Dimethyl sulfoxide, an alternative for control of Nosema ceranae infection in honey bees (Apis mellifera).

Nosema ceranae is a microsporidian parasite that threatens current apiculture. N. ceranae-infected honey bees (Apis mellifera) exhibit morbid physiological impairments and reduced honey production, malnutrition, shorter life span, and higher mortality than healthy honey bees. In this study, we found that dimethyl sulfoxide (DMSO) could enhance the survival rate of N. ceranae-infected honey bees. Therefore, we investigated the effect of DMSO on N. ceranae-infected honey bees using comparative RNA sequencing analysis. Our results revealed that DMSO was able to affect several biochemical pathways, especially the metabolic-related pathways in N. ceranae-infected honey bees. Based on these findings, we conclude that DMSO may be a useful alternative for treating N. ceranae infection in apiculture.

Investigation of the fall armyworm (Spodoptera frugiperda) gut microbiome and entomopathogenic fungus-induced pathobiome.

The gut microflora plays an important role in insect development and physiology. The gut bacterial microbiome of the fall armyworm (FAW), Spodoptera frugiperda, in both cornfield and laboratory-reared populations was investigated using a 16S metagenomic approach. The alpha- and beta-diversity of the cornfield FAW populations varied among sampling sites and were higher than those of the laboratory-reared FAW population, indicating that different diets and environments influence the gut bacterial composition. To better understand the interaction between the microbiome and entomopathogenic fungi (EPF), FAWs from organic and conventionally managed corn fields and from the laboratory-reared colony were inoculated with Beauveria bassiana NCHU-153 (Bb-NCHU-153). A longer median lethal time (LT50) was observed in the Bb-NCHU-153-infected cornfield FAW population than in the laboratory-reared FAWs. In terms of the microbiome, three Bb-NCHU-153-infected FAW groups showed different gut bacterial compositions compared to noninfected FAW. Further investigation of the cooccurrence network and linear discriminant analysis (LDA) of effect size (LEfSe) revealed that the enriched bacterial genera, such as Enterococcus, Serratia, Achromobacter, and Tsukamurella, in the gut might play the role of opportunistic pathogens after fungal infection; in contrast, some gut bacteria of Methylobacterium, Marinomonas, Paenochrobactrum, Pseudomonas, Acinetobacter, Delftia, Dietzia, Gordonia, Leucobacter, Paracoccus, and Stenotrophomonas might be probiotics against EPF infection. These results indicated that EPF infection can change the gut bacterial composition and lead to a pathobiome in the FAW and that some bacterial species might protect the FAW from EPF infection. These findings could be applied to the design of pathobiome-inducing biocontrol strategies.

Sacbrood viruses cross-infection between Apis cerana and Apis mellifera: Rapid detection, viral dynamics, evolution and spillover risk assessment.

Recent outbreaks of sacbrood virus (SBV) have caused serious epizootic disease in Apis cerana populations across Asia including Taiwan. Earlier phylogenetic analyses showed that cross-infection of AcSBV and AmSBV in both A. cerana and A. mellifera seems common, raising a concern of cross-infection intensifying the risk of disease resurgence in A. cerana. In this study, we analyzed the dynamics of cross-infection in three different types of apiaries (A. mellifera-only, A. cerana-only and two species co-cultured apiaries) over one year in Taiwan. Using novel, genotype-specific primer sets, we showed that SBV infection status varies across apiaries: AmSBV-AM and AcSBV-AC were the major genotype in the A. mellifera-only and the A. cerana-only apiaries, respectively, while AmSBV-AC and AcSBV-AC were the dominant genotypes in the co-cultured apiaries. Interestingly, co-cultured apiaries were among the only apiary type that harbored all variants and dual infections (i.e., AC and AM genotype co-infection in a single sample), indicating the interactions between hosts may form a conduit for cross-infection. The cross-infection between the two honey bee species appears to occur in a regular cycle with temporal fluctuation of AmSBV-AC and AcSBV-AC prevalence synchronized to each other in the co-cultured apiaries. Artificial infection of AcSBV in A. mellifera workers showed the suppression of viral replication, suggesting the potential of A. mellifera serving as a AcSBV reservoir that may contribute to virus spillover. Furthermore, the survival rate of A. cerana larvae was significantly reduced after artificial infections of both SBVs, indicating fitness costs of cross-infection on A. cerana and thus a high risk of disease resurgence in co-cultured apiaries. Our field and laboratory data provide baseline information that facilitates understanding of the risk of SBV cross-infection, and highlights the urgent need of SBV monitoring in co-cultured apiaries.

