The genus Entomophthora: bringing the insect destroyers into the twenty-first century

The fungal genus Entomophthora consists of highly host-specific pathogens that cause deadly epizootics in their various insect hosts. The most well-known among these is the “zombie fly” fungus E. muscae, which, like other Entomophthora species, elicits a series of dramatic behaviors in infected hosts to promote optimal spore dispersal. Despite having been first described more than 160 years ago, there are still many open questions about Entomophthora biology, including the molecular underpinnings of host behavior manipulation and host specificity. This review provides a comprehensive overview of our current understanding of the biology of Entomophthora fungi and enumerates the most pressing outstanding questions that should be addressed in the field. We briefly review the discovery of Entomophthora and provide a summary of the 21 recognized Entomophthora species, including their type hosts, methods of transmission (ejection of spores after or before host death), and for which molecular data are available. Further, we argue that this genus is globally distributed, based on a compilation of Entomophthora records in the literature and in online naturalist databases, and likely to contain additional species. Evidence for strain-level specificity of hosts is summarized and directly compared to phylogenies of Entomophthora and the class Insecta. A detailed description of Entomophthora’s life-cycle and observed manipulated behaviors is provided and used to summarize a consensus for ideal growth conditions. We discuss evidence for Entomophthora’s adaptation to growth exclusively inside insects, such as producing wall-less hyphal bodies and a unique set of subtilisin-like proteases to penetrate the insect cuticle. However, we are only starting to understand the functions of unusual molecular and genomic characteristics, such as having large > 1 Gb genomes full of repetitive elements and potential functional diploidy. We argue that the high host-specificity and obligate life-style of most Entomophthora species provides ample scope for having been shaped by close coevolution with insects despite the current general lack of such evidence. Finally, we propose six major directions for future Entomophthora research and in doing so hope to provide a foundation for future studies of these fungi and their interaction with insects.

Infection of the Western Flower Thrips, Frankliniella occidentalis, by the Insect Pathogenic Fungus Beauveria bassiana

The western flower thrips, Frankliniella occidentalis, is an aggressive agricultural insect pest causing significant damage to a wide range of fruit, vegetable, and ornamental crops. Beauveria bassiana is a broad-host-range entomopathogenic fungus capable of infecting and killing F. occidentalis. Infection of thrips by B. bassiana strain BbYT12 using a concentration of 1 × 108 conidia/mL resulted in 81.48% mortality in adults in 6 d (LT50 = 90 ± 15.1 h). Scanning electron microscopy of the infection process revealed preferential adhesion and germination of fungal spores to inter-segmental folds or grooves on the insect body surface with penetrating germlings and extended hyphae visualized during the initial stages of infection (6–24 h). Histological analyses showed the appearance of in vivo hyphal bodies in sagittal sections and the fat body as early as 24 h post-infection. Within 72 h, hyphal bodies and hyphae could be found throughout the infected organism including in the midgut, Malphigian tubules, alimentary canal, ovarioles (in females), and an extended hyphal network could be seen on insect cadavers (>72 h post-infection). Real-time RT-PCR analyses of the expression of select genes implicated in virulence including the Pr1 protease, beauvericin synthase, involved in the production of the secondary metabolite beauvericin, two cytochrome P450 monooxygenases implicated in cuticular hydrocarbon degradation, two multidrug efflux proteins, a perilipin involved in lipid storage, and the Hog1 MAP kinase and protein kinase A signaling factors revealed discrete patterns of infection-time dependent expression. These data provide basic insights into the process of B. bassiana infection of F. occidentalis.

Interaction between Metarhizium anisopliae and Its Host, the Subterranean Termite Coptotermes curvignathus during the Infection Process

Metarhizium anisopliae (Metchnikoff) Sorokin, a pathogenic fungus to insects, infects the subterranean termite, Coptotermes curvignathus Holmgren, a devastating pest of plantation trees in the tropics. Electron microscopy and proteomics were used to investigate the infection and developmental process of M. anisopliae in C. curvignathus. Fungal infection was initiated by germ tube penetration through the host’s cuticle as observed at 6 h post-inoculation (PI), after which it elongated into the host’s integumental tissue. The colonization process continued as seen from dissemination of blastospores in the hemocoel at 96 h PI. At this time point, the emergent mycelia had mummified the host and forty-eight hours later, new conidia were dispersed on the termites’ body surface. Meanwhile, hyphal bodies were observed in abundance in the intercellular space in the host’s body. The proteomes of the pathogen and host were isolated separately using inoculated termite samples withdrawn at each PI-time point and analyzed in two-dimensional electrophoresis (2-DE) gels. Proteins expressed in termites showed evidence of being related to cell regulation and the immune response, while those expressed in M. anisopliae, to transportation and fungal virulence. This study provides new information on the interaction between termites and its entomopathogen, with potential utilization for developing future biopesticide to control the termite population.

