Subterranean termite prophylactic secretions and external antifungal defenses

Termites exploit environments that make them susceptible to infection and rapid disease transmission. Gram-negative bacteria binding proteins (GNBPs) signal the presence of microbes and in some insects directly damage fungal pathogens with β-1,3-glucanase activity. The subterranean termites Reticulitermes flavipes and Reticulitermes virginicus encounter soil entomopathogenic fungi such as Metarhizium anisopliae, which can evade host immune responses after penetrating the cuticle. An external defense that prevents invasion of fungal pathogens could be crucial in termites, allowing them to thrive under high pathogenic pressures. We investigated the role of secreted β-1,3-glucanases in Reticulitermes defenses against M. anisopliae. Our results show that these termites secrete antifungal β-1,3-glucanases on the cuticle, and the specific inhibition of GNBP associated β-1,3-glucanase activity with d-δ-gluconolactone (GDL) reduces this activity and can cause significant increases in mortality after exposure to M. anisopliae. Secreted β-1,3-glucanases appear to be essential in preventing infection by breaking down fungi externally.     

The effect of ergosterol on the allogrooming behavior of termites in response to the entomopathogenic fungus Metarhizium anisopliae

Termites have physiological and behavioral immunities that make them highly resistant to pathogen infections, which complicates biocontrol efforts. However, the stimuli that trigger the pathogen-avoidance behaviors of termites are still unclear. Our study shows that workers of Coptotermes formosanus exposed to the conidia of Metarhizium anisopliae exhibited a significantly higher frequency and longer duration of allogrooming behaviors compared with untreated termites. Volatile compounds in the cuticle of control termites and termites previously exposed to a suspension of M. anisopliae conidia were analyzed and compared using a gas chromatography-mass spectrometer (GC-MS). Our results showed that the amount of ergosterol differed between the fungus-exposed and control termites. Choice tests showed that termites significantly preferred to stay on filter paper treated with ergosterol (0.05, 0.1, or 1.0 mg/mL) compared with control filter paper. In addition, termites exposed to ergosterol followed by M. anisopliae conidia were allogroomed at a significantly higher frequency and for a longer duration than termites exposed to alcohol (the solvent used with the ergosterol in the ergosterol trials) alone followed by M. anisopliae conidia. These results showed that ergosterol may enhance the allogrooming behavior of termites in the presence of entomopathogenic fungi.     

Resistance of the termite, <Emphasis Type="Italic">Coptotermes formosanus</Emphasis> Shiraki to <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> due to grooming

Termites, Coptotermes formosanus, reared individually, were highly susceptible to the entomopathogenic fungus, Metarhizium anisopliae, while termites reared in␣groups were highly resistant. When reared in groups, the termites treated with M.␣anisopliae conidia on the body surface were groomed by their nestmates and more than 80% of the conidia were removed from the cuticle within 3 h. However, there was not a significant reduction in the numbers of conidia on the body surfaces of termites reared individually. For the termites maintained in groups, conidia were found in foreguts, midguts and hindguts, but very few conidia were detected in the guts of termites reared individually. Conidia in the alimentary tracts did not germinate, but some of were alive. As a result, it seems that the removal of foreign bodies, such as fungal conidia, from the␣cuticle is one function of termite mutual grooming behavior and that conidia removed from the cuticle are eliminated through alimentary tracts. This study indicates that mutual grooming behavior is very effective in protecting these termites from M.␣anisopliae infection.     

Losing the battle against fungal infection: suppression of termite immune defenses during mycosis