Comparison of gut microbiota of healthy and diseased walking sticks, Phasmotaenia lanyuhensis.

Research on gut microbiota of phytophagous insects has shown to be important for the physiological functions of insect hosts; however, little is known about the changes in gut microbiota when they are suffering from environmental stress or pathogen infections. During rearing of Phasmotaenia lanyuhensis (Phasmatodea: Phasmatidae), sluggish locomotion was usually followed by the death of the insect with a symptom of melanization in the front part of the abdomen. Therefore, the abnormal individuals were initially classified into moribund, light- and serious-symptom based on the level of abnormal physiological circumstances and melanization. The gut microbiota of these samples were further investigated by 16S metagenomic sequencing and the differences in bacterial abundance and structure of bacterial community were analyzed. A decrease in microbiota diversity was observed in the diseased P. lanyuhensis, with the abundance of phyla Proteobacteria and Firmicute relatively higher compared to those without symptom. Interestingly, principal component analysis based on the bacterial richness was correlated to the level of melanization symptom in the diseased P. lanyuhensis, suggested the change in bacterial microbiota involved in this abnormal circumstance. However, the factor that caused the initial alternation of microbiota remains to be identified. Additionally, the lack of bacterial diversity (i.e., absence of Meiothermus and Nubsella spp.) in P. lanyuhensis might reduce the fitness for surviving. This report provided the comprehensive microbiota analysis for P. lanyuhensis and concluded that either the relative abundance or the bacterial diversity of microbiota in the insect digestive system may influence the physiological functions of phytophagous insects.

Mechanistic insight into the attenuation of gouty inflammation by Taiwanese green propolis via inhibition of the NLRP3 inflammasome.

Dysregulation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including gouty arthritis. Activation of the NLRP3 inflammasome requires priming and activation signals: the priming signal controls the expression of NLRP3 and interleukin (IL)-1ß precursor (proIL-1ß), while the activation signal leads to the assembly of the NLRP3 inflammasome and to caspase-1 activation. Here, we reported the effects of the alcoholic extract of Taiwanese green propolis (TGP) on the NLRP3 inflammasome in vitro and in vivo. TGP inhibited proIL-1ß expression by reducing nuclear factor kappa B activation and reactive oxygen species (ROS) production in lipopolysaccharide-activated macrophages. Additionally, TGP also suppressed the activation signal by reducing mitochondrial damage, ROS production, lysosomal rupture, c-Jun N-terminal kinases 1/2 phosphorylation and apoptosis-associated speck-like protein oligomerization. Furthermore, we found that TGP inhibited the NLRP3 inflammasome partially via autophagy induction. In the in vivo mouse model of uric acid crystal-induced peritonitis, TGP attenuated the peritoneal recruitment of neutrophils, and the levels of IL-1ß, active caspase-1, IL-6 and monocyte chemoattractant protein-1 in lavage fluids. As a proof of principle, in this study, we purified a known compound, propolin G, from TGP and identified this compound as a potential inhibitor of the NLRP3 inflammasome. Our results indicated that TGP might be useful for ameliorating gouty inflammation via inhibition of the NLRP3 inflammasome.

Genomic sequencing of Troides aeacus nucleopolyhedrovirus (TraeNPV) from golden birdwing larvae (Troides aeacus formosanus) to reveal defective Autographa californica NPV genomic features.

