First report of Colletotrichum fioriniae infections in brown marmorated stink bugs, Halyomorpha halys.

An epizootic caused by fungal pathogens occurred among Halyomorpha halys, brown marmorated stink bugs, while they were overwintering, with infections also occurring after overwintering. We report that one of the two pathogens responsible was Colletotrichum fioriniae (Marcelino & Gouli) Pennycook; a species well known as a plant pathogen and endophyte and which has only previously been reported naturally infecting elongate hemlock scales, Fiorinia externa. To prove pathogenicity, H. halys adults challenged with conidia died from infections and the fungus subsequently produced conidia externally on cadavers.

Entomopathogens infecting brown marmorated stink bugs before, during, and after overwintering.

The microsporidian, Nosema maddoxi Becnel, Solter, Hajek, Huang, Sanscrainte & Estep, infects brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), populations in North America and Asia and causes decreased fitness in infected insects. This host overwinters as adults, often in aggregations in sheltered locations, and variable levels of mortality occur over the winter. We investigated pathogen prevalence in H. halys adults before, during, and after overwintering. Population level studies resulted in detection of N. maddoxi in H. halys in 6 new US states, but no difference in levels of infection by N. maddoxi in autumn versus the following spring. Halyomorpha halys that self-aggregated for overwintering in shelters deployed in the field were maintained under simulated winter conditions (4°C) for 5 months during the 2021-2022 winter and early spring, resulting in 34.6 ± 4.8% mortality. Over the 2020-2021 and 2021-2022 winters, 13.4 ± 3.5% of surviving H. halys in shelters were infected with N. maddoxi, while N. maddoxi infections were found in 33.4 ± 10.8% of moribund and dead H. halys that accumulated in shelters. A second pathogen, Colletotrichum fioriniae Marcelino & Gouli, not previously reported from H. halys, was found among 46.7 ± 7.8% of the H. halys that died while overwintering, but levels of infection decreased after overwintering. These 2 pathogens occurred as co-infections in 11.1 ± 5.9% of the fungal-infected insects that died while overwintering. Increasing levels of N. maddoxi infection caused epizootics among H. halys reared in greenhouse cages after overwintering.

Batkoa major infecting the invasive planthopper Lycorma delicatula.

The entomopathogenic fungi Batkoa major and Beauveria bassiana caused co-epizootics in populations of invasive spotted lanternflies, Lycorma delicatula, in 2018 in northeastern North America. Although first described from North America in 1888, the biology and ecology of Batkoa major had not been studied since that time. This entomophthoralean fungus found infecting L. delicatula in 2018 produces conidia and rhizoids similar in appearance to the original description. We conducted laboratory bioassays to investigate infection of different ages and sexes of these planthoppers, inoculating via showered conidia. All nymphs, and male and female adults were susceptible, dying in 4.3-6.7 days. Adult males died more quickly than adult females or fourth instars. Batkoa major grew out of cadavers of adult males more frequently than adult females or fourth instar nymphs. Rhizoids that provide attachment of cadavers to substrates were produced from adult cadavers more frequently than conidia. Resting spores were not observed in vivo or in vitro in the lab, or in the field.

A pair of native fungal pathogens drives decline of a new invasive herbivore.

Two North American fungal pathogens caused a coepizootic leading to localized collapse of an outbreak population of the newly invasive planthopper pest, the spotted lanternfly (Lycorma delicatula), in the eastern United States. The pathogens partitioned the habitat, with the majority of L. delicatula on tree trunks killed by Batkoa major, while cadavers of L. delicatula killed by Beauveria bassiana were usually on the ground. The future will show whether these pathogens will be drivers in boom-bust cycles or will result in recurrent low population densities of this new invasive species.

Histologic lesions of experimental infection with Lymantria dispar multicapsid nucleopolyhedrovirus and Lymantria dispar cytoplasmic polyhedrosis virus in European gypsy moth caterpillars (Lymantria dispar dispar).

