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.     

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.     

红火蚁工蚁对绿僵菌侵染蛹的防御行为

研究了红火蚁工蚁感染绿僵菌后在蛹室的行为变化,以及健康工蚁对侵染蛹的行为保护机制.结果表明: 工蚁被绿僵菌侵染后,在蛹室的活动时间逐渐减少,由第1天的103.4 s降至第3天的38.5 s;而且育幼时间占蛹室活动时间的比例也下降,由第1天的13.6%降至第3天的3.5%.当蛹被绿僵菌侵染后,工蚁对侵染蛹的梳理总时间为对照组的5.3倍,每次梳理的平均持续时间为对照组的5.2倍.梳理行为能显著减少侵染蛹的体表分生孢子数量,在无工蚁、2只工蚁和10只工蚁存在条件下,蛹体表平均孢子数分别为103.1、51.6和31.3个.工蚁的存在能抑制蛹体表孢子的萌发,处理20 h后,无工蚁、2只工蚁和10只工蚁存在条件下,蛹体表孢子萌发率分别为95.1%、80.4%和59.9%.蛹的羽化率随着工蚁数量增加显著升高.红火蚁工蚁通过社会行为防御病原真菌侵染蛹的策略为种群的延续和发展提供了保障.     

Plasticity of grooming behavior against entomopathogenic fungus Metarhizium anisopliae in the ant Lasius japonicus

Social insects employ many types of defense mechanisms against parasites and pathogens because they face high risks from infections due to crowded living conditions with closely related nestmates. Grooming behavior, including self-grooming and allogrooming, can remove fungal spores on the cuticles of social insects and may be a behavioral defense mechanism to improve survivorship. Allogrooming between nestmates has been predicted to be especially important as a defense against ectoparasites. However, little is known about the plasticity of grooming behavior in susceptible environments. Here, we show that the ant Lasius japonicus increases the frequency of allogrooming rather than self-grooming to improve survivorship against the entomopathogenic fungus Metarhizium anisopliae. We found that increasing fungal dosage and ant group size influenced the plasticity of grooming behavior. Additionally, the survival rate of the ants over 30 days improved with increased group size. Our results suggest that social insects opt for altruistic behavior to prevent the spread of diseases. This study illustrates how ants maintain colonies through grooming behavior in the presence of fungal spores.     

Imidacloprid Inhibits Behavioral Defences of the Leaf-Cutting Ant Acromyrmex subterraneus subterraneus (Hymenoptera:Formicidae)

Leaf-cutting ants have evolved a range of defensive strategies which complicate the use of entomopathogens for their control. One of these behavioral strategies is self-grooming, which increases when ants detect the presence of fungal conidia on their integuments. We have previously shown that insecticides, when used at ultra-low concentrations, can be synergists of entomopathogenic fungi. It is possible that certain insecticides could modify ant behavior in a way that increases the chances of a fungal infection taking hold. The current study investigated the effects of the neonicotinoid insecticide Imidacloprid (IMI) and the entomopathogenic fungus Beauveria bassiana on self-grooming and locomotion behavior of Acromrymex subterraneus subterraneus workers when ants were exposed to the agents separately or together. Initially 10, 20 and 40 ng/insect IMI was topically administered to worker ants. Four hours after administration, the ants were placed into an experimental arena and self-grooming and locomotion behaviors were recorded for 10 min. Separate groups of ants were exposed to filter paper discs impregnated with conidial suspensions and 4 h later their behavior was monitored for 10 min. Subsequently, ants were treated with 10 ng/insect IMI, immediately exposed to fungal suspensions and 4 h later their behavior was monitored. The 10 ng/insect IMI treatment resulted in higher locomotor activity and lower self-grooming when compared to vehicle, 20 ng and 40 ng IMI treatments. Exposure to fungal conidia produced low locomotion and high self-grooming behaviors. The 10 ng IMI + fungal conidia treatment resulted in lower self-grooming and higher locomotion behavior. These alterations caused by a low concentration of IMI could increase ant susceptibility to infection by entomopathogenic fungi and are thus of interest for future tests in the development of integrated pest management strategies.     

Social transfer of pathogenic fungus promotes active immunisation in ant colonies

Due to the omnipresent risk of epidemics, insect societies have evolved sophisticated disease defences at the individual and colony level. An intriguing yet little understood phenomenon is that social contact to pathogen-exposed individuals reduces susceptibility of previously naive nestmates to this pathogen. We tested whether such social immunisation in Lasius ants against the entomopathogenic fungus Metarhizium anisopliae is based on active upregulation of the immune system of nestmates following contact to an infectious individual or passive protection via transfer of immune effectors among group members--that is, active versus passive immunisation. We found no evidence for involvement of passive immunisation via transfer of antimicrobials among colony members. Instead, intensive allogrooming behaviour between naive and pathogen-exposed ants before fungal conidia firmly attached to their cuticle suggested passage of the pathogen from the exposed individuals to their nestmates. By tracing fluorescence-labelled conidia we indeed detected frequent pathogen transfer to the nestmates, where they caused low-level infections as revealed by growth of small numbers of fungal colony forming units from their dissected body content. These infections rarely led to death, but instead promoted an enhanced ability to inhibit fungal growth and an active upregulation of immune genes involved in antifungal defences (defensin and prophenoloxidase, PPO). Contrarily, there was no upregulation of the gene cathepsin L, which is associated with antibacterial and antiviral defences, and we found no increased antibacterial activity of nestmates of fungus-exposed ants. This indicates that social immunisation after fungal exposure is specific, similar to recent findings for individual-level immune priming in invertebrates. Epidemiological modeling further suggests that active social immunisation is adaptive, as it leads to faster elimination of the disease and lower death rates than passive immunisation. Interestingly, humans have also utilised the protective effect of low-level infections to fight smallpox by intentional transfer of low pathogen doses ("variolation" or "inoculation").     