BACKGROUND: The golden birdwing butterfly (Troides aeacus formosanus) is a rarely observed species in Taiwan. Recently, a typical symptom of nuclear polyhedrosis was found in reared T. aeacus larvae. From the previous Kimura-2 parameter (K-2-P) analysis based on the nucleotide sequence of three genes in this isolate, polh, lef-8 and lef-9, the underlying virus did not belong to any known nucleopolyhedrovirus (NPV) species. Therefore, this NPV was provisionally named "TraeNPV". To understand this NPV, the nucleotide sequence of the whole TraeNPV genome was determined using next-generation sequencing (NGS) technology. RESULTS: The genome of TraeNPV is 125,477 bp in length with 144 putative open reading frames (ORFs) and its GC content is 40.45%. A phylogenetic analysis based on the 37 baculoviral core genes suggested that TraeNPV is a Group I NPV that is closely related to Autographa californica nucleopolyhedrovirus (AcMNPV). A genome-wide analysis showed that TraeNPV has some different features in its genome compared with other NPVs. Two novel ORFs (Ta75 and Ta139), three truncated ORFs (pcna, he65 and bro) and one duplicated ORF (38.7 K) were found in the TraeNPV genome; moreover, there are fewer homologous regions (hrs) than there are in AcMNPV, which shares eight hrs within the TraeNPV genome. TraeNPV shares similar genomic features with AcMNPV, including the gene content, gene arrangement and gene/genome identity, but TraeNPV lacks 15 homologous ORFs from AcMNPV in its genome, such as ctx, host cell-specific factor 1 (hcf-1), PNK/PNL, vp15, and apsup, which are involved in the auxiliary functions of alphabaculoviruses. CONCLUSIONS: Based on these data, TraeNPV would be clarified as a new NPV species with defective AcMNPV genomic features. The precise relationship between TraeNPV and other closely related NPV species were further investigated. This report could provide comprehensive information on TraeNPV for evolutionary insights into butterfly-infected NPV.

Characterization and functional assay of apsup (Lyxy105) from Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV).

The baculoviral anti-apoptotic genes, p35 and iap (inhibitor of apoptosis), play important roles in the initiation of viral infection. Recently, a new anti-apoptotic gene (apoptosis suppressor, apsup) was identified in Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). An apsup homolog gene, Lyxy105 (ly-apsup), was also predicted in the Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) genome. In this study, we attempt to perform a gene expression analysis and a functional assay of ly-apsup to demonstrate its anti-apoptotic activity and identify the functional domain of this protein. The transcription of the ly-apsup gene region was detected from 12 h post-infection (hpi) and increased significantly after 24-72 hpi. Comparison of the putative amino acid sequences to those of 18 baculoviral homolog proteins showed high amino acid identity to the LdMNPV sequences. Moreover, five conserved protein domains (named as domains I-V) were found. Therefore, protein functional assays were conducted on full-length proteins and different truncation clones. The overexpression of each clone was confirmed by western blot analysis, and the data revealed that a cleavage of ~ 5 kDa at the N-terminal region of the full-length, domains I-IV (1-241) and I-III (1-178), proteins occurred. The results of the functional analysis showed that full-length Ly-apsup and Ly-apsup with domain I (1-70) could inhibit Drosophila-RPR protein (D-RPR)-induced and actinomycin D (ActD)-induced apoptoses. In addition, the domains I and I-II (1-126) regions showed higher anti-apoptotic activity than the other domains in both D-RPR-induced and ActD-induced cell apoptoses. In conclusion, domain I of Ly-apsup may play an important role in the anti-apoptotic activity of this protein; cleavage of the Ly-apsup N-terminus may lead to decreased anti-apoptotic activity.

Characterization of T-DNA insertion mutants with decreased virulence in the entomopathogenic fungus Beauveria bassiana JEF-007.