European gypsy moths (Lymantria dispar dispar) are an invasive species in North America, and are listed by the International Union for the Conservation of Nature as one of the 100 most destructive invasive species worldwide. They have several known viruses, some of which are used as biological control agents. However, there are no detailed descriptions of many entomopathogenic viral infections, including in European gypsy moths, using bright-field microscopy. In this study, 11 European gypsy moth caterpillars were evaluated histologically: 4 were experimentally infected with Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV; Baculoviridae); 4 were experimentally infected with Lymantria dispar cytoplasmic polyhedrosis virus (LdCPV; Reoviridae); 3 control animals were uninfected. A complete tissue set was evaluated in all animals from all groups using bright-field microscopy, including epidermis, cuticle, striated muscle, tracheae, foregut, midgut, hindgut, Malpighian tubules, hemocytes, fat body, and nervous system. LdMNPV-infected caterpillars had marked karyomegaly and intranuclear viral inclusions in cells of the epidermis, tracheae, fat body, and hemocytes. LdMNPV-infected caterpillars also had hyperplasia and hypertrophy of epidermal and tracheal epithelial cells. LdCPV-infected caterpillars had numerous granular eosinophilic intracytoplasmic viral inclusions in midgut epithelial cells. Both LdMNPV-infected and LdCPV-infected caterpillars had atrophy of fat body adipocytes; this change was more pronounced in LdCPV-infected caterpillars. This work provides the first detailed descriptions of these viral infections in European gypsy moth caterpillars using bright-field light microscopy and provides images of normal histology from control caterpillars.

Discovery of two hypocrealean fungi infecting spotted lanternflies, Lycorma delicatula: Metarhizium pemphigi and a novel species, Ophiocordyceps delicatula.

In the eastern United States, populations of the invasive spotted lanternfly, Lycorma delicatula, can be infected by native fungal entomopathogens, including Batkoa major and Beauveria bassiana. In some areas of southeastern Pennsylvania, localized population collapses have been observed in L. delicatula populations to be caused by these pathogens. Two additional fungal pathogens were discovered infecting L. delicatula at low levels, and these were identified as Metarhizium pemphigi and Ophiocordyceps delicatula, a new species that has not been previously described. Therefore, four species of native entomopathogenic fungi have now been documented infecting this abundant, invasive planthopper that is spreading in the United States.

Genetic variability among native and introduced strains of the parasitic nematode Deladenus siricidicola.

Sirex noctilio is an invasive Eurasian woodwasp that can kill pine (Pinus spp.) trees and has been introduced to areas of the Southern Hemisphere where plantations of introduced pines are grown. The main method of control of this invasive pest has been introduction and augmentation of a parasitic nematode, Deladenus siricidicola. The strain of D. siricidicola used for biological control of S. noctilio in the Southern Hemisphere originated in Sopron, Hungary. The genotype of D. siricidicola used for biological control sterilizes females of the strain of S. noctilio present in Australia. However, different strains of S. noctilio have been introduced to different geographic areas that have been invaded and different combinations of D. siricidicola and S. noctilio genotypes vary in whether these nematodes sterilize female S. noctilio. Moreover, even in the event of sterilization, partial sterilization can occur, where not all woodwasp eggs are compromised. Sirex noctilio has now invaded North America accidentally, putatively accompanying D. siricidicola, but these host/parasite pairings do not result in female sterilization. More information is needed about the genetic diversity of D. siricidicola both where it is native and introduced. In addition, the host range of these nematodes is necessary to understand to evaluate their potential use in areas where pine communities are native. We collected and evaluated Deladenus parasitizing S. noctilio, S. juvencus, and associated insects in Hungary, Denmark, Spain, and Italy, as well as in the United States. Phylogenetic analyses were unable to fully provide fine resolution, although some community structure was evident. Many D. siricidicola samples from Hungary had identical COI and ITS sequences to the strain of D. siricidicola accidentally introduced to North America putatively when S. noctilio invaded. The same or similar strains of D. siricidicola parasitize two different Sirex species that utilize pines as well as a Sirex parasitoid and a pine-boring beetle, demonstrating some limited variability in host specificity of this species. These results highlight the genetic diversity of Deladenus siricidicola in its native range in Europe.

Impact of Nosema maddoxi on the survival, development, and female fecundity of Halyomorpha halys.