The dampwood termite, Zootermopsis angusticollis is known to generate humoral immune responses to the entomopathogenic fungus Metarhizium anisopliae. However, little is known about how the termite's cellular immune system reacts to fungal infection. To test the effect of conidia exposure on cellular immunity, we quantified the number and types of hemocytes in the hemolymph of naïve nymphs and compared their circulating counts with those of nestmates exposed to 0, 2 × 10³, 2 × 10⁶ or 2 × 10⁸ conidia/ml doses. These termites were then bled and their hemocytes counted on days 1, 2, 3, 4, 7 post-exposure. Our results show, first, that naïve Z. angusticollis nymphs have three different blood cell types tentatively identified as granular hemocytes, prohemocytes and plasmatocytes. In these individuals, plasmatocytes were on average 13.5 and 3.3 times more numerous than granular hemocytes and prohemocytes, respectively. Second, a full factorial general linear analysis indicated that hemocyte type, time elapsed since conidia exposure and conidia dosage as well as all their interactions explained 43% of the variability in hemocyte density. The numbers of prohemocytes and particularly plasmatocytes, but not granular hemocytes, appear to be affected by the progression of disease. The decline in hemocyte numbers coincided with the appearance of hyphal bodies and the onset of “sluggish” termite behavior that culminated in the insect's death. Hemocyte counts of infected males and females were affected to the same extent. Hence, M. anisopliae overtakes the cellular immune responses of Z. angusticollis mainly by destroying the host's most abundant hemocyte types.     

Fipronil enhanced natural occurrence of Fusarium solani (Hypocreales: Nectriaceae) on building infesting termite Heterotermes indicola Wasmann (Blattodea: Rhinotermitidae)

The Southeast Asian subterranean termite, Heterotermes indicola Wasmann (Blattodea; Rhinotermitidae), is recognized as a building infesting lower termite species in urban environment. The extensive use of chemical termiticides against aerial mud tubes and underground nests of H. indicola beneath the buildings could not suppress its infestation; however, it enhanced the environmental contamination and insecticide resistance. In the present study, we tried to control termites using naturally occurring entomopathogenic fungi Fusarium solani (Mart.) Sacc., along with sublethal concentrations of termiticide fipronil in no-choice feeding pathogenecity bioassay for 20?days. Termite mortality after 20?days of continuous exposure to highest fungal treatment 1?×?10⁹?conidia/mL was 10% exclusively, whereas 100% mortality was calculated just after 16?days of concurrently exposure to 5?ppm of fipronil and highest rate of fungus 1?×?10⁹?conidia/mL. These results indicated that insecticidal stress declined the immune response of termites and reduced the repellency of termites against fungal conidia by breaching the primary defense mechanism (allogrooming). This co-application of F. solani at suitable sublethal concentrations of fipronil showed the promising potential against termites that may reduce the selection pressure of pesticides and resistance risk by targeted pests, but further investigations are necessary for developing field trials.     

Fungistatic activity of the sternal gland secretion of the dampwood termite <Emphasis Type="Italic">Zootermopsis angusticollis</Emphasis>

Summary In vivo and in vitro studies indicate that cuticular chemicals from the ventral region of the abdomen where the sternal gland of the dampwood termite Zootermopsis angusticollis is located have fungistatic properties. Germination rates of conidia of the entomopathogenic fungus Metarhizium anisopliae were significantly reduced from 91% (controls) to 38.5% after nymphs walked over conidia-seeded agar medium, but did not differ from controls when the sternal gland and surrounding cuticle were sealed with nail polish. In vitro studies show that germination of fungal conidia was also significantly reduced following incubation with cuticular extracts of either sternal or tergal segments suggesting that cuticular exudates in general may have antifungal properties. Extracts of sternites had greater fungistatic activity than extracts of tergites, but the difference was not statistically significant. Extracts of the sternal gland significantly reduced germination rates by up to 9%. Germination rates were significantly reduced when conidia were incubated with n-hexanoic acid, or its vapor. n-Hexanoic acid has been recovered from whole body extracts of Zootermopsis nevadensis and may indeed be a component of the sternal gland of Z. angusticollis. Here we suggest that sternal gland secretions in termites may have had the original function of controlling microbes within the nest and their prominent role in communication may have evolved secondarily.Received 18 April 2003; revised 20 November and 17 December 2003; accepted 19 January 2004.     