The bean bug, Riptortus pedestris, is a major agricultural pest that reduces crop quality and value. Chemical pesticides have contributed to pest management, but resistance to these chemicals has significantly limited their use. Alternative strategies with different modes of action, such as entomopathogenic fungi, are therefore of great interest. Herein, we explored how entomopathogenic fungi can potentially be used to control the bean bug and focused on identifying virulence-related genes. Beauveria bassiana (JEF isolates) were assayed against bean bugs under laboratory conditions. One isolate, JEF-007, showed >80 % virulence by both spray and contact exposure methods. Agrobacterium tumefaciens-mediated transformation (AtMT) of JEF-007 generated 249 random transformants, two of which (B1-06 and C1-49) showed significantly reduced virulence against Tenebrio molitor and R. pedestris immatures. Both species were used for rapid screening of virulence-reduced mutants. The two transformants had different morphologies, conidial production, and thermotolerance than the wild type. To determine the localization of the randomly inserted T-DNA, thermal asymmetric interlaced (TAIL) PCR was conducted and analysis of the two clones found multiple T-DNA insertions (two in B1-06 and three in C1-49). Genes encoding complex I intermediate-associated protein 30 (CIA30) and the autophagy protein (Atg22) were possibly disrupted by the T-DNA insertion and might be involved in the virulence. This work provides a strong platform for future functional genetic studies of bean bug-pathogenic B. bassiana. The genes putatively involved in fungal virulence should be experimentally validated by knockdown in future studies.

A novel picorna-like virus, Riptortus pedestris virus-1 (RiPV-1), found in the bean bug, R. pedestris, after fungal infection.

A viral genome was assembled de novo from next-generation sequencing (NGS) data from bean bugs, Riptortus pedestris, infected with an entomopathogenic fungus, Beauveria bassiana (Bb), and was further confirmed via the RACE method. This is a novel insect positive-sense single-stranded RNA virus, which we named Riptortus pedestris virus-1 (RiPV-1) (GenBank accession no. KU958718). The genome of RiPV-1 consists of 10,554 nucleotides (nt), excluding the poly(A) tail, which contains a single large open reading frame (ORF) of 10,371 nt encoding a polyprotein (3456 aa) and flanked by 71 and 112 nt at the 5' and 3' untranslated regions (UTR), respectively. RiPV-1 genome organization from the 5' end contains a consensus organization of picorna-like RNA virus helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp), in addition to two putative structural proteins located at the 3' region and a poly(A) tail at the 3' end. The viral particles were approximately 30nm in diameter with some dispersal distinctive surface projections. Based on the phylogenetic analysis of the RdRp sequences, RiPV-1 was clustered in the unassigned insect RNA viruses with two other viruses, APV and KFV. These three viruses were suggested to constitute a new group of insect RNA viruses. RiPV-1 could be found in all stages of lab-reared bean bugs and was detected abundantly in the thorax, abdomen, midgut and fat body, but not in the reproductive organs and muscle. Interestingly, RiPV-1 replication was increased dramatically in bean bugs 2-6days after fungal infection. In conclusion, a novel insect RNA virus was found by NGS data assembly. This virus can provide further insight into the interaction between virus, fungus and the host.

A new spiroplasma isolate from the field cricket (Gryllus bimaculatus) in Taiwan.

We briefly described the morphology and transmission pathway of a Spiroplasma sp. isolated from the field cricket, Gryllus bimaculatus in Taiwan, followed by the phylogenetic analysis based on the 16S rRNA gene sequence. The cricket spiroplasma infected the hemolymph, gut, muscle tissues and tracheal cells; therefore we suggest that the pathogen invaded tissues and organs from the hemolymph through the tracheal system and the endoplasmic reticular system. Based on 16S rRNA gene sequences and the phylogeny, this spiroplasma was most closely related to Spiroplasma platyhelix (Identity=95%) isolated from the dragonfly Pachydiplax longipennis and belongs to the Ixodetis clade.