Nosema maddoxi Becnel, Solter, Hajek, Huang, Sanscrainte, & Estep, a microsporidian species native to the United States, has been found infecting the invasive brown marmorated stink bug, Halyomorpha halys (Stål). Microsporidian infections in insects often shorten lifespans, decrease fecundity, prolong development, and stunt growth. This study was conducted to determine the impact of N. maddoxi on H. halys fitness. Adult females (2 doses) and nymphs (1 dose) drank suspensions of N. maddoxi spores to promote infection. Adult females receiving a high dose died faster than the controls. Nosema maddoxi infections impacted female egg production and egg viability at both doses compared with the controls. Infections were transmitted to 34.9% of adult males caged with infected females. As the number of days after inoculation increased, infection intensity (# spores found within an infected individual) for both adult treatments transitioned from low-intensity to high-intensity. Infected nymphs died significantly sooner than the controls. Of the treated nymphs, 55.9% died before molting into the fourth instar and only 26.5% eclosed to adults. Nymphal development rate and size were not impacted by N. maddoxi infection. These results indicate that N. maddoxi infection can negatively impact the lifespan of adult females, female fecundity, egg viability, and nymphal survival, which we hypothesize would negatively impact H. halys population densities.

Virulence of Commercialized Fungal Entomopathogens Against Asian Longhorned Beetle (Coleoptera: Cerambycidae).

Nine strains of five species of entomopathogenic hypocrealean fungi were tested against adults of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky). These strains have been developed as commercial biopesticide products in the United States, Brazil, South Korea, or the European Union (EU). Metarhizium anisopliae (Metschnikoff) (Hypocreales: Clavicipitaceae) ESALQ E-9 and Metarhizium brunneum (Petch) F52 (formerly M. anisopliae F52) (Hypocreales: Clavicipitaceae) killed 100% of treated beetles with the shortest survival times. Virulence differed among the five strains of Beauveria bassiana (Balsamo) (Hypocreales: Cordycipitaceae) tested, ranging from 0 to 77.3% mortality within 28 d. Two Isaria fumosorosea (Wize, 1904) (Hypocreales: Cordycipitaceae) (formerly Paecilomyces fumosoroseus) strains and the Lecanicillium muscarium (Petch) Zare & Gams (Hypocreales: Cordycipitaceae) strain used in Mycotal were not pathogenic to A. glabripennis adults. Within the entomopathogenic fungi tested, the Metarhizium strains may be the most appropriate for further evaluation.

Asian longhorned beetle bioassays to evaluate formulation and dose-response effects of Metarhizium microsclerotia.

Asian longhorned beetles (ALB; Anoplophora glabripennis), are invasive wood borers susceptible to Metarhizium brunneum. This fungus can be prepared as dried microsclerotia which, after rehydration, produce infective conidia within weeks. Wood samples coated with formulated microsclerotia were attached to trees in the Ohio USA ALB-eradication zone and collected after 4-week periods. Adult ALB exposed to these samples had 100% mortality. In an experiment comparing formulations with or without humectant hydrogel, hydrogel did not significantly increase mortality of exposed ALB. In a dose-response experiment with 5 application rates, ALB survival decreased with increasing application rate and conidial density.

Nosema maddoxi sp. nov. (Microsporidia, Nosematidae), a Widespread Pathogen of the Green Stink Bug Chinavia hilaris (Say) and the Brown Marmorated Stink Bug Halyomorpha halys (Stål).

We describe a unique microsporidian species that infects the green stink bug, Chinavia hilaris; the brown marmorated stink bug, Halyomorpha halys; the brown stink bug, Euschistus servus; and the dusky stink bug, Euschistus tristigmus. All life stages are unikaryotic, but analysis of the consensus small subunit region of the ribosomal gene places this microsporidium in the genus Nosema, which historically has been characterized by diplokaryotic life stages. It is also characterized by having the reversed arrangement of the ribosomal gene (LSU -ITS- SSU) found in species within the "true Nosema" clade. This microsporidium is apparently Holarctic in distribution. It is present in H. halys both where it is native in Asia and where it is invasive in North America, as well as in samples of North American native C. hilaris collected prior to the introduction of H. halys from Asia. Prevalence in H. halys from mid-Atlantic, North America in 2015-2016 ranged from 0.0% to 28.3%, while prevalence in C. hilaris collected in Illinois in 1970-1972 ranged from 14.3% to 58.8%. Oral infectivity and pathogenicity were confirmed in H. halys and C. hilaris. Morphological, ultrastructural, and ecological features of the microsporidium, together with a molecular phylogeny, establish a new species named Nosema maddoxi sp. nov.

Symbiont Spillover from Invasive to Native Woodwasps.