Recombinant β-1,3-1,4-glucanase from Theobroma cacao impairs Moniliophthora perniciosa mycelial growth

In this work, we identified a gene from Theobroma cacao L. genome and cDNA libraries, named TcGlu2, that encodes a β-1,3-1,4-glucanase. The TcGlu2 ORF was 720 bp in length and encoded a polypeptide of 239 amino acids with a molecular mass of 25.58 kDa. TcGlu2 contains a conserved domain characteristic of β-1,3-1,4-glucanases and presented high protein identity with β-1,3-1,4-glucanases from other plant species. Molecular modeling of TcGlu2 showed an active site of 13 amino acids typical of glucanase with β-1,3 and 1,4 action mode. The recombinant cDNA TcGlu2 obtained by heterologous expression in Escherichia coli and whose sequence was confirmed by mass spectrometry, has a molecular mass of about 22 kDa (with His-Tag) and showed antifungal activity against the fungus Moniliophthora perniciosa, causal agent of the witches’ broom disease in cacao. The integrity of the hyphae membranes of M. perniciosa, incubated with protein TcGlu2, was analyzed with propidium iodide. After 1 h of incubation, a strong fluorescence emitted by the hyphae indicating the hydrolysis of the membrane by TcGlu2, was observed. To our knowledge, this is the first study of a cacao β-1,3-1,4-glucanase expression in heterologous system and the first analysis showing the antifungal activity of a β-1,3-1,4-glucanase, in particular against M. perniciosa.     

Behavioral and electrophysiological responses of Coptotermes formosanus Shiraki towards entomopathogenic fungal volatiles

Termites adjust their response to entomopathogenic fungi according to the profile of fungal volatile organic compounds (VOCs). This study demonstrates the pathogenicity of Metarhizium anisopliae, Beauveria bassiana and Isaria fumosorosea (=Paecilomyces fumosoroseus) towards the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Using no-choice assays, M. anisopliae was found to be highly virulent (LT₅₀ 3.10 d) when compared to B. bassiana (LT₅₀ 6.62 d) and I. fumosorosea (LT₅₀ 12.39 d). Also using choice assays, the foraging behavior of C. formosanus was determined in the presence of pathogenic fungi. The highly pathogenic fungi (M. anisopliae) elicited a repellent response, causing most of the termites to forage in a safe zone farthest from the fungal source. This repellency resulted in relatively low mortality similar to the controls. The repellency of M. anisopliae conidia can be used to protect human belongings and timber from termites. While I. fumosorosea cultures were not repellent to C. formosanus workers, the termites were highly susceptible to infection. Electroantennographic responses of workers showed approximately 47% and 78% lower level of response to conidia of B. bassiana and I. fumosorosea, respectively, as compared to M. anisopliae. The VOC profile of repellent cultures of M. anisopliae mainly consisted of paraffins (60.97%), while the major proportion of the I. fumosorosea profile consisted of branched and cyclic alkanes (84.41%). From the above findings, we conclude that the incorporation of I. fumosorosea may increase the control potential of bait.     

Degradative GH5 β-1,3-1,4-glucanase PpBglu5A for glucan in Paenibacillus polymyxa KF-1

A novel β-1,3-1,4-glucanase in the glycoside hydrolase family 5 (GH5) has been identified in the secretome of Paenibacillus polymyxa KF-1. The recombinant GH5 enzyme PpBglu5A shows broad substrate specificity, with strong lichenase activity, medium β-1,3-glucanase activity, and minimal cellulase activity. Barley β-glucan, lichenan, curdlan, and carboxymethyl cellulose are hydrolyzed to varying degrees by PpBglu5A, with the highest catalytic activity being observed with barley β-glucan. Hydrolysates from barley β-glucan or lichenan are primarily glucan oligosaccharides with degrees of polymerization from 2 to 4. PpBglu5A also hydrolyzes oat bran into oligosaccharides mainly consisted of di-, tri-, and tetra- oligosaccharides that are useful in the preparation of gluco-oligosaccharides. In addition to hydrolytic activity, transglycosylation was also observed with PpBglu5A and cellotriose as substrate. An in vitro assay indicated that the recombinant PpBglu5A has antifungal activity and can inhibit the growth of Canidia albicans. These results suggest that PpBglu5A exhibits unique properties and may be useful as an antifungal agent.     

Termite pathogens: Effects of ingested Metarhizium,beauveria, and Gliocladium conidia on worker termites (Reticulitermes sp.)

