Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls.

Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls, in which insect larva can develop while being sheltered and feeding on the plant. In particular, cynipid (Hymenoptera: Cynipidae) wasps have evolved to form morphologically complex galls and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is cell wall deposition that dictates the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how valley oak (Quercus lobata) leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form unique structures and organs.

The relationship between resource abundance and insect herbivory on islands.

We examined the relationship between resource abundance and the feeding activity of phytophagous insects on three common island plants. The aim was to investigate the correlation between phytophagous insects' abundance and availability of food and island geography. We collected 30,835 leaves from three tree species groups (Mallotus japonicus, Prunus species, and Quercus species) on 18 islands in southwest Korea. The number of plant resources for herbivores varied: the number of leaves per shoot was the highest in Mallotus, leaf weight and the water content per leaf was significantly lower in Quercus species. External feeding was higher for Prunus and Quercus species, whereas the internal feeding type was significantly higher for Quercus species. Geography (area and distance), elevation and food resource (elevation, number of plant species, and the forest cover rate) had a variable effect on phytophagous insects feeding activities: distance and the number of plant species were more explainable to the external feeding guild. In contrast, area and forest cover were more to the internal feeding guild.

Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with A Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein.

We present a reproducible procedure for transforming somatic embryos of cork oak with the CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the CsTL1 gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.

Assessing the climate suitability and potential economic impacts of Oak wilt in Canada.

We assess risks posed by oak wilt-a disease caused by the fungal pathogen Bretziella fagacearum. Though not currently found in Canada, our distribution models indicate that suitable climate conditions currently occur in southern Ontario for B. fagacearum and two of its main insect dispersal vectors, Colopterus truncatus and Carpophilus sayi. Climate habitat for these species is projected to expand northward under climate change, with much of the oak range in eastern Canada becoming climatically suitable within the next two decades. Potential costs for the removal and replacement of oak street trees ranged from CDN$266 to $420 million, with variation related to uncertainty in costs, rate of tree replacement, and city-level estimates of oak street tree density. The value of standing oak timber in eastern Canada was estimated at CDN$126 million using provincial stumpage fees and as a CDN$24 million annual contribution to national Gross Domestic Product (GDP) when calculated using a combination of economic and forestry product statistics. These values can help inform the scale of eradication and/or management efforts in the event of future oak wilt introductions.

From Inquilines to Gall Inducers: Genomic Signature of a Life-Style Transition in Synergus Gall Wasps.

Gall wasps (Hymenoptera: Cynipidae) induce complex galls on oaks, roses, and other plants, but the mechanism of gall induction is still unknown. Here, we take a comparative genomic approach to revealing the genetic basis of gall induction. We focus on Synergus itoensis, a species that induces galls inside oak acorns. Previous studies suggested that this species evolved the ability to initiate gall formation recently, as it is deeply nested within the genus Synergus, whose members are mostly inquilines that develop inside the galls of other species. We compared the genome of S. itoensis with that of three related Synergus inquilines to identify genomic changes associated with the origin of gall induction. We used a novel Bayesian selection analysis, which accounts for branch-specific and gene-specific selection effects, to search for signatures of selection in 7,600 single-copy orthologous genes shared by the four Synergus species. We found that the terminal branch leading to S. itoensis had more genes with a significantly elevated dN/dS ratio (positive signature genes) than the other terminal branches in the tree; the S. itoensis branch also had more genes with a significantly decreased dN/dS ratio. Gene set enrichment analysis showed that the positive signature gene set of S. itoensis, unlike those of the inquiline species, is enriched in several biological process Gene Ontology terms, the most prominent of which is "Ovarian Follicle Cell Development." Our results indicate that the origin of gall induction is associated with distinct genomic changes, and provide a good starting point for further characterization of the genes involved.

Diurnal effects on sporangium and zoospore production by Phytophthora ramorum on Rhododendron 'Cunningham's White'.

We evaluated sporangium and zoospore production by three isolates of Phytophthora ramorum on Rhododendron 'Cunningham's White' leaves under light and dark conditions at both variable and constant (14 C) temperature. P. ramorum-infected leaves were detached and placed in funnels inside of a 62-L plastic storage container located in a growth chamber. Cool mist was introduced to the container to create a high-humidity environment. Sporangia and zoospores were collected over a 4-day period by misting leaves with 5 mL of distilled water, which was collected in conical test tubes that also contained runoff from the misting. Spores were collected daily just before a 13-h light period and again just before an 11-h dark period. Sporangia and zoospores in the collection tubes were counted using a dissecting microscope following staining with lactoglycerin/aniline blue. Large differences in sporangium and zoospore numbers observed for the dark versus light periods were observed on days 2, 3, and 4. A diurnal effect has been observed for production of propagules of other oomycetes, but such effects have not been previously reported for P. ramorum. This information will help provide a better understanding of patterns of inoculum production by P. ramorum and resulting fluctuations in inoculum density that will influence sudden oak death epidemics in forest ecosystems in the United States and other countries where it occurs.