Monitoring the Season-Prevalence Relationship of Vairimorpha ceranae in Honey Bees (Apis mellifera) over One Year and the Primary Assessment of Probiotic Treatment in Taichung, Taiwan.

Microsporidiosis, which is caused by the pathogen Vairimorpha ceranae, is a prevalent disease in the honey bee (Apis mellifera) and might lead to significant adult honey bee mortality. In this study, we conducted an annual survey of the mature spore load of V. ceranae in the guts of nurse bees and forager bees in the apiary of National Chung Hsing University (NCHU) in Taiwan. The results indicated that, on average, honey bees hosted approximately 2.13 × 106 mature spore counts (MSCs)/bee in their guts throughout the entire year. The highest number of MSCs was 6.28 × 106 MSCs/bee, which occurred in April 2020, and the lowest number of MSCs was 5.08 × 105 MSCs/bee, which occurred in November 2020. Furthermore, the guts of forager bees had significantly higher (>58%) MSCs than those of nurse bees. To evaluate the potential of the probiotic to treat microsporidiosis, the lactic acid bacterium Leuconostoc mesenteroides TBE-8 was applied to honey bee colonies. A significant reduction (>53%) in MSCs following probiotic treatment was observed, indicating the potential of probiotic treatment for managing microsporidiosis. This research provided information on V. ceranae MSCs in the honey bee gut at NCHU in Taiwan and the MSCs' correlation with the annual season. Furthermore, a potential probiotic treatment for microsporidiosis was assessed for future management.

A new microsporidium, Triwangia caridinae gen. nov., sp. nov. parasitizing fresh water shrimp, Caridina formosae (Decapoda: Atyidae) in Taiwan.

A new microsporidium was isolated from the endemic, Taiwanese shrimp, Caridina formosae (Decapoda, Atyidae) from northern Taiwan. A conspicuous symptom of infection was presence of opaque white xenomas located in the proximity of the alimentary tract, the surface of the hepatopancreas, and the gills. A fully developed xenoma consisted of a hard, thick capsule filled with sporophorous vesicles containing multiple spores. Microsporidia developed synchronously within the same sporophorous vesicle, although the stage of parasite development differed among the vesicles. Fresh spores were pyriform, mononucleated and measured 6.53 × 4.38 µm. The polar filament was anisofilar with 9-11 coils. Phylogenetic analysis based on the small subunit ribosomal DNA sequence showed that the isolate is most similar to the fish microsporidian clade containing the genera Kabatana, Microgemma, Potaspora, Spraguea, and Teramicra. The highest sequence identity, 80%, was with Spraguea spp. Based on pathogenesis, life cycle and phylogenetic analysis, we erect a new genus and species, Triwangia caridinae for the novel microsporidium.

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.).

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.

Whole-genome DNA methylome analysis of different developmental stages of the entomopathogenic fungus Beauveria bassiana NCHU-157 by nanopore sequencing.

