Microbiome changes in Akanthomyces attenuatus JEF-147-infected two-spotted spider mites.

The two-spotted spider mite (Tetranychus urticae Koch) is an agriculturally serious polyphagous pest that has acquired strong resistance against acaricides because of its short life cycle and continuous exposure to acaricides. As an alternative, mite-pathogenic fungi with different modes of action could be used to control the mites. The spider mite has symbiotic microorganisms that could be involved in the physiological and ecological adaptations to biotic stresses. In this study, mite-pathogenic fungi were used to control female adults, and the microbiomes changes in the fungus-infected mites were analyzed. The acaricidal activity of 77 fungal isolates was tested, and Akanthomyces attenuatus JEF-147 exhibited the highest acaricidal activity. Subsequently a dose-response assay and morphological characterization was undertaken For microbiome analysis in female adults infected with A. attenuatus JEF-147, 16S rDNA and ITS1 were sequenced using Illumina Miseq. Infected mite showed a higher Shannon index in bacterial diversity but lower index in fungal diversity. In beta diversity using principal component analysis, JEF-147-treated mites were significantly different from non-treated controls in both bacteria and fungi. Particularly in bacterial abundance, arthropod defense-related Rickettsia increased, but arthropod reproduction-associated Wolbachia decreased. The change in major bacterial abundance in the infected mites could be explained by a trade-off between reproduction and immunity against the early stage of fungal attack. In fungal abundance, Akanthomyces showed up as expected. Foremost, this work reports microbiome changes in a fungus-infected mite and suggests a possible trade-off in mites against fungal pathogens. Future studies will focus on gene-based investigations related to this topic.

Detection of viral genes in Metarhizium anisopliae JEF-290-infected longhorned tick, Haemaphysalis longicornis using transcriptome analysis.

Ticks are carriers of viruses that can cause disease in humans and animals. The longhorned ticks (Haemaphysalis longicornis; LHT), for example, mediates the severe fever with thrombocytopenia syndrome virus (SFTSV) in humans, and the population of ticks is growing due to increases in temperature caused by climate change. As ticks carry primarily RNA viruses, there is a need to study the possibility of detecting new viruses through tick virome analysis. In this study, viruses in LHTs collected in Korea were investigated and virus titers in ticks exposed to the entomopathogenic fungus Metarhizium anisopliae JEF-290 were analyzed. Total RNA was extracted from the collected ticks, and short reads were obtained from Illumina sequencing. A total of 50,024 contigs with coding capacity were obtained after de novo assembly of the reads in the metaSPAdes genome assembler. A series of BLAST-based analyses using the GenBank database was performed to screen viral contigs, and three putative virus species were identified from the tick meta-transcriptome, such as Alongshan virus (ALSV), Denso virus and Taggert virus. Measurements of virus-expression levels of infected and non-infected LHTs failed to detect substantial differences in expression levels. However, we suggest that LHT can spread not only SFTSV, but also various other disease-causing viruses over large areas of the world. From the phylogenetic analysis of ALSV glycoproteins, genetic differences in the ALSV could be due to host differences as well as regional differences. Viral metagenome analysis can be used as a tool to manage future outbreaks of disease caused by ticks by detecting unknown viruses.

Gene-disruption of the entomopathogenic fungus Beauveria bassiana incubated with dsRNA.

The species of Beauveria bassiana is widely used for the management of agricultural insect pests. In this study, we integrated egfp-double-stranded RNA (dsRNA) to a previously generated egfp-expressing B. bassiana transformant (Bb-egfp#3) using a protoplast integration method. The Bb-egfp#3 protoplast was mixed with the dsRNA under PEG/CaCl2 conditions and liquid-cultured in Sabouraud dextrose broth for 5 days. A control culture followed the same procedure without dsRNA. Bb-egfp#3/egfp-dsRNA cultures showed very low fungal growth (OD630 = 0.2) compared to the control culture, Bb-egfp#3 only (OD630 = 1.1). Screening of possible transformants on Sabouraud dextrose agar revealed a transformant T3, without egfp signal. T3 was confirmed as B. bassiana through sequencing of conserved genes and insect bioassays. Interestingly, the genomic egfp fragment of T3 was disrupted, and the egfp signal was not detected over four subcultures, which was also confirmed by RNA-seq of Bb-egfp#3 and T3. This study provides an interesting observation that protoplast integration with dsRNA could possibly generate significantly reduced gene expression in B. bassiana and it is stable across several generations.

Beauveria bassiana ERL836 and JEF-007 with similar virulence show different gene expression when interacting with cuticles of western flower thrips, Frankniella occidentalis.

