First record of Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) in Slovakia

The entomopathogenic fungus Entomophaga maimaiga was found for the first time in Slovakia in 2013. Late instar larvae of gypsy moth, Lymantria dispar, from two sites with different population densities were dissected to evaluate the presence of pathogens. The presence of conidia and resting spores of E. maimaiga in gypsy moth cadavers was confirmed from both sites.     

First record of Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) in Georgia

In 2005, high levels of mortality occurred in an outbreak of the gypsy moth population in Georgia. Resting spores typical of entomophthoralean fungi were found within larval cadavers and molecular analyses confirmed that the pathogen was Entomophaga maimaiga. This is the first record of this entomopathogen in Georgia and in this part of Europe.     

Different susceptibility of indigenous populations of Lymantria dispar to the exotic entomopathogen Entomophaga maimaiga

The recovery of the host‐specific entomopathogen Entomophaga maimaiga is still limited to certain world areas, although it is recently spreading to Eastern Europe. This study evaluated the effectiveness and fitness of an E. maimaiga isolate from Balkans against Lymantria dispar populations collected along the Italian peninsula and main islands, where the fungus has never been reported. As a result of different bioassays, the pathogenicity against gypsy moth larvae was generally confirmed, although significant differences among insects feeding upon diverse forest plant species were observed. The lack of significant susceptibility of other lepidopteran species from the same areas is also reported.     

The potential for Entomophaga maimaiga to regulate gypsy moth Lymantria dispar (L.) (Lepidoptera: Erebidae) in Europe

Gypsy moth, Lymantria dispar L., is one of the most important pests of deciduous trees in Europe. In regular cycles, it causes large‐scale defoliation mostly of oak, Quercus spp., forests. Government authorities in the most infested countries in Europe conduct large‐scale applications of pesticides against gypsy moth. In 1999, a new natural enemy, the entomopathogenic fungus Entomophaga maimaiga, was successfully introduced into a gypsy moth population in Bulgaria. Recent investigations suggest that now E. maimaiga is quickly spreading in Europe. Herein, past studies are reviewed regarding this fungus with special emphasis on its potential for becoming an important factor regulating gypsy moth populations in Europe, focusing on the host's population dynamics in relation to the fungus, the influence of environmental conditions on fungal activity, the influence of E. maimaiga on the native entomofauna, including other gypsy moth natural enemies, and spread of the fungus. Based on this analysis, the potential of E. maimaiga for providing control in European gypsy moth populations is estimated.     

The genome ofEntomophaga aulicae (Entomophthorales,Zygomycetes): Base composition and size

The DNA of the fungusEntomophaga aulicae was examined to obtain preliminary information on the genetic apparatus of a potentially important biological control agent and to investigate the hypothesis that the presence of an extensive amount of condensed chromatin in its nucleus is correlated with an unusually large genome. From thermal denaturation studies the base composition of the DNA was determined to be 38% G + C. The DNA content of protoplast nuclei was determined by two independent techniques. The results indicate that the haploid genome size ofE. aulicae is two orders of magnitude greater than that generally attributed to members of the Fungi. This is supported by ultrastructural observations which show large amounts of condensed chromatin in these nuclei. Further ultrastructural evidence suggests that a large mitochondrial DNA pool, polyploidy, and extensive differential replication or gene amplification in protoplast nuclei do not account for the high DNA values reported here.     

Distribution of resting spores of the Lymantria dispar pathogen Entomophaga maimaiga in soil and on bark

Cadavers of late instar Lymantria dispar (gypsy moth) larvae killed by the fungal pathogen Entomophaga maimaiga predominantly contain resting spores (azygospores). These cadavers frequently remain attached to tree trunks for several weeks before they detach and fall to the ground. Density gradient centrifugation was used to quantify resting spores in the soil and on tree bark. Titers of resting spores were extremely high at 0–10 cm from the base of the tree and the number decreased with distance from the trunk of the tree. Titers were also highest in the organic layer of the soil with numbers decreasing precipitously with increasing depth in the soil. While resting spores were obtained from tree bark, densities per unit area were much lower than those found in the organic soil layer at the base of the tree. Field bioassays were conducted with caged L. dispar larvae to compare infection levels with distance from the tree trunk as well as on the trunk. Highest infection levels were found at 50cm from the tree base with lowest infection on the tree trunk at 0.5 m height, although we expected the highest infection levels among larvae caged at the bases of trees, where highest spore titers occurred. Laboratory experiments demonstrated that L. dispar larvae exposed to resting spore- bearing soil at the soil surface became infected while larvae exposed to soil with resting spores buried at least 1 cm below the surface did not become infected.     