Separate groups of subterranean termites (Reticulitermes sp.) were exposed to whole cultures of Beauveria bassiana, Gliocladium virens, or Metarhizium anisopliae. Individuals were removed after varying time intervals and hindgut contents were plated onto potato dextrose agar. Viable spores first appeared in the hindguts within 8 hr of exposure. Fungi reisolated from the hindguts of diseased termites were pathogenic of healthy termites. Histological examination showed that invasion of the hemocoel by M. anisopliae occurred exclusively through direct invasion of the integument ca. 24 hr after death. B. bassiana invaded, primarily through the alimentary tract, ca. 12 hr prior to termite death.     

Cellular encapsulation in the eastern subterranean termite, Reticulitermes flavipes (Isoptera), against infection by the entomopathogenic fungus Metarhizium anisopliae

Reticulitermes flavipes workers were topically inoculated with ≈10,000 conidia of the entomopathogenic fungus Metarhizium anisopliae. After being kept in groups of 20 individuals for 1-9 d, histopathological examination showed that termites had an individual immune reaction. The nodule formation at the point of entrance of the fungal hyphae was identified as a cellular encapsulation and the different steps in the nodule formation are described. The relative number of hemocytes per termite increased 24 h after fungal exposure and remained high in the hemolymph for at least 3 d before decreasing back to pre-exposure levels. The role of an individual immune cellular reaction in social insects is discussed.     

Dissemination of Metarhizium anisopliae infection among the population of Odontotermes obesus (Isoptera: Termitidae) by augmenting the fungal conidia with attractants

Long-term biocontrol strategies for termite management have limited success as the behavioral response exhibited by termites in the presence of entomopathogenic fungi was different. To minimize these responses, a study was conducted to attract the termites towards the treated area by augmenting fungal conidia with a mixture of attractants such as sugarcane bagasse, sawdust and cardboard powder. In laboratory experiments, mortality due to the horizontal transmission of Metarhizium anisopliae infection among Odontotermes obesus workers and soldiers was 50 to 98% and 16 to 78% for the five isolates tested. The foraging activity of workers and soldiers increased from 23 to 58% for IWST-Ma13 when conidia were mixed with attractants. In a field study, the weight loss of stakes treated with conidial attractant decreased to 10.9 g compared to dry conidial treatments (19.3 g) for IWST-Ma13 after five months. Similarly, the width of mud galleries covering the tree bark was reduced when treated with conidial baits and gunny bags containing conidial attractant. In treated mounds, as the Acoustic emission signal voltage decreased from the threshold voltage level, the relative magnitude signal (RMS) remains low after five months of treatments with that of the control. From this study it was observed that the mixing of conidia with attractants could augment the dissemination potential of fungal infection among the termites. By adapting attractant based Metarhizium baits and dusting of conidial attractant mixture inside mounds may augment the dissemination potential in epizootic transmission of fungal infection.     

Gut Bacterial Community of the Xylophagous Cockroaches Cryptocercus punctulatus and Parasphaeria boleiriana

Cryptocercus punctulatus and Parasphaeria boleiriana are two distantly related xylophagous and subsocial cockroaches. Cryptocercus is related to termites. Xylophagous cockroaches and termites are excellent model organisms for studying the symbiotic relationship between the insect and their microbiota. In this study, high-throughput 454 pyrosequencing of 16S rRNA was used to investigate the diversity of metagenomic gut communities of C. punctulatus and P. boleiriana, and thereby to identify possible shifts in symbiont allegiances during cockroaches evolution. Our results revealed that the hindgut prokaryotic communities of both xylophagous cockroaches are dominated by members of four Bacteria phyla: Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Other identified phyla were Spirochaetes, Planctomycetes, candidatus Saccharibacteria (formerly TM7), and Acidobacteria, each of which represented 1–2% of the total population detected. Community similarity based on phylogenetic relatedness by unweighted UniFrac analyses indicated that the composition of the bacterial community in the two species was significantly different (P < 0.05). Phylogenetic analysis based on the characterized clusters of Bacteroidetes, Spirochaetes, and Deltaproteobacteria showed that many OTUs present in both cockroach species clustered with sequences previously described in termites and other cockroaches, but not with those from other animals or environments. These results suggest that, during their evolution, those cockroaches conserved several bacterial communities from the microbiota of a common ancestor. The ecological stability of those microbial communities may imply the important functional role for the survival of the host of providing nutrients in appropriate quantities and balance.     

