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.     

Opposing effects of allogrooming on disease transmission in ant societies

To prevent epidemics, insect societies have evolved collective disease defences that are highly effective at curing exposed individuals and limiting disease transmission to healthy group members. Grooming is an important sanitary behaviour—either performed towards oneself (self-grooming) or towards others (allogrooming)—to remove infectious agents from the body surface of exposed individuals, but at the risk of disease contraction by the groomer. We use garden ants (Lasius neglectus) and the fungal pathogen Metarhizium as a model system to study how pathogen presence affects self-grooming and allogrooming between exposed and healthy individuals. We develop an epidemiological SIS model to explore how experimentally observed grooming patterns affect disease spread within the colony, thereby providing a direct link between the expression and direction of sanitary behaviours, and their effects on colony-level epidemiology. We find that fungus-exposed ants increase self-grooming, while simultaneously decreasing allogrooming. This behavioural modulation seems universally adaptive and is predicted to contain disease spread in a great variety of host–pathogen systems. In contrast, allogrooming directed towards pathogen-exposed individuals might both increase and decrease disease risk. Our model reveals that the effect of allogrooming depends on the balance between pathogen infectiousness and efficiency of social host defences, which are likely to vary across host–pathogen systems.     

Methane Oxidation in Termite Hindguts: Absence of Evidence and Evidence of Absence

A steep oxygen gradient and the presence of methane render the hindgut internal periphery of termites a potential habitat for aerobic methane-oxidizing bacteria. However, methane emissions of various termites increased, if at all, only slightly when termites were exposed to an anoxic (nitrogen) atmosphere, and ¹⁴CH₄ added to the air headspace over live termites was not converted to ¹⁴CO₂. Evidence for the absence of methane oxidation in living termites was corroborated by the failure to detect pmoA, the marker gene for particulate methane monooxygenase, in hindgut DNA extracts of all termites investigated. This adds robustness to our concept of the degradation network in the termite hindgut and eliminates the gut itself as a potential sink of this important greenhouse gas.     

Tropheryma whipplei in the Environment: Survey of Sewage Plant Influxes and Sewage Plant Workers

We studied the prevalence of Tropheryma whipplei in influxes to 46 sewage treatment plants and in stool, mouthwash fluids, and dental plaques of 64 healthy workers in those facilities and 146 disease control patients. T. whipplei was found in sewage water, in stool of healthy individuals, and significantly more often in stool of workers exposed to sewage water.     

A common antifungal defense strategy in Cryptocercus woodroaches and termites

We identified and characterized Gram-negative bacteria binding proteins (GNBPs) and their predicted antifungal activity in the woodroach Cryptocercus punctulatus. C. punctulatus is likely to share many characteristics with the subsocial ancestor of the Isoptera, including allogrooming, which facilitated the evolution of termite sociality. Based on a phylogenetic analysis, an ancestral GNBP with an intact β-1,3-glucanase active site appears to have duplicated in a common ancestor of subsocial Cryptocercus woodroaches and termites. In termites, the secreted β-1,3-glucanase activity of GNBPs provides important prophylactic protection from fungal pathogens such as Metarhizium anisopliae, which can evade the immune system after entering the insect. Here, we identify β-1,3-glucanase activity on the cuticular surface of C. punctulatus that originates from the salivary gland and is likely spread by allogrooming. Cuticular washes have antifungal activity against M. anisopliae conidia that is suppressed by an inhibitor (GDL) of termite GNBP β-1,3-glucanase activity. C. punctulatus nymphs that are treated with GDL and subsequently exposed to M. anisopliae conidia show significantly greater mortality than the untreated nymphs exposed to conidia. A molecular evolutionary analysis of GNBPs in two species of Parcoblatta, Periplaneta americana, C. punctulatus and representative termites indicates that selection-directed change in a glycosylphosphatidylinositol (GPI) anchor region. Modification of the GPI anchor region may have been instrumental in the evolution of an antifungal defense strategy that depends on the external secretion of GNBPs from the salivary gland and their dissemination by grooming. This strategy may have helped compensate for the vulnerability of a subsocial woodroach-like ancestor to fungal disease that results from prolonged development with a thin cuticle and facilitated the transition to termite eusociality.     