Epizootics and behavioral alteration in the arctiid caterpillar Chionarctia nivea (Lepidoptera: Arctiidae) caused by an entomopathogenic fungus, Entomophaga aulicae (Zygomycetes: Entomophthorales)

We observed epizootics and behavioral alteration in the arctiid caterpillar Chionarctia nivea after infection by an entomopathogenic fungus, Entomophaga aulicae, in April at a riverbank in Kyoto Prefecture, central Japan. The density of arctiid cadavers infected with E. aulicae was 1.31 individuals/m². The critically ill caterpillars crawled up the dead stems of grasses and herbs such as the common reed Phragmites communis and the Japanese mugwort Artemisia indica var. maximowiczii to die at and near the highest parts of the stems, while the healthy larvae usually wandered on the ground and fed on the leaves of small herbs. This behavioral difference could be caused by infection with E. aulicae to enhance dispersal of conidia by active discharge in Entomophthorales with the aid of wind and rainfall.     

Deposition and germination of conidia of the entomopathogen Entomophaga maimaiga infecting larvae of gypsy moth,Lymantria dispar

Germination of conidia of Entomophaga maimaiga, an important fungal pathogen of gypsy moth, Lymantria dispar, was investigated on water agar and larval cuticle at varying densities. Percent germination was positively associated with conidial density on water agar but not on larval cuticle. When conidia were showered onto water agar, the rate of germination was much slower than on the cuticle of L. dispar larvae. From the same conidial showers, the resulting conidial densities on water agar were much higher than those on larval cuticle in part because many conidia adhered to setae and did not reach the cuticle. A second factor influencing conidial densities on larval cuticle was the location conidia occurred on larvae. Few conidia were found on the flexible intersegmental membranes in comparison with the areas of more rigid cuticle, presumably because conidia were physically dislodged from intersegmental membranes when larvae moved. Conidia were also exposed to heightened CO(2) to evaluate whether this might influence germination. When conidia on water agar were exposed to heightened CO(2) levels, germinating conidia primarily formed germ tubes while most conidia exposed to ambient CO(2) rapidly formed secondary conidia.     

Effect of conidial dispersal of the fungal pathogen Entomophaga maimaiga (Zygomycetes: Entomophthorales) on survival of its gypsy moth (Lepidoptera: Lymantriidae) host

Several researchers have developed a one-generational computer model that simulates infection prevalence of gypsy moth, Lymantria dispar, caterpillars by its fungal pathogen, Entomophaga maimaiga. Inputs required are temperature, humidity, and rainfall records, a measure of fungus resting spore load in the soil, and an estimate of gypsy moth larval density. In a previous study, the model accurately tracked fungal-induced host mortality as long as airborne fungal conidia were allowed to disperse freely over a local area. In 2002, dispersal of conidia and its influence on the impact of the fungus on the gypsy moth was investigated. Gypsy moth densities and fungus resting spore loads were measured in 15 plots within a 3 km area. In 7 of the plots, prevalence of fungal disease was determined weekly by collecting and rearing gypsy moth larvae. Different strategies were used to disperse conidia within the model, and resulting simulated prevalence rates were compared to actual data. Model output was most accurate when airborne conidia were permitted to disperse equally to all plots. Thus, to accurately assess the impact of the fungus in one location, it is necessary to take into account fungal activity throughout the local area.     

Distribution of the entomopathogenic fungus Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) at the northern edge of its range in Europe

The gypsy moth Lymantria dispar is a serious economic pest in European broadleaf forests. However, the entomopathogenic fungus Entomophaga maimaiga, which has a great potential to regulate gypsy moth numbers, has recently spread in the Central and Eastern European area of the moth's range. In the current study, 39 plots in oak forests in the Slovak Republic and the Czech Republic were monitored for E. maimaiga from 2014 to 2016. These plots were located along the northern edge of the E. maimaiga range where gypsy moth outbreaks have occurred in the past. The fungus was detected in 16 of the 39 plots. The results thus confirm that E. maimaiga is quite widespread along the northern edge of its range in Europe and can be considered to be established in that area.     