Changes in oak (Quercus robur) photosynthesis after winter moth (Operophtera brumata) herbivory are not explained by changes in chemical or structural leaf traits.

Insect herbivores have the potential to change both physical and chemical traits of their host plant. Although the impacts of herbivores on their hosts have been widely studied, experiments assessing changes in multiple leaf traits or functions simultaneously are still rare. We experimentally tested whether herbivory by winter moth (Operophtera brumata) caterpillars and mechanical leaf wounding changed leaf mass per area, leaf area, leaf carbon and nitrogen content, and the concentrations of 27 polyphenol compounds on oak (Quercus robur) leaves. To investigate how potential changes in the studied traits affect leaf functioning, we related the traits to the rates of leaf photosynthesis and respiration. Overall, we did not detect any clear effects of herbivory or mechanical leaf damage on the chemical or physical leaf traits, despite clear effect of herbivory on photosynthesis. Rather, the trait variation was primarily driven by variation between individual trees. Only leaf nitrogen content and a subset of the studied polyphenol compounds correlated with photosynthesis and leaf respiration. Our results suggest that in our study system, abiotic conditions related to the growth location, variation between tree individuals, and seasonal trends in plant physiology are more important than herbivory in determining the distribution and composition of leaf chemical and structural traits.

Effect of Neuroterus quercusbaccarum (L.) galls on physiological and biochemical response of Quercus robur leaves.

Gall formation is associated with multiple changes in plant cells, which still requires a better understanding. In this study, galls caused by sexual generation (♀♂) of Neuroterus quercusbaccarum (L.) (Hymenoptera: Cynipidae) on pedunculate oak trees (Quercus robur L.) were used as a model. Cytoplasmic membrane condition, concentration of hydrogen peroxide (H2O2), the activity of antioxidant enzymes and amino acid decarboxylase as well as chlorophyll fluorescence parameters were determined. Changes in physiological and biochemical parameters were analyzed in foliar tissues with galls and gall tissues themselves and compared to control. The presence of galls on oak leaves caused an increase of lipid peroxidation level. A significant decline in H2O2 and TBARS content with the reduction of guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) activity were observed in gall tissues. The activity amino acid decarboxylase, i.e., LDC, ODC and TyDC varied between samples, which may affect the content of amino acids. The presence of N. quercusbaccarum galls caused an insignificant increase of the chlorophylls, carotenoids and anthocyanin contents, while the content of pigments and their ratios in gall tissues was extremely low. Moreover, photosynthetic parameters (F0, Fm, Fv/Fm, Y, qP) were significantly decreased. Data generated in this study indicate that the development of N. quercusbaccarum galls on pedunculate oak leaves has a negative effect on host plant related to the disruption of cell membrane integrity, disturbance of photosynthesis and reduction of the antioxidant potential of the host plant.

Genomic dissection of an extended phenotype: Oak galling by a cynipid gall wasp.

Galls are plant tissues whose development is induced by another organism for the inducer's benefit. 30,000 arthropod species induce galls, and in most cases the inducing effectors and target plant systems are unknown. Cynipid gall wasps are a speciose monophyletic radiation that induce structurally complex galls on oaks and other plants. We used a model system comprising the gall wasp Biorhiza pallida and the oak Quercus robur to characterise inducer and host plant gene expression at defined stages through the development of galled and ungalled plant tissues, and tested alternative hypotheses for the origin and type of galling effectors and plant metabolic pathways involved. Oak gene expression patterns diverged markedly during development of galled and normal buds. Young galls showed elevated expression of oak genes similar to legume root nodule Nod factor-induced early nodulin (ENOD) genes and developmental parallels with oak buds. In contrast, mature galls showed substantially different patterns of gene expression to mature leaves. While most oak transcripts could be functionally annotated, many gall wasp transcripts of interest were novel. We found no evidence in the gall wasp for involvement of third-party symbionts in gall induction, for effector delivery using virus-like-particles, or for gallwasp expression of genes coding for plant hormones. Many differentially and highly expressed genes in young larvae encoded secretory peptides, which we hypothesise are effector proteins exported to plant tissues. Specifically, we propose that host arabinogalactan proteins and gall wasp chitinases interact in young galls to generate a somatic embryogenesis-like process in oak tissues surrounding the gall wasp larvae. Gall wasp larvae also expressed genes encoding multiple plant cell wall degrading enzymes (PCWDEs). These have functional orthologues in other gall inducing cynipids but not in figitid parasitoid sister groups, suggesting that they may be evolutionary innovations associated with cynipid gall induction.