Seasonal prevalence and histopathology of Beauveria bassiana infecting larvae of the leopard moth, Zeuzera pyrina L. (Lepidoptera: Cossidae)

The natural epizootic potential of the fungus, Beauveria bassiana, against larvae of the leopard moth, Zeuzera pyrina L. (Lepidoptera: Cossidae) was investigated under natural conditions in two different locations for two seasons (2015–2016 and 2016–2017) in Rasheed, Behiara Governorate, Egypt. In an orchard, located in the old cultivated land, Beauveria infected 1.9–5.6% of larvae of Z. pyrina in autumn (September to November), increased to 3.4–7.2% in winter (December to February). The infection did not exceed 3.1% in spring (March to May), and no infection was recorded in summer (June to August). While in an olive farm, located in a newly reclaimed land, in both seasons of survey, no infections were recorded from May to September, while in winter, the epizootics ranged from 1.4–3.1%, from 0.4 to 1.3% in spring, and from 0.3 to 2.1% in autumn. Histopathological analysis revealed that Beauveria most commonly penetrated larvae directly through the intersegmental integument and the first symptoms were observed 3 to 4 days later. Hyphae radiated inward from the point of ingress and the fungus developed sparsely within the body cavity prior to the death of the larva. Hyphal bodies were concentrated around the point of ingress, but some were found at distance from this site. Only in the vicinity of penetration, the hypodermis showed signs of histolysis. The saprophytic phase developed rapidly and the fungus invaded all internal organs. The conidiophores seemed to rupture the cuticle by mechanical pressure and emerged all over the host cadaver, with sporulation occurring 48 h later. Pathological changes in the hemolymph appeared to be the primary cause of larval mortality.

Artificial Cultivation of the Chinese Cordyceps From Injected Ghost Moth Larvae

The Chinese cordyceps, regarded as the ‘Himalayan Viagra’, is highly valued for its medicinal benefits. The decline of its yield due to over-exploitation and increased market demand have stimulated efforts to artificially cultivate Chinese cordyceps for over half a century. However, successful cultivation of Chinese cordyceps through caterpillar infection by the fungus Ophiocordyceps sinensis (Berk.) and the induction of the fruiting body from each mummified cadaver remains difficult for its complex life cycle. Herein, we report the developmental dynamics of hyphal bodies in hemolymph of injected Thitarodes xiaojinensis (Tu, Ma & Zhang) larvae and the success in artificial cultivation of sexual fruiting bodies from the mummified cadavers in the low-altitude area. We find that not only the numbers of hyphal bodies but also the conversion of hyphal bodies into hyphae played important roles in the mummification of the injected larvae. This cultivation will be beneficial for sustainable utilization of natural resources and provides the possibility for further research on the mechanism of the interaction between pathogenic fungus and host insect.

First record of entomopathogenic fungus Entomophaga aulicae in the populations of browntail moth in Bosnia and Herzegovina

Browntail moth, is a well-known pest of broadleaf forests of Bosnia and Herzegovina. Although it is extremely polyphagous, it prefers to consume the leaves of various species of oaks. Browntail moth occurs periodically in high numbers (outbreak). Entomopathogenic fungus Entomophaga aulicae (Reichardt and Bail) Humber (Zygomycotina: Entomophtorales, Entomophtoraceae) is widespread Holarctic species, with many host insects from order Lepidoptera, where are some of the most economically harmful, outbreaking species of forest defoliators. In sessile oak forests of Eastern Bosnia and Herzegovina, the population density of browntail moth was determined by using route measurement during the growing season in the period 2015-2016. Browntail moth newly litters (40) were collected in four oak stands located in the region of Foča, Višegrad and Rogatica (PE Forests of the Republic of Srpska, Forest Estates Maglić, Panos and Sjemeć). In the litters, there were an average of 3,1 of dead old caterpillars and 4.7 pupae.The evaluation of E. aulicae infections was recorded as positive when hyphal bodies, primary conidia, or resting spores were detected on the surface of cadavers and puparia or in their tissues. The species identification was based on the size, shape and structural characteristics of different life forms of the fungus. By the microscopical studies of the causes of the mortality of the browntail moth larvae and pupae, the presence of hyphal bodies, primary conidia and resting spores of the E. aulicae were confirmed in them. The dimension of the resting spores (n=257) are 32.4 - 48.5 µm, a.v. 44.1 µm, primary conidia (n=54) 26.7-38.6 x 21.0-43.1 µm, a.v. 34.1-29.3 µm. Hyphal bodies were not measured. As entomopathogenic fungus on two development stages of the host, larvae and pupae, presented results indicate that E. aulicae is a promising microbial control agent.