The entomopathogenic fungus (EPF), Beauveria bassiana, is an important and commonly used EPF for microbial control. However, the role of DNA methylation has not been thoroughly studied. Therefore, the whole genomic DNA methylome of one promising EPF isolate, B. bassiana NCHU-157 (Bb-NCHU-157), was investigated by Oxford Nanopore Technologies (ONT). First, the whole genome of Bb-NCHU-157 was sequenced by next-generation sequencing (NGS) and ONT. The genome of Bb-NCHU-157 contains 16 contigs with 34.19 Mb and 50% GC content, which are composed of 10,848 putative protein-coding genes. Two putative DNA methyltransferases (DNMTs) were found, including Dim-2 and C-5 cytosine-specific DNA methylases. Both DNMTs showed higher expression levels in the mycelium stage than in the conidia stage, indicating that development of DNA methylation in Bb-NCHU-157 might occur in the mycelium stage. The global methylation level of the mycelium stage (5 mC = 4.56%, CG = 3.33%, CHG = 0.74%, CHH = 0.49%) was higher than that of the conidial stage (5 mC = 2.99%, CG = 1.99%, CHG = 0.63%, CHH = 0.37%) in both the gene and transposable element (TE) regions. Furthermore, the TE regions showed higher methylation frequencies than the gene regions, especially for CHH site methylation, suggesting regulation of genomic stabilization during mycelium development. In the gene regions, high methylation frequencies were found around the transcription start site (TSS) and transcription end site (TES). Moreover, CG and CHG methylation mainly occur in the promoter and intergenic regions, while CHH methylation occurs in the TE region. Among the methylated regions, 371, 661, and 756 differentially DNA methylated regions (DMRs) were hypermethylated in the mycelium in CG, CHG, and CHH, while only 13 and 7 DMRs were hypomethylated in the mycelium in CHG, and CHH, respectively. Genes located in the DMR shared the GO terms, DNA binding (GO: 0003677), and sequence-specific DNA binding (GO: 0043565) for hypermethylation in the mycelium, suggesting that methylation might regulate gene expression from the initial process. Evaluation of the DNA methylome in Bb-NCHU-157 by ONT provided new insight into this field. These data will be further validated, and epigenetic regulation during the development of B. bassiana will be explored.

Low-Level Fluvalinate Treatment in the Larval Stage Induces Impaired Olfactory Associative Behavior of Honey Bee Workers in the Field.

Fluvalinate is a widely used insecticide for varroa mite control in apiculture. While most beekeepers have ignored the effects of low levels of fluvalinate on bees, this study aims to demonstrate its effects at very low concentrations. We first used fluvalinate doses ranging from 0.4 to 400 ng/larva to monitor the capping, pupation, and emergence rates of larval bees. Second, we used the honey bees' proboscis extension reflex reaction to test the learning ability of adult bees that were exposed to fluvalinate doses from 0.004 to 4 ng/larva in the larval stage. The brood-capped rate of larvae decreased dramatically when the dose was increased to 40 ng/larva. Although no significant effect was observed on brood-capping, pupation, and eclosion rates with a dose of 4 ng/larva, we found that the olfactory associative behavior of adult bees was impaired when they were treated with sublethal doses from 0.004 to 4 ng/larva in the larval stage. These findings suggest that a sublethal dose of fluvalinate given to larvae affects the subsequent associative ability of adult honey bee workers. Thus, a very low dose may affect the survival conditions of the entire colony.

A Novel Application of 3D Printing Technology Facilitating Shell Wound Healing of Freshwater Turtle.

Numerous cases and a shortage of resources usually limit wild animal rescue. New technology might save these severely injured wild animals from euthanasia by easing the requirement of intensive medication. Three-dimensional (3D) technologies provide precise and accurate results that improve the quality of medical applications. These 3D tools have become relatively low-cost and accessible in recent years. In the medical field of exotic animals, turtle shell defects are highly challenging because of inevitable water immersion. This report is the first attempt to apply the combination of 3D scanning, computer-aided design (CAD), and 3D printing to make a device that protects the wound from exposure to water or infection sources. The presented techniques successfully extricate a wild freshwater turtle from an extensive shell defect within a short period. Integration of multiple sciences to 3D technology can provide a facile model for veterinary medical applications.

The complete mitochondrial genome of Attacus atlas formosanus Villiard, 1969 (Lepidoptera: Saturniidae).

The complete mitochondrial genome (mitogenome) of Attacus atlas formosanus (Villiard, 1969) is 15,280 bp in length, with the typical gene content and arrangement usually observed in Insecta. It contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one AT-rich region. The overall nucleotide composition of the mitogenome was 39.8% A, 12.9% C, 7.7% G, and 39.6% T, with an A + T bias of 79.4%. Phylogenetic analyses of 23 species in Saturniidae and 3 species in Bombycidae by Bayesian inference showed that A. atlas formosanus belonged to the Tribe Attacini, closely related to Tribe Saturniini. Besides, A. atlas formosanus is closely related to A. atlas with 99% sequence identity. This result well supported the taxonomic position of Saturniidae and their close relationship with the family Bombycidae.