Home economics in an oak gall: behavioural and chemical immune strategies against a fungal pathogen in Temnothorax ant nests.

Nest architecture is a fundamental character shaping immune strategies of social insects. The arboreal ant Temnothorax unifasciatus nests in cavities such as oak galls where the entire colony lives in a unique small chamber. In these conditions, physiological and behavioural strategies likely prevail over compartmentalisation and are presumably tuned with colony size. We designed two experiments to study chemical and behavioural immune strategies against the entomopathogenic fungus Metarhizium anisopliae in colonies of different sizes. First, we compared spore germination and length of germinal tubes inside artificial nests, designed to impede the contact between the ants and the fungus, in colonies of different size. In the absence of direct contact, Temnothorax unifasciatus colonies inhibit fungal growth inside their nests, presumably through volatile compounds. The analysis revealed a positive correlation between fungistatic activity and colony size, indicating that workers of smaller colonies do not invest a higher per capita effort in producing such substances compared to larger colonies. Second, we performed a removal experiment of contaminated and non-contaminated items introduced inside the nests of colonies of different size. Small colonies challenged with contaminated fibres showed an increased removal of all the items (both contaminated and non-contaminated) compared to small colonies challenged with non-contaminated fibres only. Conversely, larger colonies moved items regardless of the presence of the spores inside the nest. Colony size qualitatively affected removal of waste items showing a pathogen elicited reaction in small colonies to optimise the reduced workforce, while the removal behaviour in larger colonies revealed to be expressed constitutively.

Variation in Susceptibility of Tanoak to the NA1 and EU1 Lineages of Phytophthora ramorum, the Cause of Sudden Oak Death.

Phytophthora ramorum, the cause of sudden oak death (SOD), kills tanoak (Notholithocarpus densiflorus) trees in southwestern Oregon and California. Two lineages of P. ramorum are now found in wildland forests of Oregon (NA1 and EU1). In addition to the management of SOD in forest ecosystems, disease resistance could be used as a way to mitigate the impact of P. ramorum. The objectives of this study were to (i) characterize the variability in resistance of N. densiflorus among families using lesion length; (ii) determine whether lineage, isolate, family, or their interactions significantly affect variation in lesion length; and (iii) determine whether there are differences among isolates and among families in terms of lesion length. The parameters isolate nested within lineage (isolate[lineage]) and family × isolate(lineage) interaction explained the majority of the variation in lesion length. There was no significant difference between the NA1 and EU1 lineages in terms of mean lesion length; however, there were differences among the six isolates. Lesions on seedlings collected from surviving trees at infested sites were smaller, on average, than lesions of seedlings collected from trees at noninfested sites (P = 0.0064). The results indicate that there is potential to establish a breeding program for tanoak resistance to SOD and that several isolates of P. ramorum should be used in an artificial inoculation assay.

Host plant associations and geography interact to shape diversification in a specialist insect herbivore.

Disentangling the processes underlying geographic and environmental patterns of biodiversity challenges biologists as such patterns emerge from eco-evolutionary processes confounded by spatial autocorrelation among sample units. The herbivorous insect, Belonocnema treatae (Hymenoptera: Cynipidae), exhibits regional specialization on three plant species whose geographic distributions range from sympatry through allopatry across the southern United States. Using range-wide sampling spanning the geographic ranges of the three host plants and genotyping-by-sequencing of 1,217 individuals, we tested whether this insect herbivore exhibited host plant-associated genomic differentiation while controlling for spatial autocorrelation among the 58 sample sites. Population genomic structure based on 40,699 SNPs was evaluated using the hierarchical Bayesian model entropy to assign individuals to genetic clusters and estimate admixture proportions. To control for spatial autocorrelation, distance-based Moran's eigenvector mapping was used to construct regression variables summarizing spatial structure inherent among sample sites. Distance-based redundancy analysis (dbRDA) incorporating the spatial variables was then applied to partition host plant-associated differentiation (HAD) from spatial autocorrelation. By combining entropy and dbRDA to analyse SNP data, we unveiled a complex mosaic of highly structured differentiation within and among gall-former populations finding evidence that geography, HAD and spatial autocorrelation all play significant roles in explaining patterns of genomic differentiation in B. treatae. While dbRDA confirmed host association as a significant predictor of patterns of genomic variation, spatial autocorrelation among sites explained the largest proportion of variation. Our results demonstrate the value of combining dbRDA with hierarchical structural analyses to partition spatial/environmental patterns of genomic variation.