Histopathological events and detection of Metarhizium anisopliae using specific primers in infected immature stages of the fruit fly Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae)

The fungus Metarhizium anisopliae is used on a large scale in Brazil as a microbial control agent against the sugar cane spittlebugs, Mahanarva posticata and M. fimbriolata (Hemiptera., Cercopidae). We applied strain E9 of M. anisopliae in a bioassay on soil, with field doses of conidia to determine if it can cause infection, disease and mortality in immature stages of Anastrepha fraterculus, the South American fruit fly. All the events were studied histologically and at the molecular level during the disease cycle, using a novel histological technique, light green staining, associated with light microscopy, and by PCR, using a specific DNA primer developed for M. anisopliae capable to identify Brazilian strains like E9. The entire infection cycle, which starts by conidial adhesion to the cuticle of the host, followed by germination with or without the formation of an appressorium, penetration through the cuticle and colonisation, with development of a dimorphic phase, hyphal bodies in the hemocoel, and death of the host, lasted 96 hours under the bioassay conditions, similar to what occurs under field conditions. During the disease cycle, the propagules of the entomopathogenic fungus were detected by identifying DNA with the specific primer ITSMet: 5' TCTGAATTTTTTATAAGTAT 3' with ITS4 (5' TCCTCCGCTTATTGATATGC 3') as a reverse primer. This simple methodology permits in situ studies of the infective process, contributing to our understanding of the host-pathogen relationship and allowing monitoring of the efficacy and survival of this entomopathogenic fungus in large-scale applications in the field. It also facilitates monitoring the environmental impact of M. anisopliae on non-target insects.

Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence

ABSTRACT Beauveria bassiana is an important entomopathogenic fungus widely used as a biological agent to control insect pests. A gene (B. bassiana JEN1 [BbJEN1]) homologous to JEN1 encoding a carboxylate transporter in Saccharomyces cerevisiae was identified in a B. bassiana transfer DNA (T-DNA) insertional mutant. Disruption of the gene decreased the carboxylate contents in hyphae, while increasing the conidial yield. However, overexpression of this transporter resulted in significant increases in carboxylates and decreased the conidial yield. BbJEN1 was strongly induced by insect cuticles and highly expressed in the hyphae penetrating insect cuticles not in hyphal bodies, suggesting that this gene is involved in the early stage of pathogenesis of B. bassiana. The bioassay results indicated that disruption of BbJEN1 significantly reduced the virulence of B. bassiana to aphids. Compared to the wild type, ΔBbJEN1 alkalinized the insect cuticle to a reduced extent. The alkalinization of the cuticle is a physiological signal triggering the production of pathogenicity. Therefore, we identified a new factor influencing virulence, which is responsible for the alkalinization of the insect cuticle and the initiation of fungal pathogenesis in insects.

Uptake of the fluorescent probe FM4-64 by hyphae and haemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana is under intensive study as a pest biological control agent. B. bassiana produces several distinct single-cell types that include aerial conidia, in vitro blastospores and submerged conidia. Under appropriate nutrient conditions these cells can elaborate germ tubes that form hyphae, which in turn lead to the formation of a fungal mycelium. In addition, B. bassiana displays a dimorphic transition, producing in vivo specific yeast-like hyphal bodies during growth in the arthropod haemolymph. The amphiphilic styryl dye FM4-64 was used to investigate internalization and morphological features of in vitro and in vivo insect haemolymph-derived B. bassiana cells. In vitro blastospores and submerged conidia displayed a punctate pattern of internal labelling, whereas aerial conidia failed to internalize the dye under the conditions tested. FM4-64 was also taken up into both apical and subapical compartments of living hyphae in a time-dependent manner, with clearly observable vesicle labelling. Internalization, where occurring, was reversibly disrupted by lowering the temperature of the assay or by treatment with azide/fluoride and latrunculin A. Treatment with cytochalasin D and monensin also caused abnormal vesicle trafficking, although some staining of vesicles was noted. Fungal cells derived from infected Heliothis virescens haemolymph (in vivo cells) actively internalized FM4-64. The in vivo blastospores or hyphal bodies displayed bright membrane and internal vesicle staining, although diffuse staining of internal structures was also visible. These results suggest active uptake by different developmental stages of B. bassiana, including haemolymph-derived cells that can evade the insect immune system.

Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host–pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simplicifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.