Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi

Termites, Coptotermes formosanus Shiraki, reared individually, were highly susceptible to entomopathogenic fungi, Paecilomyces fumosoroseus and Beauveria brongniartii and Metarhizium anisopliae, while termites reared in groups were highly resistant. Quantitative assays with an epifluoresent microscope revealed a significant difference in the number of conidia attachments among three entomopathogenic fungi. The conidia of B. brongniartii and P. fumosoroseus bound to termite cuticles more effectively than M. anisopliae conidia. Our results also suggested that self-grooming behavior is less effective, but mutual grooming is very effective in the removal of conidia from cuticles of their nestmates. Statistical analysis of removal rates indicated that conidia of P. fumosoroseus and B. brongniartii were removed more rapidly than M. anisopliae conidia from termite cuticles.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Fungus Exposed Solenopsis invicta Ants Benefit from Grooming

We investigated aspects of resistance to entomopathogenic fungi in the social insect Solenopsis invicta, the red imported fire ant (RIFA). RIFA reared individually were significantly more susceptible to the entomopathogenic fungi Metarhizium anisopliae var. anisopliae M09 than reared in groups. Fungus exposed ants performed more self-grooming behavior when isolated as individuals and received more allo-grooming when accompanied with four healthy nestmates. Using fluorescence microscopy, we counted the number of fluorescein isothiocyanate (FITC)-labeled conidia on the cuticle of fungus exposed ants reared individually or as groups. The number of conidia on the surface of grouped ants decreased more rapidly than on isolated individuals. Allo-grooming behavior appears to be important in removing the conidia on the surface of RIFA. Individuals help fungus exposed ants by performing intensive grooming behaviors, which either risk infecting themselves or get them immunized as social immunity. We show evidence that contacting with fungus exposed ants would decrease susceptibility of nestmates to the fungus. All these results indicate that RIFA benefit from grooming behavior to fight against the fungal pathogens. Future advances in biological control of RIFA with entomopathogenic fungi are also discussed.     

Inhibition of Metarhizium anisopliae in the alimentary tract of the eastern subterranean termite Reticulitermes flavipes

Reticulitermes flavipes workers were individually inoculated with 10,000 conidia of the entomopathogenic fungus Metarhizium anisopliae. After being kept in groups of 20 individuals for 1-6 d, histopathological approach showed that most of the inoculated conidia were groomed from the surface of the cuticle by nestmates within 24 h, and that a large number of conidia was subsequently found in different parts of the gut of the groomers. Our observations showed that, among thousands of conidia found in the termite’s gut, conidial germination never occurred in all inspected specimens, even when the conidia had the chance to bind to the surface of the cuticular lining of the gut. In addition, when termites were left for decomposition several days after death caused by an external infection of M. anisopliae, the hyphal growth was generalized in the body cavity of the cadaver, but was never observed in the lumen of the gut even 2 d post-mortem. Our observation suggests that the putative biochemicals involved in the termite’s gut defense against fungal pathogens are from multiple origins.     

Morphology and molecular taxonomy of Evlachovaea-like fungi,and the status of this unusual conidial genus

The entomopathogenic anamorphic genus Evlachovaea was described to differ from other fungi in forming its conidia obliquely to the axis of the conidiogenous cell and with successive conidia having alternate orientations with a zipper- or chevron-like arrangement resulting in flat, ribbon-like chains. Morphological and molecular studies of six Evlachovaea-like isolates baited from Central Brazilian soils using Triatoma infestans (a vector of Chagas disease) and of other entomopathogens with Evlachovaea-like conidiogenesis led to a re-evaluation of the status of this little known fungal genus. The Brazilian isolates formed two distinct groups based on gene sequences for both the internal transcribed spacer (ITS) and translation elongation factor (EF-1α) genes, morphology, and growth patterns; both groups also differed from the type species, Evlachovaea kintrischica. More detailed studies of these fungi indicated that the alternatingly oblique orientations of forming conidia are neither a stable nor invariant character (even on single phialides). Furthermore, the molecular cladistic analysis unambiguously placed the Evlachovaea isolates firmly within the genus Isaria (Hypocreales: Cordycipitaceae). The ITS sequences of E. kintrischica were very similar or even identical to those of Isaria amoenerosea and Isaria cateniobliqua, thereby suggesting that E. kintrischica is a synonym of one of these species, and that the genus Evlachovaea must be treated as a later synonym of Isaria, which must now be recognized to include several highly divergent modes of conidiogenesis. These taxonomic findings are discussed in the context of dramatic changes recently imposed on the nomenclatural standards used to determine the correct names of all pleomorphic fungi.     