Updated Assembly of Phytophthora ramorum pr102 Isolate Incorporating Long Reads from PacBio Sequencing.

The NA1 clonal lineage of Phytophthora ramorum is responsible for sudden oak death, an epidemic that has devastated California coastal forest ecosystems. An NA1 isolate, Pr102, derived from coast live oak in California, was previously sequenced and reported with a 65-Mb assembly containing 12 Mb of gaps in 2,576 scaffolds. Here, we report an improved 70-Mb genome in 1,512 scaffolds with 6,752 bp of gaps after incorporating PacBio P5-C3 long reads. This assembly contains 19,494 gene models (average gene length of 2,515 bp) compared with 16,134 genes (average gene length of 1,673 bp) in the previous version. We predicted 29 new RXLR genes and 76 new paralogs of a total 392 RXLR genes from this assembly. We predicted 35 CRN genes compared with 19 in an earlier version with six paralogs. Our long non-coding RNA prediction identified 255 candidates. This new resource will be invaluable for future evolution studies on the invasive plant pathogen.

Three types of gut bacteria collaborating to improve Kui Jie'an enema treat DSS-induced colitis in mice.

A rising amount of evidences show that gut microbiota is an important factor in mediating the growth of ulcerative colitis (UC), the major product of colon bacteria fermentation butyrate as mediator have effects on the mucosal immune system by expanding regulatory T cells (Treg) in the colon. Turkish galls, an insect gall parasitized on the tree branches of Quercus infectoria Oliv., exhibiting the promising prospect in treating the remedy of UC by regulating the bacteria, whereas its mechanism remains unclear. Here, this work found that three types of gut bacteria collaborating to improve DSS-induced UC in mice after Turkish galls intervention, including putative SCFAs-producing bacteria (PCPB), anti-inflammatory bacteria and harmful bacteria. The Helicobacter, Bilophila, Acinetobacter and Odoribacter, which belong to the harmful bacteria were dramatically increased in UC group, whereas the harmful bacteria were reduced after treatment with Turkish galls. The Allobaculum, Bacteriodes, Blautia, Butyricimonas, belonging to PCPB, were significantly increased after Turkish galls and butyrate intervention, and we also observed that the concentration of butyrate increased with the grows of PCPB. The Bifidobacterium, Lactococcus, which belong to the anti-inflammatory bacteria, were also significantly increased after Turkish galls intervention. Meanwhile, rectal administration of Turkish galls and butyrate could increase mucosa inflammation and diarrhea. The expression of cytokines in the colon was improved by butyrate and Turkish galls treatment group. The percentage of Treg out of CD4+ population was evaluated by flow cytometry after Turkish galls and butyrate intervention. The results suggested that Turkish galls alleviated UC by modulating three types of the gut microbiota, and butyrate may be used to relieve inflammatory. This study may help us to understand the mechanism of Turkish galls in treating UC from the perspective of intestinal flora and also offered a mechanism reference for UC treatment using an insect gall in rich of polyphenolic compounds.

Quantifying pine processionary moth defoliation in a pine-oak mixed forest using unmanned aerial systems and multispectral imagery.

Pine processionary moth (PPM) feeds on conifer foliage and periodically result in outbreaks leading to large scale defoliation, causing decreased tree growth, vitality and tree reproduction capacity. Multispectral high-resolution imagery acquired from a UAS platform was successfully used to assess pest tree damage at the tree level in a pine-oak mixed forest. We generated point clouds and multispectral orthomosaics from UAS through photogrammetric processes. These were used to automatically delineate individual tree crowns and calculate vegetation indices such as the normalized difference vegetation index (NDVI) and excess green index (ExG) to objectively quantify defoliation of trees previously identified. Overall, our research suggests that UAS imagery and its derived products enable robust estimation of tree crowns with acceptable accuracy and the assessment of tree defoliation by classifying trees along a gradient from completely defoliated to non-defoliated automatically with 81.8% overall accuracy. The promising results presented in this work should inspire further research and applications involving a combination of methods allowing the scaling up of the results on multispectral imagery by integrating satellite remote sensing information in the assessments over large spatial scales.