Hosts and their associated microbes are being increasingly introduced around the world, which can lead to novel host/microbe associations via new sympatries. Woodwasps (Hymenoptera: Siricidae) are able to utilize wood for its nutrients due to obligate mutualistic associations with white rot fungi in the genus Amylostereum and when invasive woodwasps are introduced to new areas, their symbionts accompany them. There is increasing evidence that woodwasp-fungus associations previously believed to be highly specific are actually flexible. We show that in North America, both Urocerus albicornis and Urocerus cressoni, which develop in trees in the Pinaceae, usually use Amylostereum chailletii but sometimes carry an Amylostereum areolatum strain putatively introduced to North America by the invasive woodwasp Sirex noctilio. Symbiont spillover from invasive to native hosts is a source of new host/introduced symbiont associations that could result in changes in microbes and host fitness with the potential to impact communities.

Phytophagous larvae occurring in Central and Southeastern European oak forests as a potential host of Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) - A field study.

We evaluated the presence and impact of Entomophaga maimaiga on both target and non-target phytophagous larvae. All six study plots, with low gypsy moth population density, were situated in Central and Southeastern European oak forests and E. maimaiga had previously been reported from these plots. Totally, 45 of 4,045 (1.13%) collected non-target larvae died due to fungal infections. No non-target insect specimen was infected by E.maimaiga, although the presence of the pathogen could not be fully excluded in three cadavers. Out of 1,780L.dispar larvae collected, 15individuals (0.84%) were infected by E.maimaiga.

Modification of a Pollen Trap Design To Capture Airborne Conidia of Entomophaga maimaiga and Detection of Conidia by Quantitative PCR.

The goal of this study was to develop effective and practical field sampling methods for quantification of aerial deposition of airborne conidia of Entomophaga maimaiga over space and time. This important fungal pathogen is a major cause of larval death in invasive gypsy moth (Lymantria dispar) populations in the United States. Airborne conidia of this pathogen are relatively large (similar in size to pollen), with unusual characteristics, and require specialized methods for collection and quantification. Initially, dry sampling (settling of spores from the air onto a dry surface) was used to confirm the detectability of E. maimaiga at field sites with L. dispar deaths caused by E. maimaiga, using quantitative PCR (qPCR) methods. We then measured the signal degradation of conidial DNA on dry surfaces under field conditions, ultimately rejecting dry sampling as a reliable method due to rapid DNA degradation. We modified a chamber-style trap commonly used in palynology to capture settling spores in buffer. We tested this wet-trapping method in a large-scale (137-km) spore-trapping survey across gypsy moth outbreak regions in Pennsylvania undergoing epizootics, in the summer of 2016. Using 4-day collection periods during the period of late instar and pupal development, we detected variable amounts of target DNA settling from the air. The amounts declined over the season and with distance from the nearest defoliated area, indicating airborne spore dispersal from outbreak areas.IMPORTANCE We report on a method for trapping and quantifying airborne spores of Entomophaga maimaiga, an important fungal pathogen affecting gypsy moth (Lymantria dispar) populations. This method can be used to track dispersal of E. maimaiga from epizootic areas and ultimately to provide critical understanding of the spatial dynamics of gypsy moth-pathogen interactions.

Zombie soldier beetles: Epizootics in the goldenrod soldier beetle, Chauliognathus pensylvanicus (Coleoptera: Cantharidae) caused by Eryniopsis lampyridarum (Entomophthoromycotina: Entomophthoraceae).

Adult goldenrod soldier beetles, Chauliognathus pensylvanicus, were found infected by the fungus Eryniopsis lampyridarum (Entomophthoromycotina) in Arkansas during September - October (1996, 2001, 2015 and 2016). Living and dead infected beetles were found on flowering frost aster, Symphyotrichum pilosum, common boneset, Eupatorium perfoliatum, and Canada goldenrod, Solidago canadensis. Live and dead beetles (n=446) were collected in 1996 from S. pilosum flowers and held individually in the laboratory for determination of fungal prevalence. Of the beetles collected, 281 (63%) were males and 165 (37%) were females. A total of 90 beetles were infected with E. lampyridarum, an overall prevalence of 20.2%. Prevalence in males was 19.6% (n=55 infected/281 males total) and prevalence in females was 21.2% (n=35 infected /165 females total). Conidia were produced from 57% of the infected beetles, 23% of the infected beetles produced resting spores, and 20% contained the hyphal body stage. Infected beetles produced either conidia or resting spores but never both in the same host. Post-mortem morphological changes in the hosts due to E. lampyridarum were observed periodically for 24h. Shortly before death, by unknown mechanisms, dying infected beetles tightly clamped their mandibles into flower heads and ca. 15-22h later (between 2400 and 0700h) the fungus caused dead beetles to raise their elytra and expand their metathoracic wings.