BACKGROUND: Insect-killing fungal species, Beauveria bassiana, is as an environment-friendly pest management tool, and many isolates are on the track of industrialization. However, some of B. bassiana isolates show similar morphology and virulence against insect pests, and so it is hard to differentiate them. Herein we used two patented isolates, ERL836 and JEF-007, and investigated their virulence against western flower thrips, Frankliniella occidentalis, and further analyzed genome structures and transcriptional responses when interacting with cuticles of thrips to see possible differences on the initial step of fungal infection. RESULTS: The two isolates showed no significant differences in fungal growth, conidial production, and virulence against thrips, and they were structurally similar in genome. But, in transcription level, ERL836 appeared to infect thrips easily, while JEF-007 appeared to have more difficulty. In the GO analysis of ERL836 DEGs (differentially expressed genes), the number of up-regulated genes was much larger than that of down-regulated genes, when compared to JEF-007 DEGs (more genes down-regulated). Interestingly, in the enrichment analysis using shared DEGs between two infecting isolates, plasma membrane-mediated transporter activity and fatty acid degradation pathway including cytochrome P450 were more active in infecting ERL836. CONCLUSION: The two B. bassiana isolates had similar morphology and virulence as well as genome structure, but in transcription level they differently interacted with the cuticle of western flower thrips. This comparative approach using shared DEG analysis could be easily applied to characterize the difference of the two B. bassiana isolates, JEF-007 and ERL836.

Pathogenesis-related genes of entomopathogenic fungi.

All living things on Earth experience various diseases such as those caused by viruses, bacteria, and fungi. Insects are no exception to this rule, and fungi that cause disease in insects are called entomopathogenic fungi. These fungi have been developed as microbial insecticides and are used to control various pests. Generally, the mode of action of entomopathogenic fungi is divided into the attachment of conidia, germination, penetration, growth, and generation of secondary infectious conidia. In each of these steps, that entomopathogenic fungi use genes in a complex manner (specific or diverse) has been shown by gene knock-out and RNA-sequencing analysis. In this review, the information mechanism of entomopathogenic fungi was divided into six steps: (1) attachment of conidia to host, (2) germination and appressorium, (3) penetration, (4) fungal growth in hemolymph, (5) conidia production on host, and (6) transmission and dispersal. The strategy used by the fungi in each step was described at the genetic level. In addition, an approach for studying the mode of action of the fungi is presented.

Evaluation of spray applications of Metarhizium anisopliae, Metarhizium brunneum and Beauveria bassiana against larval winter ticks, Dermacentor albipictus.

Dermacentor albipictus (Acari: Ixodidae), the winter tick, is a one-host tick that parasitizes large ungulates. They can dramatically affect moose, Alces alces (Artiodactyla: Cervidae), causing significant physiological and metabolic stress and mortality among heavily parasitized individuals. Entomopathogenic fungi in the genera Metarhizium (Hypocreales: Clavicipitaceae) and Beauveria (Hypocreales: Cordycipitaceae) are promising tick biological control agents. We examined the pathogenicity of experimental and commercially formulated isolates of M. anisopliae, M. brunneum and B. bassiana sprayed at concentrations of 106, 107 and 108 conidia/mL against the larval stage of D. albipictus and assessed the efficacy of spraying the commercial product Met52®EC, containing M. brunneum, strain F52, under laboratory conditions. Results showed larval D. albipictus mortality was significantly higher and occurred earlier when treated with M. anisopliae and M. brunneum isolates compared to B. bassiana at 106, 107 and 108 conidia/mL. Mortality was observed as early as 3 days in the M. anisopliae and M. brunneum treatments and after 6 days in the B. bassiana treatments. After 21 days, larval mortality ranged from 74-99% when ticks were treated with M. anisopliae and M. brunneum isolates at 106, 107 and 108 and conidia/mL. In contrast, mortality of ticks treated with B. bassiana ranged from 30 to 64%. When larvae were treated with the commercial product Met52, mortality was ~ 45% after 3 days and ~ 96% after 9 days. These results demonstrate the effectiveness of M. anisopliae and M. brunneum against D. albipictus.

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.

Use of Metarhizum aniopliae s.l. to control soil-dwelling longhorned tick, Haemaphysalis longicornis.

The longhorned tick (bush tick),Haemaphysalis longicornis (Ixodida: Ixodidae), is a serious pest; it transmits the severe fever with thrombocytopenia syndrome (SFTS) virus to humans and has a wide distribution. The use of chemical control is not favored for environmental and health reasons, so more environmentally sound management methods need to be developed. Herein, we describe the use of an entomopathogenic fungal library to develop a fungus-mediated tick management system. Field-collected nymphs were assayed for their susceptibility to entomopathogenic fungi belonging to genera Beauveria, Metarhizium, Cordyceps, and Akanthomyces. Three M. anisopliae s.l. isolates, JEF-214, -279, and -290 showed high virulence in a dose-dependent manner. One Cordyceps isolate was pathogenic but virulence was much lower than the M. anisopliae isolates. Beauveria isolates were not pathogenic to the tick. Because the longhorned tick dwells on the soil surface except for blood-feeding periods, the soil surface was sprayed with conidial suspensions of the isolates after the release of longhorned ticks. The treatments resulted in 60-90% mortality after 30 days. M. anisopliae s.l. isolates were highly virulent against longhorned tick, and the application of fungus-based biopesticides on the soil surface could be an effective control strategy to reduce the tick population for long-term tick management.