Diurnal pattern of death and sporulation in Entomophaga maimaiga-infected Lymantria dispar

Entomophaga maimaiga Humber, Shimazu, et Soper (Zygomycotina: Entomophthoraceae) is a naturally occurring obligate fungal pathogen specific to gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae) larvae. This fungus is considered the most important natural enemy of this pest insect in North America and Asia. A critically important step for the development of E. maimaiga epizootics is the transmission of propagules to healthy larvae, a process known to require high humidity. Some pathogens are known to manipulate the time of day that hosts die so that propagules are produced to maximize chances of survival and thus enhance transmission. The objective of this study was to assess whether E. maimaiga manipulates L. dispar to die at a certain time of day. Laboratory bioassays were conducted at 15 and 20 °C to record the 24‐h activity pattern of death and sporulation exhibited under an L14:D10 photoperiod and 100% r.h. by four isolates of E. maimaiga in its host L. dispar. Events were recorded every 4 h. Our results clearly demonstrate that E. maimaiga‐infected L. dispar larvae die mainly in the afternoon and that the fungus sporulates during the night. The rhythm was independent of the fungal isolate tested and type of spores produced after larval death. By raising the temperature from 15 to 20 °C, the peak death time narrowed and sporulation was initiated earlier at night.     

Pathogenic fungi of insects from Argentina (Zygomycetes: Entomophthorales)

Pathogenic fungi of insects from Argentina (Zygomycetes: Entomophthorales). Three species of Entomophthorales entomopathogenic fungi (Zygomycotina: Zygomycetes) have been identified from insects in agricultural crops (Buenos Aires Province, Argentina): Zoophthora radicans Batko (Brefeld); Entomophthora planchoniana Cornu and Pandora gammae (Weiser) Humber. Fungal structure measurements are reported.     

Virulence and fitness of the fungal pathogen Entomophaga maimaiga in its host Lymantria dispar, for pathogen and host strains originating from Asia, Europe, and North America

In this study, we tested (1) whether non-North American gypsy moth strains are susceptible to North American isolates of Entomophaga maimaiga and (2) the potential for erosion in the efficacy of E. maimaiga in controlling gypsy moth. We used bioassays to assess the variability in virulence (measured as time to death) as well as fitness of the pathogen (measured as spore production) in four gypsy strains challenged with six E. maimaiga isolates, using host and pathogen strains originating from Asia, Europe, and North America. We found that all E. maimaiga isolates tested were pathogenic to all strains of Lymantria dispar, regardless of the geographical origin of the fungal isolate, with at least 86% mortality for all combinations of fungal isolate and gypsy moth strain. We therefore conclude that Asian gypsy moths are susceptible to North American strains of E. maimaiga. No significant interactions between fungal isolates and gypsy moth strains with regard to time to death were found, indicating that each fungal isolate had the same overall effect on all the gypsy moth strains tested. However, fungal isolates differed significantly with regard to virulence, with a Russian isolate being the slowest to kill gypsy moth (5.1+/-0.1 days) and a Japanese isolate being the overall fastest to kill its host (4.0+/-0.1 days). Fungal isolates also differed in fitness, with variability in types of spores produced. These differences in virulence and fitness were, however, not correlated with geographical origin of the fungal isolate. Gypsy moth strains had no or only little effect on fungal virulence and fitness. Based on our studies with laboratory-reared gypsy moth strains, erosion of successful control of gypsy moth by E. maimaiga seems unlikely.     

Pathology and Epizootiology of Entomophaga maimaiga Infections in Forest Lepidoptera

The insect-pathogenic fungal pathogen Entomophaga maimaiga is endemic to northeastern Asia and was first found in North America in 1989. Due to repeated epizootics and spread within populations of the major forest defoliator in northeastern North America, the gypsy moth (Lymantria dispar), this pathogen has gained much notoriety. Although this pathogen was purposely introduced to North America for biological control of L. dispar in 1910 to 1911, it is questionable whether it became established at the time of release and then remained at innocuous levels until relatively recently. Alternatively, the fungal strain present in North America today could be a more recent accidental introduction. DNA analysis demonstrates that this pathogen differs significantly from North American members of the same species complex (the Lepidoptera-specific Entomophaga aulicae species complex), and, to date, isolates of this introduced pathogen display little heterogeneity in North America. Nonsusceptible lepidopteran larvae have been identified, and either E. maimaiga is unable to penetrate the cuticle or the fungus cannot survive within the hemocoel. In the latter case, although E. maimaiga grows as protoplasts lacking cell walls in the host hemolymph, glycoproteins on plasma membranes of the protoplasts could lead to host recognition. Epizootiological studies demonstrate a clear association between fungal activity and environmental moisture but little association with host density under hypothesized conditions of high fungal density. Prediction of the occurrence of epizootics is not yet possible. E. maimaiga is easily established in new areas by releasing azygospores, but the ability to use this pathogen further for biological control will depend, in large part, on the development of mass production systems.     