Effects of insect infestation on rodent-mediated dispersal of Quercus aliena: results from field and enclosure experiments.

Rodents influence plant establishment and regeneration by functioning as both seed predators and dispersers. However, these rodent-plant interactions can vary significantly due to various environmental conditions and the activity of other insect seed predators. Here, we use a combination of both field and enclosure (i.e. individual cage and semi-natural enclosure) experiments, to determine whether rodents can distinguish sound seeds from those infested with insects. We also demonstrate how such responses to insects are influenced by food abundance and other environmental factors. We presented rodents with 2 kinds of Quercus aliena seeds (sound and insect-infested seeds) in a subtropical forest in the Qinling Mountains, central China, from September to November of 2011 to 2013. The results showed that rodents preferred to hoard and eat sound seeds than infested seeds in the field and semi-natural enclosure, while they preferred to eat infested seeds over sound seeds in the individual cages. In addition, both hoarding and eating decisions were influenced by food abundance. Rodents hoarded more sound seeds in years of high food abundance while they consumed more acorns in years of food shortage. Compared with field results, rodents reduced scatter-hoarding behavior in semi-natural enclosures and ate more insect-infested seeds in smaller individual cages. These results further confirm that rodents distinguish infested seeds from non-infested seeds but demonstrate that this behavior varies with conditions (i.e. environment and food abundance). We suggest that such interactions will influence the dispersal and natural regeneration of seeds as well as predation rates on insect larvae.

Screening for active constituents in Turkish galls against ulcerative colitis by mass spectrometry guided preparative chromatography strategy: in silico, in vitro and in vivo study.

Turkish galls have been reported to exhibit remedial effects in ulcerative colitis (UC). However, the active constituents of Turkish galls for the treatment of UC remain unclear. The objective of this study was to screen for anti-inflammatory active constituents and clarify their associated molecular mechanisms. Therefore, systems pharmacology was developed to predict the relationship between constituents and the corresponding targets as well as pathways. In addition, mass spectrometry-guided preparative chromatography technique was used for preparing constituents to evaluate the anti-inflammatory activities and the therapeutic efficacy against UC. In silico, active constituents exhibited a remedial effect on UC possibly by regulating multiple pathways and attacking multiple targets, of which those involved mainly in the NF-κB pathway were selected for verification. In vitro, 5 categories of constituents were screened as active constituents by comparing the cytotoxicity and detecting the level of the pro-inflammatory factors of 9 category constituents. In vivo, dextran sulfate sodium (DSS)-induced UC was significantly ameliorated in active constituents-fed mice. The results indicated that the active fraction comprising methyl gallate, digallic acid, di-O-galloyl-ß-d-glucose, and tri-O-galloyl-ß-d-glucose primarily contributed to the treatment of UC. Moreover, active fraction could also inhibit the phosphorylation level of IKKß, thus inhibiting the downstream NF-κB signaling pathway. The approach developed in this study not only clarifies the anti-inflammation effect of Turkish galls but also provides a beneficial reference for the discovery of the base material and functional mechanism of this herbal medicine.

Three new Leptographium spp. (Ophiostomatales) infecting hardwood trees in Norway and Poland.

Species of Leptographium are characterized by mononematous or synnematous conidiophores and are commonly associated with different arthropods. Some of them also produce a sexual state characterised by globose ascomata with elongated necks. Compared to investigations on coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various hardwood tree species in Norway and Poland, three unusual species, which fit in the broader morphological description of Leptographium spp., were found in association with Trypodendron domesticum, Trypodendron signatum and Dryocoetes alni, and from wounds on a variety of hardwoods. Phylogenetic analyses of sequence data for six different loci (ITS1-5.8 S-ITS2, ITS2-LSU, ACT, ß-tubulin, CAL, and TEF-1α) showed that these Leptographium species are phylogenetically closely related to the species of the Grosmannia olivacea complex. The first species forms a well-supported lineage that includes Ophiostoma brevicolle, while the two other new taxa resided in a separate lineage; possibly affiliated with Grosmannia francke-grosmanniae. All the new species produce perithecia with necks terminating in ostiolar hyphae and orange-section shaped ascospores with cucullate, gelatinous sheaths. These species also produce dark olivaceous mononematous asexual states in culture. In addition, two of the newly described species have a second type of conidiophore with a short and non-pigmented stipe. The new Leptographium species can be easily distinguished from each other by their appearance and growth in culture. Based on novel morphological characters and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium tardum sp. nov., Leptographium vulnerum sp. nov., and Leptographium flavum sp. nov. are provided.

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.