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.     

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.     

Pathogenicity of Beauveria bassiana ANU1 to the red imported fire ant,Solenopsis invicta workers in Korea

The red imported fire ant (RIFA), Solenopsis invicta, is one of the globalized invasive pests. This study focused on pathogenicity and virulence of entomopathogenic fungi as one of the biological control agents to RIFA workers under different temperatures. The fungal pathogen, Beauveria bassiana ANU1 was isolated from Korea in 2015 and showed the pathogenicity to RIFA workers. A conidial suspension (1 × 10⁷ conidia/ml) induced a low mortality from day 2 after treatment and reached to 100% mortality at day 7 and day 8 after treatment for major and minor workers, respectively. The median lethal concentrations of B. bassiana ANU1 were calculated as 3.9 × 10³ for major and 4.6 × 10³ for minor workers at day 7 after treatment. Low temperatures decreased a virulence of B. bassiana ANU1 (1 × 10⁷ conidia/ml) to RIFA and showed mortality of 26.6% for major and 20% for minor workers. Based on bioassay results, this study provides one of possibilities of effective and successful strategy for controlling RIFA by entomopathogenic fungi.     

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.     

Quarantining behaviour in ants: are Myrmica aphid milkers able to detect and get rid of fungus-contaminated aphids?

Fungal infections are highly dangerous for social insects including ants. Close trophobiotic interaction between ants and aphids promotes infection transmission, as aphids can be a disease vector. The ability of ants to detect fungus-infected aphids and get rid of them is important to the prosperity of both symbiotic partners. However, the diversity of quarantining behaviour among ants is still poorly studied. Here, the behaviour of honeydew foragers of two ant species – Myrmica rubra L. and Myrmica scabrinodis Nylander (Hymenoptera: Formicidae, Myrmicinae, Myrmicini) – was studied in laboratory towards Schizaphis graminum (Rondani) (Hemiptera: Aphididae, Aphidini) aphids contaminated with the generalist fungal pathogen Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales). This fungus attacks a wide range of hosts including aphids and ants. The removal of conidia-contaminated aphids from the host plant was found not to be typical of the ants studied. Aphid milkers of M. rubra and M. scabrinodis usually displayed non-aggressive behaviour (tolerance, antennation, honeydew collection, grooming) towards the experimental aphids regardless of whether they were covered with conidia or not. Neither ant species, nor the number of milkers had significant effects on their behaviour towards ‘infected’ aphids. However, some individuals were found to demonstrate quarantining behaviour in full. They quickly detected and removed contaminated aphids, placing them at some distance from the plant. Moreover, in addition to the simple carrying of ‘infected’ aphids down, the more effective technique of dropping them from the plant was noted as well. Ants of the genus Myrmica appear to have a tendency to perform a certain sequence of actions to remove conidia-contaminated aphids from the plant. It is likely that in larger colonies or under conditions of increased risk of infection with entomopathogenic fungi, some Myrmica ants are able to deploy and actively use the behavioural pattern of quarantining behaviour to increase their viability.     

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.     

Ant Colonies Prefer Infected over Uninfected Nest Sites

During colony relocation, the selection of a new nest involves exploration and assessment of potential sites followed by colony movement on the basis of a collective decision making process. Hygiene and pathogen load of the potential nest sites are factors worker scouts might evaluate, given the high risk of epidemics in group-living animals. Choosing nest sites free of pathogens is hypothesized to be highly efficient in invasive ants as each of their introduced populations is often an open network of nests exchanging individuals (unicolonial) with frequent relocation into new nest sites and low genetic diversity, likely making these species particularly vulnerable to parasites and diseases. We investigated the nest site preference of the invasive pharaoh ant, Monomorium pharaonis, through binary choice tests between three nest types: nests containing dead nestmates overgrown with sporulating mycelium of the entomopathogenic fungus Metarhizium brunneum (infected nests), nests containing nestmates killed by freezing (uninfected nests), and empty nests. In contrast to the expectation pharaoh ant colonies preferentially (84%) moved into the infected nest when presented with the choice of an infected and an uninfected nest. The ants had an intermediate preference for empty nests. Pharaoh ants display an overall preference for infected nests during colony relocation. While we cannot rule out that the ants are actually manipulated by the pathogen, we propose that this preference might be an adaptive strategy by the host to “immunize” the colony against future exposure to the same pathogenic fungus.     