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.     

Selective isolation of agents of chromoblastomycosis from insect-associated environmental sources

Chromoblastomycosis is a neglected disease characterized by cutaneous, subcutaneous or disseminated lesions. It is considered an occupational infectious disease that affects mostly rural workers exposed to contaminated soil and vegetal matter. Lesions mostly arise after a traumatic inoculation of herpotrichiellaceous fungi from the Chaetothyriales order. However, the environmental niche of the agents of the disease remains obscure. Its association with insects has been predicted in a few studies. Therefore, the present work aimed to analyze if social insects, specifically ants, bees, and termites, provide a suitable habitat for the fungi concerned. The mineral oil flotation method was used to isolate the microorganisms. Nine isolates were recovered and phylogenetic analysis identified two strains as potential agents of chromoblastomycosis, i.e., Fonsecaea pedrosoi CMRP 3076, obtained from a termite nest (n = 1) and Rhinocladiella similis CMRP 3079 from an ant exoskeleton (n = 1). In addition, we also identified Fonsecaea brasiliensis CMRP 3445 from termites (n = 1), Exophiala xenobiotica CMRP 3077 from ant exoskeleton (n = 1), Cyphellophoraceae CMRP 3103 from bees (n = 1), Cladosporium sp. CMRP 3119 from bees (n = 1), Hawksworthiomyces sp. CMRP 3102 from termites (n = 1), and Cryptendoxyla sp. from termites (n = 2). The environmental isolate of F. pedrosoi CMRP 3076 was tested in two animal models, Tenebrio molitor and Wistar rat, for its pathogenic potential with fungal retention in T. molitor tissue. In the Wistar rat, the cells resembling muriform cells were observed 30 d after inoculation.     

Cryoprotection in dampwood termites (Termopsidae, Isoptera)

In contrast to the majority of the Order, the dampwood termites of the family Termopsidae found in colder regions can experience frost and snow, either in cool temperate areas at high latitudes (45°), or alpine areas at high elevations (>1000 m). This suggests that dampwood termites are adapted to cold climates. We investigated this hypothesis in two dampwood termites, Porotermes adamsoni Froggatt and Stolotermes victoriensis Hill. We measured nest temperatures and atmospheric temperatures of their alpine habitat during winter, and measured survival and recovery at subzero temperatures. We also determined the minimum temperature at which these species remain active and the LT50 values. We used a novel gas chromatographic strategy to examine eight metabolites from individuals of both species collected in winter and summer to identify possible cryoprotectants. Both P. adamsoni and S. victoriensis had significantly higher levels of trehalose, a known cryoprotectant, in winter than in summer; in addition S. victoriensis also had higher levels of unsaturated fatty acid ligands in winter than in summer, consistent with patterns observed for cold adaptation in other organisms. These results are the first to reveal that dampwood termites are adapted to cold climates and use trehalose and unsaturated lipids as cryoprotectants.     

The Trojan horse approach for managing invasive ants: a study with Asian needle ants, <Emphasis Type="Italic">Pachycondyla chinensis</Emphasis>