Evaluation of the Potential Entomopathogenic Fungi Purpureocillium lilacinum and Fusarium verticillioides for Biological Control of Forcipomyia taiwana (Shiraki).

Forcipomyia taiwana (Diptera: Ceratopogonidae) is a nuisance blood-sucking pest to humans in Taiwan. An F. taiwana bite causes itching and redness and usually causes serious harassment to human outdoor activity. In terms of F. taiwana control, chemical pesticides are ineffective. Therefore, other efforts are needed. Fungal mycosis in the larvae, pupae, and emerging F. taiwana adults was found during the rearing of F. taiwana. In this study, six fungal isolates were isolated from infected cadavers and subjected to molecular identification. In addition, their biocontrol potential was evaluated against different life stages of F. taiwana. Based on the pathogenicity screening, two fungal isolates, NCHU-NPUST-175 and -178, which caused higher mortality on the fourth instar larvae of F. taiwana, were selected for virulence tests against different life stages of F. taiwana larvae. The results of the phylogenetic analysis indicated that the NCHU-NPUST-175 and -178 belonged to Purpureocillium lilacinum and Fusarium verticillioides, respectively. Bioassay against different life stages of F. taiwana with different spore concentrations (5 × 105 to 5 × 107 conidia/mL) revealed a dose-dependent effect on larvae for both fungal isolates, while only 38% and 50% mortality was found in highest concentration (5 × 107 conidia/mL) at fourth instar larvae by Pl-NCHU-NPUST-175 and Fv-NCHU-NPUST-178, respectively. Moreover, reductions in egg-hatching rate and adult emergence rate were found, when the last stage of F. taiwana was inoculated with both fungal isolates, indicating the ovicidal potential and the impact of entomopathogenic fungi on the development of F. taiwana. In conclusion, Pl-NCHU-NPUST-175 and Fv-NCHU-NPUST-178 showed larvicidal activity, ovicidal activity, and impact on adult emergence on F. taiwana.

Transcriptome of Nosema ceranae and Upregulated Microsporidia Genes during Its Infection of Western Honey Bee (Apis mellifera).

Nosema ceranae is one of the fungal parasites of Apis mellifera. It causes physical and behavioral effects in honey bees. However, only a few studies have reported on gene expression profiling during A. mellifera infection. In this study, the transcriptome profile of mature spores at each time point of infection (5, 10, and 20 days post-infection, d.p.i.) were investigated. Based on the transcriptome and expression profile analysis, a total of 878, 952, and 981 differentially expressed genes (DEGs) (fold change ≥ 2 or ≤ -2) were identified in N. ceranae spores (NcSp) at 5 d.p.i., 10 d.p.i., and 20 d.p.i., respectively. Moreover, 70 upregulated genes and 340 downregulated genes among common DEGs (so-called common DEGs) and 166 stage-specific genes at each stage of infection were identified. The Gene Ontology (GO) analysis indicated that the DEGs and corresponding common DEGs are involved in the functions of cytosol (GO:0005829), cytoplasm (GO:0005737), and ATP binding (GO:0005524). Furthermore, the pathway analysis found that the DEGs and common DEGs are involved in metabolism, environmental information processing, and organismal systems. Four upregulated common DEGs with higher fold-change values, highly associated with spore proteins and transcription factors, were selected for validation. In addition, the stage-specific genes are highly involved in the mechanism of pre-mRNA splicing according to GO enrichment analysis; thus, three of them showed high expression at each d.p.i. and were also subjected to validation. The relative gene expression levels showed a similar tendency as the transcriptome predictions at different d.p.i., revealing that the gene expression of N. ceranae during infection may be related to the mechanism of gene transcription, protein synthesis, and structural proteins. Our data suggest that the gene expression profiling of N. ceranae at the transcriptomic level could be a reference for the monitoring of nosemosis at the genetic level.

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.