Symbiotic adaptation of bacteria in the gut of Reticulitermes speratus: Low endo-β-1,4-glucanase activity

The termite is a good model of symbiosis between microbes and hosts and possesses an effective cellulose digestive system. Oxygen-tolerant bacteria, such as Dyella sp., Chryseobacterium sp., and Bacillus sp., were isolated from Reticulitermes speratus gut. Notably, the endo-β-1,4-glucanase (EG) activity of all 16 strains of isolated bacteria was low. Due to the combined activity of EG from the termites and their symbiotic protozoa, the bacteria might not be compelled to express EG. This observation demonstrates how well intestinal bacteria have assimilated themselves into the efficient cellulose digestive systems of termites.     

Extended disease resistance emerging from the faecal nest of a subterranean termite

Social insects nesting in soil environments are in constant contact with entomopathogens but have evolved a range of defence mechanisms, resulting in both individual and social immunity that reduce the chance for epizootics in the colony, as in the case of subterranean termites. Coptotermes formosanus uses its faeces as building material for its nest structure that result into a ‘carton material’, and here, we report that the faecal nest supports the growth of Actinobacteria which provide another level of protection to the social group against entomopathogens. A Streptomyces species with in vivo antimicrobial activity against fungal entomopathogens was isolated from the nest material of multiple termite colonies. Termite groups were exposed to Metarhizium anisopliae, a fungal entomopathogen, during their foraging activity and the presence of Streptomyces within the nest structure provided a significant survival benefit to the termites. Therefore, this report describes a non-nutritional exosymbiosis in a termite, in the form of a defensive mutualism which has emerged from the use of faecal material in the nesting structure of Coptotermes. The association with an Actinobacteria community in the termite faecal material provides an extended disease resistance to the termite group as another level of defence, in addition to their individual and social immunity.     

Antifungal activity of the termite alkaloid norharmane against the mycelial growth of Metarhizium anisopliae and Aspergillus nomius

Antifungal activity of norharmane, a β-carboline alkaloid found in termites (Isoptera, Rhinotermitidae) was tested against two entomopathogenic fungi, Metarhizium anisopliae and Aspergillus nomius. It was determined that, at physiological concentration (10 μg ml⁻¹), norharmane had no significant effect on A. nomius mycelial growth rate but reduced M. anisopliae growth rate by 11.9%. Contrary to previous findings, we suggest that norharmane has a limited role in disease resistance against fungal pathogens in individual subterranean termites, and we discuss the potential role of this chemical at a colony level.     

Characterization of a β-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to β-glucan-binding proteins

Spodoptera frugiperda β-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against β-1,3-glucan (laminarin), but cannot hydrolyze yeast β-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive β-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of β-1,3-glucanases and β-glucan-binding protein supports the assumption that the β-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived β-1,3-glucanases by the loss of an extended N-terminal region and the β-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.     

Odor Aversion and Pathogen-Removal Efficiency in Grooming Behavior of the Termite Coptotermes formosanus

The results of biocontrol with entomopathogens in termites have been discouraging because of the strong social hygiene behavior for removing pathogens from termite colonies. However, the mechanism of pathogen detection is still unclear. For the successful application of biopesticides to termites in nature, it would be beneficial to identify substances that could disrupt the termite’s ability to perceive pathogens. We hypothesized that termites can perceive pathogens and this ability plays an important role in effective hygiene behavior. In this study, pathogen-detection in the subterranean termite Coptotermes formosanus was investigated. We performed quantitative assays on conidia removal by grooming behavior using epifluoresence microscopy and Y-maze tests to examine the perception of fungal odor by termites. Three species each of high- and low-virulence entomopathogenic fungi were used in each test. The results demonstrated that termites removed conidia more effectively from a nestmate’s cuticle if its odor elicited stronger aversion. Highly virulent pathogens showed higher attachment rates to termite surfaces and their odors were more strongly avoided than those of low-virulence isolates in the same species. Moreover, termites appeared to groom each other more persistently when they had more conidia on their bodies. In brief, insect perception of pathogen-related odor seems to play a role in the mechanism of their hygiene behavior.     

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.