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.

Conidiogenesis-related DNA photolyase gene in Beauveria bassiana.

Beauveria bassiana is an entomopathogenic fungi used in environmentally mindful pest management. Its main active ingredient, conidia, is commercially available as a fungal biopesticide. Many studies of conidia production have focused on how to optimize culture conditions for maximum productivity and stability against unfavorable abiotic factors. However, understanding of how conidiogenesis-related genes provide improved conidial production remains unclear. In this study, we focus on identifying conidiogenesis-related genes in B. bassiana ERL1170 using a random mutagenesis technique. Transformation of ERL1170 using restriction enzyme-mediated integration generated one morphologically different transformant, ERL1170-pABeG #163. The transformant was confirmed to represent B. bassiana, and the binary vector was successfully integrated into the genome of ERL1170. Compared to the wild type, transformant #163 showed very slow hyphal growth and within 6 days only produced <1 × 106 conidia/0.28 cm2 agar block (wild type: 6.2 × 107 conidia/agar block). Transformant #163 also exhibited different morphology than the wild type, including thicker hyphae with some club-shaped parts. In contrast, the typical morphology of wild type B. bassiana exhibits thread-like hyphae and conidiophore structures and circular conidia. To determine the location of the randomly inserted DNA, we conducted thermal asymmetric interlaced (TAIL) PCR and Escherichia coli cloning to clearly sequence the disrupted region. We identified one colony (colony No. 7) with an insertion site identified as DNA photolyase. This was confirmed through a gene knock-out study. It is possible the gene that encodes for DNA photolyase was disrupted during the insertion process and might be involved in fungal conidiogenesis. This work serves as a platform for exploring the function of a variety of B. bassiana genes involved in pest management and their downstream processing.

IL-12 and IL-23 Production in Toxoplasma gondii- or LPS-Treated Jurkat T Cells via PI3K and MAPK Signaling Pathways.

IL-12 and IL-23 are closely related in structure, and have been shown to play crucial roles in regulation of immune responses. However, little is known about the regulation of these cytokines in T cells. Here, we investigated the roles of PI3K and MAPK pathways in IL-12 and IL-23 production in human Jurkat T cells in response to Toxoplasma gondii and LPS. IL-12 and IL-23 production was significantly increased in T cells after stimulation with T. gondii or LPS. T. gondii and LPS increased the phosphorylation of AKT, ERK1/2, p38 MAPK, and JNK1/2 in T cells from 10 min post-stimulation, and peaked at 30-60 min. Inhibition of the PI3K pathway reduced IL-12 and IL-23 production in T. gondii-infected cells, but increased in LPS-stimulated cells. IL-12 and IL-23 production was significantly reduced by ERK1/2 and p38 MAPK inhibitors in T. gondii- and LPS-stimulated cells, but not in cells treated with a JNK1/2 inhibitor. Collectively, IL-12 and IL-23 production was positively regulated by PI3K and JNK1/2 in T. gondii-infected Jurkat cells, but negatively regulated in LPS-stimulated cells. And ERK1/2 and p38 MAPK positively regulated IL-12 and IL-23 production in Jurkat T cells. These data indicate that T. gondii and LPS induced IL-12 and IL-23 production in Jurkat T cells through the regulation of the PI3K and MAPK pathways; however, the mechanism underlying the stimulation of IL-12 and IL-23 production by T. gondii in Jurkat T cells is different from that of LPS.

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.

Production of antibacterial Bombyx mori cecropin A in mealworm-pathogenic Beauveria bassiana ERL1170.

Efforts are underway to produce antimicrobial peptides in yellow mealworms (Tenebrio molitor), which can be developed as more effective and safer animal feed additives. In this work, we expressed Bombyx mori (Bm) cecropin-A in mealworms by the infection of transformed entomopathogenic Beauveria bassiana ERL1170. The active domain of Bm cecropin A gene was tagged with a signal sequence of B. bassiana for extracellular secretion, and the fragment was inserted into ERL1170 by the restriction enzyme-mediated integration method. Transformant D-6 showed antibacterial activity against Bacillus subtilis and Listeria monocytogenes. Against T. molitor larvae, D-6 had similar mortality to wild-type, and D6-infected mealworm suspension showed strong antibacterial activity against the two bacteria, but not in the wild-type-infected mealworms, thereby increasing the value of mealworms as animal feed additives.