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.     

Differential fluctuation in virulence and VOC profiles among different cultures of entomopathogenic fungi

Insect-passaged cultures of entomopathogenic fungi grown on potato dextrose agar media have been shown to have altered virulence and profiles of volatile compounds. The present study demonstrated the pathogenic status of FS₀ (in vitro) and FS₁ and FS₂ (insect-passaged cultures grown on PDA) cultures of Metarhizium anisopliae (strains 406 and 02049) and Beauveria bassiana by a non-choice assay, in which filter paper was inoculated with fungal spores at a concentration of 1 × 10⁷ spores/ml. The FS₁ and FS₂ cultures of M. anisopliae strain 02049 and B. bassiana produced conidia with high virulence, and the volatile profiles of these conidia comprised relatively lower percentages of branched-alkanes than conidia from the FS₀ cultures. In contrast, the conidia from an FS₀ culture of M. anisopliae strain 406 had somewhat elevated virulence levels, but their volatile profile had <2% branched-alkanes. The FS₁ and FS₂ cultures of M. anisopliae strain 406 did not gain virulence, and these cultures showed a decline in virulence along with major alteration of their volatile profiles. Their volatile profiles mainly comprised branched-alkanes. The volatile profiles of the FS₁ and FS₂ cultures lacked n-tetradecane, which was an important component of all the virulent cultures. Four compounds, 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane, were detected only from the virulent cultures, suggesting that low LT₅₀ values were probably due to the production of these compounds. This is the first report to characterize volatiles from FS₀, FS₁ and FS₂ cultures of entomopathogenic fungi; its utility in different aspects opens an interesting area for further investigations.     

Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface

Conidial germination and the formation of appressoria are important events in the interactions between entomopathogenic fungi and their arthropod hosts. In this study, we demonstrate the effects of lipids extracted from tick epicuticle and the surface of a mammalian host (calf) on conidial germination and the development of appressoria in two subspecies of Metarhizium anisopliae, M. anisopliae var. anisopliae (M.an.an.-7) and M. anisopliae var. acridum (M.an.ac.-5), which have different levels of virulence toward ticks. Pentane extracts of epicuticles of ticks susceptible and resistant to fungal infection always stimulated the germination of M.an.an.-7 conidia and the development of their appressoria; whereas the effects of dichloromethane (DCM) extracts of tick epicuticle varied depending on the tick. The DCM extracts from most of the tick species and developmental stages stimulated conidial germination and/or the formation of appressoria in M.an.an.-7. However, a DCM extract of lipids from the most resistant tick, engorged Hyalomma excavatum female, inhibited the germination of M.an.an.-7 conidia. Conidia of the non-virulent M.an.ac.-5 did not germinate on agarose amended with any of the examined tick extracts. However, when the tick extracts were placed on bactoagar, conidial germination increased 7- to 8-fold. Extracts from the skin, hair and ear secretions of a calf stimulated conidial germination and the formation of appressoria in M.an.an.-7, but not M.an.ac.-5. This study demonstrates that lipids from tick epicuticles and mammalian skin selectively affect the germination of conidia of entomopathogenic fungi. The effects of these lipids may explain the variability in tick control these fungi provide for different hosts.     

Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites?

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.     