Ants are among the most ecologically and economically significant biological invaders and are notoriously difficult to eradicate once established. Invasive ants are typically managed with toxic baits which are often unattractive to the target species, toxic to non-targets, and environmentally persistent. The current study evaluated a novel Trojan horse approach for managing invasive ants in natural habitats based on the use of poisoned prey. Eastern subterranean termites (Reticulitermes flavipes) were topically exposed to fipronil and presented to Asian needle ants (Pachycondyla chinensis) which are a significant invader in natural and disturbed habitats in the eastern United States. In laboratory assays, P. chinensis colonies were offered fipronil-treated termites within experimental arenas. The termites were readily attacked and consumed and results demonstrate that a single termite exposed to 25 ppm fipronil for 1 h is capable of killing 100 P. chinensis workers in 9 h. To evaluate population effects, field studies were conducted in forested areas invaded by P. chinensis. Fipronil-treated termites scattered on the forest floor provided rapid control of P. chinensis and ant densities throughout the treated plots declined by 98 ± 5 % within 28 days. I demonstrate that the poison baiting approach based on fipronil-treated termite prey is highly effective against P. chinensis and may offer an effective alternative to traditional bait treatments against other invasive ants, especially those with predatory and generalist feeding habits. Furthermore, I demonstrate that the poison baiting approach offers environmental benefits by delivering substantially less toxicant to the environment relative to current control methods which rely on commercial bait formulations. In summary, the poison baiting approach evaluated in this study appears highly suitable for controlling invasive ants and should be further tested against other invasive ants.     

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.     

Impact of a juvenile hormone analogue on the anatomy and the frontal gland secretion of Prorhinotermes simplex (Isoptera: Rhinotermitidae)

In termites, juvenile hormone plays a key role in soldier differentiation. To better understand the evolutionary origin of the soldiers, we studied the external and inner morphology of pseudergate-soldier intercastes and neotenic-soldier intercastes formed artificially by the application of juvenile hormone analogue in Prorhinotermes simplex. A majority of these intercastes had a soldier phenotype, whereas the inner anatomy had an intermediary form between two castes or a form specific to intercastes. Our experiments showed that traits of neotenics and soldiers can be shared by the same individuals, although such individuals do not exist naturally in P. simplex, and they have not been reported in other species but in some Termopsidae. Our results reinforce the hypothesis that soldiers may have emerged from soldier neotenics during the evolution of termites.     

Low temperature enhances the ability of the termite-egg-mimicking fungus Athelia termitophila to compete against wood-decaying fungi

A unique symbiosis exists between subterranean termites and the sclerotium-forming fungus Athelia termitophila, which forms termite-egg-mimicking sclerotia called ‘termite balls’. While the sclerotia gain a competitor-free habitat by being harboured by termite eggs, A. termitophila mycelia have to compete with wood-decay fungi in the life stage without termites. To understand its relationship with termites, the factors that affect the ability of A. termitophila to compete with other wood-decay fungi must be clarified. Here, we show that A. termitophila is competitive against other wood-decay fungi at low temperatures. In Petri dish experiments to evaluate the effects of the physicochemical conditions, that A. termitophila experiences in termite nests, on its competitive ability, A. termitophila overcame surrounding fungi in the winter, when termites are less active. Further studies quantifying the effects of A. termitophila on termites in winter will help us to understand this relationship.     

Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.     

Behavioral variation among tunnelers in the Formosan subterranean termite

Division of labor is a common feature of insect societies and has been theorized to account for much of their success. Asymmetries in the work of individuals, whose aggregate labor results in the completion of a task, can lead to the emergence of key individuals that dominate or govern the task. Coptotermes formosanus Shiraki excavate in a series of alternating workers whose efforts combine not only to elongate tunnels, but also to guide the direction of propagation. When groups of 100 termites were presented with a single tunnel, only ～ 16% of termites entered. Of those that entered, the level of excavation was not uniform, with 20.6% of termites responsible for over half of the total excavation. These termites, a small percentage of the total available workforce, act as key individuals, producing the majority of labor and possibly guiding the efforts of others. An examination of the excavation patterns of individuals shows that some individuals excavate sporadically, but at a very high rate (number of excavation events per time). By focusing their effort over a short period, these highly active individuals may influence the orientation of a tunnel and the formation of branches to a degree out of proportion to the total amount of digging they engage in.     

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.     