Survival of Neozygites cf. floridana (Zygomycetes: Entomophthorales) in mummified cassava green mites and the viability of its primary conidia

The survival of Neozygites cf. floridana (Weiser and Muma) as dry hyphal bodies in mummified cassava green mites, Mononychellus tanajoa (Bondar), at 5.0% RH in the dark was affected by storage temperature. Survival of the fungus in mummies kept at 24±1.0°C could be demonstrated for 6–7 months. When stored at 4°C, the fungus sporulated from 90% of the mummies liberating an average of 186.9 primary conidia per mummy even after a storage period of 16 months, when the experiment was terminated. The temperature, humidity and light condition significantly affected the viability of primary conidia. The percent viability across all factors dropped from 98.4% after 0 h (beginning of the experiment) to 23.4% after a 1 h exposure to the conditions tested. Lower temperatures maintained higher viabilities with 86.3% of the conidia surviving after 18 h at 18°C, whereas almost all conidia died after 12 h at 33°C. Conidia survived less than 1 h when exposed to SDs (saturation deficit) of 2.0 mm Hg or higher at any tested temperature.     

Formulation and application of the entomopathogenic fungus: Zoophthora radicans (Brefeld) Batko (Zygomycetes: Entomophthorales)

Aims: To isolate and formulate a native strain of Zoophthora radicans naturally infecting larvae of diamondback moth, Plutella xylostella, existing in South Australia and to provide evidence that formulation of the fungus is effective against P. xylostella larvae, and therefore, it could be used as a tool in pest management of this insect. Methods and Results: Dose–response bioassays using formulated and unformulated forms of the fungus strain were carried out against third instar larvae of P. xylostella. Results obtained have indicated a significant increase in the larval mortality when higher concentrations of a formulated form of the fungus strain were applied compared to the treatments with the unformulated form (85·0 vs 57·5% of larval mortality, respectively, at the top concentration of 10⁷ conidia/ml). The median lethal concentration (LC50) for a formulated form was 100 times less than that of the unformulated form when they were applied against the third instar larvae of P. xylostella. In addition, the formulation used in the present bioassays has preserved the viability of introduced fungus conidia for longer time in comparison with the unformulated conidia. Conclusions: The effective application of a formulated fungus strain against P. xylostella larvae constitutes the first step towards its use in pest management of this insect. Significance and Impact of the Study: The formulated fungus in inverted emulsion could be used as an alternative tool to insecticides in pest management of P. xylostella larvae because of the development of resistance to insecticides in the treated larvae.     

Inheritance of female flight in Lymantria dispar (Lepidoptera: Lymantriidae)

A clinal female flight polymorphism exists in the gypsy moth, Lymantria dispar L., where female flight diminishes from east to west across Eurasia. A Russian population where females are capable of sustained ascending flight and a North American population with females incapable of flight were crossed: parentals, reciprocal F(1) hybrids, double reciprocal F(2) hybrids, and all possible backcrosses to both the parental lines were compared. Heritabilities were estimated using a threshold model, female offspring on female parent regressions, and joint-scaling analyses. Heritability of female flight capability measured using a free flight test was at least 0.60, and variation in wing size, muscle strength, and flight behaviors contributed to the flight polymorphism. Relative wing size varied continuously and had a heritability of 0.70. Environmental variation accounted for >90% of the variation in female preflight weight and relative flight muscle strength, as estimated by an inverted female's ability to right herself. Preflight walking behavior and early deposition of eggs were each inherited through a single gene with two co-dominant alleles. There was no evidence for sex-linkage or maternal effects in female flight capability or associated traits. Continued vigilance to exclude and eradicate introductions of strains capable of female flight in North America is warranted even in areas where no females fly, because some of the alleles needed for full flight capability may not be present in the North American populations, and some flight capability is maintained in the hybrids that could increase the rate of spread of L. dispar.     

Entomophthora ferdinandii (Zygomycetes: Entomophthorales) Causing Natural Infections of Musca domestica (Diptera: Muscidae) in Argentina

The identity and activity of an entomopathogenic fungus belonging to the Entomophthora muscae species complex and infecting Musca domestica in poultry houses from La Plata, Argentina, is reported. Entomophthora caused natural infections between September 2001 and September 2003. Primary conidia of this fungus were on average 29.5 ± 1.2 × 23.4 ± 2.4 μm and contained, on average, 10.5 ± 0.1 nuclei (range: 7–15) with an average diameter of 4.8 ± 0.1 μm. This fungus is identified as E. ferdinandii Keller (this specific epithet includes a nomenclaturally required spelling correction); this is a first record of E. ferdinandii in South America and of any member of the E. muscae species complex from flies in Argentina.