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.

Biodegradation of pesticide in agricultural soil employing entomopathogenic fungi: Current state of the art and future perspectives.

Pesticides play a pivotal role in agriculture for the effective production of various crops. The indiscriminate use of pesticides results in the significant bioaccumulation of pesticide residues in vegetables. This situation is beyond the control of consumers and poses a serious health issue for human beings. Occupational exposure to pesticides may occur for farmers, agricultural workers, and industrial producers of pesticides. This occupational exposure primarily causes food and water contamination that gets into humans and environmental pollution. Depending on the toxicity of pesticides, the causes and effects differ in the environment and in human health. The number of criteria used and the method of implementation employed to assess the effect of pesticides on humans and the environment have been increasing, as they may provide characterization of pesticides that are already on the market as well as those that are on the way. The biological control of pests has been increasing nowadays to combat all these effects caused by synthetic pesticides. Myco-biocontrol has received great attention in research because it has no negative impact on humans, the environment, or non-target species. Entomopathogenic fungi are microbes that have the ability to kill insect pests. Fungi also make enzymes like the lytic enzymes, esterase, oxidoreductase, and cytochrome P450, which react with chemical residues in the field and break them down into nontoxic substances. In this review, the authors looked at how entomopathogenic fungi break down insecticides in the environment and how their enzymes break down insecticides on farms.

The Combination of Enzymes and Conidia of Entomopathogenic Fungi against Aphis gossypii Nymphs and Spodoptera frugiperda Larvae.

The slow action of fungi is one of the biggest challenges in using entomopathogenic fungi. A promising alternative to reduce the time of action is to combine conidia with extracellular enzymes. This study aimed to characterize the production of Pr1 subtilisin protease and lipases by Beauveria bassiana and Metarhizium anisopliae in different culture media and to evaluate the efficiency of the enzymatic treatment against Aphis gossypii and Spodoptera frugiperda. The isolates were cultivated in five different liquid cultures, and, after 7 days, the culture was filtered and centrifuged, and the activity of the Pr1 and lipases was measured. The fungi cultured in a Luria-Bertani broth medium had the highest activity of proteases and lipases. The mortality of A. gossypii nymphs treated with conidia 7 days after the treatment was 39% (JEF-410), 76.5% (JEF-492), 74.8% (ERL-836), and 70.9% (JEF-214). The B. bassiana JEF-410 supernatant combined with conidia increased the fungal virulence at day 5 and day 6 after treatment. When S. frugiperda larvae were treated with B. bassiana JEF-492 conidia combined with its supernatant, the time of infection was shorter compared to the larvae treated with conidia only. Once the supernatant was incubated at 37 °C, the relative activity decreased from 100% to 80% after 2 h and to 45% after 24 h. The results suggest that the supernatant of entomopathogenic fungi may be formulated and used as a biopesticide in an efficient strategy for the biological control of pests.

NeuroBactrus, a novel, highly effective, and environmentally friendly recombinant baculovirus insecticide.

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin-cry1-5-polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin-Cry1-5-polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an ∼65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.

Pathogenesis and defense mechanism while Beauveria bassiana JEF-410 infects poultry red mite, Dermanyssus gallinae.

The poultry red mite, Dermanyssus gallinae (Mesostigmata: Dermanyssidae), is a major pest that causes great damage to chicken egg production. In one of our previous studies, the management of red mites using entomopathogenic fungi was evaluated, and the acaricidal fungus Beauveria bassiana JEF-410 was selected for further research. In this study, we tried to elucidate the pathogenesis of B. bassiana JEF-410 and the defense mechanisms of red mites at a transcriptome level. Red mites collected from a chicken farm were treated with B. bassiana JEF-410. When the mortality of infected red mites reached 50%, transcriptome analyses were performed to determine the interaction between B. bassiana JEF-410 and red mites. Uninfected red mites and non-infecting fungus served as controls. In B. bassiana JEF-410, up-regulated gene expression was observed in tryptophan metabolism and secondary metabolite biosynthesis pathways. Genes related to acetyl-CoA synthesis were up-regulated in tryptophan metabolism, suggesting that energy metabolism and stress management were strongly activated. Secondary metabolites associated with fungal up-regulated DEGs were related to the production of substances toxic to insects such as beauvericin and beauveriolide, efflux pump of metabolites, energy production, and resistance to stress. In red mites, physical and immune responses that strengthen the cuticle against fungal infection were highly up-regulated. From these gene expression analyses, we identified essential factors for fungal infection and subsequent defenses of red mites. These results will serve as a strong platform for explaining the interaction between B. bassiana JEF-410 and red mites in the stage of active infection.