The first entomophthoralean killing millipedes, Arthrophaga myriapodina n. gen. n. sp., causes climbing before host death.

A new species and genus of entomophthoralean fungus, Arthrophaga myriapodina kills polydesmid millipedes. This species was first seen over a century ago but never described. It is the first millipede pathogen known from the order Entomophthorales, species of which are best known as pathogens of a wide diversity of insects. The fungus induces pre-death climbing behavior in its hosts, enabling the fungus to broadcast its forcibly-discharged conidia from a high vantage, which presumably increases the fitness of the fungus. Study of herbarium specimens and photographic discoveries on the internet suggest the fungus occurs widely in eastern North America.

Fatal diseases and parasitoids: from competition to facilitation in a shared host.

Diverse parasite taxa share hosts both at the population level and within individual hosts, and their interactions, ranging from competitive exclusion to facilitation, can drive community structure and dynamics. Emergent pathogens have the potential to greatly alter community interactions. We found that an emergent fungal entomopathogen dominated pre-existing lethal parasites in populations of the forest defoliating gypsy moth,Lymantria dispar The parasite community was composed of the fungus and four parasitoid species that only develop successfully after they kill the host, and a virus that produces viable propagules before the host has died. A low-density site was sampled over 17 years and compared with 66 sites across a range of host densities, including outbreaks. The emergent fungal pathogen and competing parasitoids rarely co-infected host individuals because each taxa must kill its host. The virus was not present at low host densities, but successfully co-infected with all other parasite species. In fact, there was facilitation between the virus and one parasitoid species hosting a polydnavirus. This newly formed parasite community, altered by an emergent pathogen, is shaped both by parasite response to host density and relative abilities of parasites to co-inhabit the same host individuals.

Influence of mating and age on susceptibility of the beetle Anoplophora glabripennis to the fungal pathogen Metarhizium brunneum.

The age and life history of an insect can influence its susceptibility to pathogens. Reproduction can be costly and may trade off with immunity while it is generally assumed that immunity will decrease with increasing age through a process called immunosenescence. Fungal pathogens are used as biological control agents for a variety of insect pests, and Metarhizium brunneum is being developed to control the Asian longhorned beetle (Anoplophora glabripennis), an invasive wood-borer. Because adult female A. glabripennis take 1-2weeks to mature after eclosion and both sexes can be long-lived, we investigated how age and mating status would influence susceptibility of A. glabripennis to M. brunneum. Young (6.5day-old) unmated, mature (27-33day-old) mated and unmated, and old (57-71day-old) unmated and mated adults were inoculated with a lethal dose of M. brunneum. The presence of M. brunneum in the hemolymph was quantified and beetle mortality was monitored daily. There was a cost to reproduction for mated mature male and female beetles which died a median of 1.6-1.9days earlier than unmated beetles, while there was no effect of mating on susceptibility for old beetles. We found no evidence for immunosenescence in old beetles, as they did not die faster than young or mature beetles. Young unmated males however were more susceptible than mature or old unmated males, while there was no effect of age on susceptibility of unmated females.

Growth of the Sirex-parasitic nematode Deladenus siricidicola on the white rot fungus Amylostereum.

The Kamona strain of the nematode Deladenus siricidicola has been extensively used as a biological control agent against invasive Sirex noctilio woodwasps in the Southern Hemisphere, where it sterilizes female hosts. In North America, a non-sterilizing (NS) strain of D. siricidicola, thought to have been introduced with S. noctilio, is commonly found parasitizing this invasive woodwasp. Species of Deladenus that parasitize Sirex have a parasitic form, as well as a mycophagous form. The mycophagous form feeds on Sirex fungal symbionts in the genus Amylostereum. The goal of this study was to compare reproduction of NS and Kamona D. siricidicola when feeding on four isolates of Amylostereum areolatum (three introduced and one native in North America) and one native strain of Amylostereum chailletii isolated from Sirex nigricornis. Mycophagous forms of the two D. siricidicola strains displayed relatively similar production of offspring when feeding on most of the A. areolatum found associated with S. noctilio in this continent, except for strain BD on which NS produced more offspring than the biological control strain Kamona. Growth of both nematodes was greater on the introduced versus the native A. areolatum isolates.