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.     

A Low-cost Contamination Device for Infecting Adult Tsetse Flies, Glossina spp., with the Entomopathogenic Fungus Metarhizium anisopliae in the Field

A low-cost device for infecting adult tsetse fly, Glossina fuscipes fuscipes , with the entomopathogenic fungus Metarhizium anisopliae was designed and tested in the field. Tsetse flies that are attracted to the trap entered the contamination device and ultimately became infected with the fungus. Traps exposed to the sun attracted more flies than did the ones placed in the shade. The time spent by single flies in the contamination device varied between 5- 189 s, and the subsequent number of conidia collected varied between 1.6 ×10 5 conidia and 40.5 ×10 5 conidia per fly, and largely depended on the behavior of individual flies. Dry conidia of M. anisopliae in the device retained their viability for 31 days in the field, and efficacy against G. fuscipes was not affected.     

Adhesion of the Entomopathogenic Fungus Beauveria (Cordyceps) bassiana to Substrata

The entomopathogenic fungus Beauveria bassiana produces at least three distinct single-cell propagules, aerial conidia, vegetative cells termed blastospores, and submerged conidia, which can be isolated from agar plates, from rich broth liquid cultures, and under nutrient limitation conditions in submerged cultures, respectively. Fluorescently labeled fungal cells were used to quantify the kinetics of adhesion of these cell types to surfaces having various hydrophobic or hydrophilic properties. Aerial conidia adhered poorly to weakly polar surfaces and rapidly to both hydrophobic and hydrophilic surfaces but could be readily washed off the latter surfaces. In contrast, blastospores bound poorly to hydrophobic surfaces, forming small aggregates, bound rapidly to hydrophilic surfaces, and required a longer incubation time to bind to weakly polar surfaces than to hydrophilic surfaces. Submerged conidia displayed the broadest binding specificity, adhering to hydrophobic, weakly polar, and hydrophilic surfaces. The adhesion of the B. bassiana cell types also differed in sensitivity to glycosidase and protease treatments, pH, and addition of various carbohydrate competitors and detergents. The outer cell wall layer of aerial conidia contained sodium dodecyl sulfate-insoluble, trifluoroacetic acid-soluble proteins (presumably hydrophobins) that were not present on either blastospores or submerged conidia. The variations in the cell surface properties leading to the different adhesion qualities of B. bassiana aerial conidia, blastospores, and submerged conidia could lead to rational design decisions for improving the efficacy and possibly the specificity of entomopathogenic fungi for host targets.     

红火蚁病原真菌分离鉴定及致病性研究

红火蚁Solenopsis invicta是世界上最具破坏力和威胁性的入侵检疫害虫之一,对农林业生产安全、公共设施以及人体健康都造成了巨大影响。为筛选对红火蚁有致病性的病原真菌,本研究通过组织分离法对自然罹病的红火蚁进行致病菌分离,结合病原形态学及分子生物学的方法对病原真菌进行鉴定并采用喷雾法测定了病原真菌对红火蚁的致病力。结果表明：从罹病红火蚁上分离出3株病原真菌HHYSFJ01、HHYSFJ02和HHYSFJ05,分别为红绶曲霉Aspergillus nomius、淡紫拟青霉Purpureocillium lilacinum和球孢白僵菌Beauveria bassiana。致病性测定,这3株病原菌对红火蚁均具有较强的致病性,且3株病原菌致病性间差异显著。经1.0×10^8孢子/mL孢子悬浮液喷雾处理红火蚁10 d后,球孢白僵菌菌株HHYSFJ05的致病性最强,红火蚁工蚁的校正死亡率为84.71%,LT50为4.67 d;红绶曲霉菌株HHYSFJ01和淡紫拟青霉菌株HHYSFJ02次之,红火蚁工蚁的校正死亡率分别为78.82%和73.29%,LT50分别为4.99 d和5.20 d。     

Selection of an Isolate of the Insect Pathogenic Fungus Metarhizium anisopliae Virulent to the Lettuce Root Aphid, Pemphigus bursarius

The virulence of hyphomycete entomopathogenic fungi was measured in laboratory bioassays against the lettuce root aphid, Pemphigus bursarius, a serious pest of field lettuce grown in the UK. Of 25 isolates of fungi examined, only one isolate, Metarhizium anisopliae 391.93, killed lettuce root aphids consistently. This fungus was isolated originally from the closely related saltmarsh aphid, P. trehernei. The median lethal concentration of conidia at 10 days post6- 1 inoculation estimated from five independent bioassays was 2.45 × 10⁶ conidia ml^-1. The fungus had no significant effect on the mean number of offspring/aphid produced, but it sporulated 6 profusely on host cadavers, producing approximately 4 × 10⁶ conidia/cadaver 14 days after treatment, and diseased aphids died attached to plant roots. It thus has the potential to spread through densely packed colonies of P. bursarius feeding on the roots of susceptible or partially resistant plants.