Susceptibility and behavioral responses of the dampwood termite Zootermopsis angusticollis to the entomopathogenic nematode Steinernema carpocapsae

Termites exploit microbially rich resources such as decayed wood and soil that are colonized by potentially pathogenic and parasitic fungi, bacteria, viruses, and nematodes. In colonies composed of thousands of individuals, the risk of infection among nestmates is significant, and individual and social behavior could involve various adaptations to resist disease and parasitism. Here we show that the dampwood termite Zootermopsis angusticollis (Hagen) exhibits a dosage dependent susceptibility to the soil nematode Steinernema carpocapsae (Weiser) (Mexican strain) and that this social insect significantly alters its behavior in response to this entomopathogenic roundworm. Relative to their baseline behavior, termites exposed to infective juveniles increased the frequency and duration of allogrooming and vibratory displays as well as two other novel behaviors, abdominal tip-raising and self-scratching. Whereas the first two behaviors likely reflect general adaptations to reduce susceptibility to a variety of pathogens and parasites, the latter behaviors might be specific to nematodes because they have never been observed in Z. angusticollis in any other pathogenic context. Our results support the hypotheses that behavioral responses in termites are important in the control of pathogenic and parasitic microorganisms and that termite susceptibility is socially mediated.     

The Use of Entomopathogenic Fungi for Control of Termites

Termites are major pests of timber and timber products which live in warm, humid environments that are conducive to the development and spread of entomopathogenic fungi. Laboratory studies have shown that termite species are highly susceptible to entomopathogenic fungi from genera including the most commonly studied species, Metarhizium anisopliae and Beauveria bassiana . There appears to be very little host specificity among fungal isolates with many isolates being highly virulent to many species of termites. The grooming and other social interactions between termites are seen to have the potential to spread the fungus through the colony, allowing for colony control by the treatment of remote feeding sites. However, factors such as avoidance of the fungus conidia by the termites, the removal and burial of fungus-killed termites, together with defensive secretions and inhibitory components in termite frass and the possibility of humoral resistance may limit the spread of the disease in the colony. Field studies have shown mixed results. Direct application of fungi to nests has resulted in complete colony mortality, but studies where feeding sites or bait stations have been treated with fungus have yet to show similar success. The effectiveness of termite control in urban pest management, particularly in structural timber and dwellings, has yet to be reported in detail. Such studies require the examination of the complex relationships between dose, speed of kill, virulence, horizontal transmission and ultimate colony death, combined with avoidance and recognition factors, and survival of the fungi under field conditions. There is currently one commercial product available in the USA.     

Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni

Termites encounter a diverse array of potentially useful and harmful fungi in their subterranean habitats. These vary from symbiotic to harmful species with varying levels of virulence. How these hemiedaphic insects survive in habitats with infective fungi is not well understood. Possible mediation of olfactory signals in avoiding contact with entomopathogenic fungi has been explored by a number of workers. In the present study, we initially found that Macrotermes michaelseni detected a virulent isolate of Metarhizium anisopliae from some distance and avoided direct physical contact. We hypothesized that there may be a relationship between virulence and repellency of different isolates of M. anisopliae and Beauveria bassiana to the termite. We compared these for selected isolates of the two fungi. Positive correlations between the two parameters for both sets of isolates of the fungi were obtained. The results show an interesting co-evolutionary phenomenon in which the termite's response to either M. anisopliae or B. bassiana is directly related to potential harm these fungi can inflict on the insect and that the virulent strains are more likely to be recognized from some distance and avoided.     

Entomopathogenic fungi recorded from the harlequin ladybird, Harmonia axyridis

Entomopathogenic fungi were recorded from field samples of the harlequin ladybird Harmonia axyridis, an invasive coccinellid that has recently arrived in Denmark. Larvae, pupae and adults were found to be infected by Isaria farinosa, Beauveria bassiana and species of Lecanicillium. This is the first record of entomopathogenic fungi infecting larvae and pupae. Winter mortality due to fungal infection reached 17.9% in adults collected at one location. The larval stage was most susceptible to fungal infection, as confirmed through bioassay with I. farinosa.     

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.