, winged adults of the Formosan subterranean termite, Coptotermes formosanus lose their wings and form tandem pairs. These dealates or primary reproductives then form incipient colonies. Topical application of 5 microg of the non-steroidal ecdysone agonist RH-0345 (halofenozide) in 0.1 microl DMSO to the primary reproductives during the 2000 season resulted in significant reduction in the number of eggs laid. There was however complete recovery of the treated females. Apparently the non-treated partner removed the treatment chemical while grooming indicating oral activity. In 2001 both topical application as well as feeding methods were tried. Significant effects were observed only in the topical treatment group, perhaps because of inconsistency in feeding. In this group, total progeny, the number of ovarioles in ovaries and the size of the female's corpora allata (CA) were all significantly reduced. Ultrastructure of the CA of treated females showed extensive vacuolation near the surface of the gland. The experiment was repeated in 2002 using both topical application and an improved oral feeding method. Whereas there was apparent recovery in the topically treated group after 90 days, the oral treatment was more persistent in its effect perhaps due to a higher amount of halofenozide consumed during feeding.