Inbreeding and disease resistance in a social insect: effects of heterozygosity on immunocompetence in the termite Zootermopsis angusticollis

Recent research has shown that low genetic variation in individuals can increase susceptibility to infection and group living may exacerbate pathogen transmission. In the eusocial diploid termites, cycles of outbreeding and inbreeding characterizing basal species can reduce genetic variation within nestmates during the life of a colony, but the relationship of genetic heterogeneity to disease resistance is poorly understood. Here we show that, one generation of inbreeding differentially affects the survivorship of isolated and grouped termites (Zootermopsis angusticollis) depending on the nature of immune challenge and treatment. Inbred and outbred isolated and grouped termites inoculated with a bacterial pathogen, exposed to a low dose of fungal pathogen or challenged with an implanted nylon monofilament had similar levels of immune defence. However, inbred grouped termites exposed to a relatively high concentration of fungal conidia had significantly greater mortality than outbred grouped termites. Inbred termites also had significantly higher cuticular microbial loads, presumably due to less effective grooming by nestmates. Genetic analyses showed that inbreeding significantly reduced heterozygosity and allelic diversity. Decreased heterozygosity thus appeared to increase disease susceptibility by affecting social behaviour or some other group-level process influencing infection control rather than affecting individual immune physiology.     

Intra- and Inter-colonial Agonistic Behavior in the Termite, Microcerotermes fuscotibialis Sjostedt (Isoptera: Termitidae: Termitinae)

Pairing termite workers from cultures obtained from same colony of Microcerotermes fuscotibialis Sjostedt did not result in agonistic behavior or mortality of the paired individuals. Termite pairings from different colonies located at different distances resulted in serious encounters and aggressive behavior leading to significant mortality of the individuals. Subtle agonistic behavior was observed when colony mates were reunited after months of separation, but this did not result in any significant mortality. These results confirmed observations of non-overlap of foraging trails of these termites in the field which averts possible encounters and conflict. Therefore these termites are able to recognize their colony mates irrespective of period of separation, a factor for colony affiliation, kinship and recognition.     

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.     

Analysis of the Airway Microbiota of Healthy Individuals and Patients with Chronic Obstructive Pulmonary Disease by T-RFLP and Clone Sequencing

Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways. Objective: The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.     

Do Termites Avoid Carcasses? Behavioral Responses Depend on the Nature of the Carcasses

Background

Undertaking behavior is a significant adaptation to social life in enclosed nests. Workers are known to remove dead colony members from the nest. Such behavior prevents the spread of pathogens that may be detrimental to a colony. To date, little is known about the ethological aspects of how termites deal with carcasses.

Methodology and Principal Findings

In this study, we tested the responses to carcasses of four species from different subterranean termite taxa: Coptotermes formosanus Shiraki and Reticulitermes speratus (Kolbe) (lower termites) and Microcerotermes crassus Snyder and Globitermes sulphureus Haviland (higher termites). We also used different types of carcasses (freshly killed, 1-, 3-, and 7-day-old, and oven-killed carcasses) and mutilated nestmates to investigate whether the termites exhibited any behavioral responses that were specific to carcasses in certain conditions. Some behavioral responses were performed specifically on certain types of carcasses or mutilated termites. C. formosanus and R. speratus exhibited the following behaviors: (1) the frequency and time spent in antennating, grooming, and carcass removal of freshly killed, 1-day-old, and oven-killed carcasses were high, but these behaviors decreased as the carcasses aged; (2) the termites repeatedly crawled under the aging carcass piles; and (3) only newly dead termites were consumed as a food source. In contrast, M. crassus and G. sulphureus workers performed relatively few behavioral acts. Our results cast a new light on the previous notion that termites are necrophobic in nature.

Conclusion

We conclude that the behavioral response towards carcasses depends largely on the nature of the carcasses and termite species, and the response is more complex than was previously thought. Such behavioral responses likely are associated with the threat posed to the colony by the carcasses and the feeding habits and nesting ecology of a given species.