Dimethyl sulfoxide and ascarosides improve the growth and yields of entomopathogenic nematodes in liquid cultures.

Increasing the infective juvenile (IJ) yields of entomopathogenic nematodes in monoxenic culture systems would reduce their production cost for the market. Ascarosides act as universal nematode pheromones with developmental and behavioral effects of nematodes. Dimethyl sulfoxide (DMSO) is unexpectedly found to enhance the IJ yields of entomopathogenic nematodes on fortified nutrient broth plates. In this study, the influence of selected ascarosides (ascr#7, ascr#9 and ascr#11) and DMSO in three concentrations on the IJ yields of S. carpocapsae All and H. bacteriophora H06 in liquid culture flasks was determined, and the critical development parameters (IJ recovery rate, number of hermaphrodites, number of visible eggs in a hermaphrodite) were examined for H. bacteriophora H06. The results demonstrated that IJ yields were significantly improved in the liquid medium containing 0.01 % DMSO, and 0.02 nM ascr#11 for S. carpocapsae All, and 0.1 % and 0.01 % DMSO and 0.02 pM ascr#11 for H. bacteriophora H06 in proper concentrations. Furthermore, it was discovered that increased recovery rate, hermaphrodite numbers and eggs in the hermaphrodites may contribute to the improved IJ yields of H. bacteriophora H06 in DMSO-supplemented liquid medium. Compared with the control flasks, the IJ yields from the flasks containing 0.01 % DMSO were 15 % and 35 % higher for S. carpocapsae All and H. bacteriophora H06 respectively in 15 days. The cost for ascarosides and DMSO is almost negligible. The results would provide practical technology for low-cost commercial production of these nematodes for pest management program.

Influence of the ascarosides on the recovery, yield and dispersal of entomopathogenic nematodes.

Recovery, yield, and dispersal are crucial developmental and behavioral indices for the infective juveniles of entomopathogenic nematodes, which are used as biocontrol agents against a variety of agricultural pests. Ascarosides and isopropylstilbene (ISO) function as nematode pheromones with developmental and behavioral effects. In this study, 11 synthesized ascarosides identified from Caenorhabditis elegans, together with ISO identified from Photorhabdus luminescens, were used to determine their influence on the IJ recovery, growth on agar plates, and dispersal of S. carpocapsae All, H. bacteriophora H06 and H. indica LN2 nematodes. Compared with the controls, significant differences in IJ recovery of three nematode species were detected from the supernatants of their corresponding bacterial cultures with almost all ascarosides or isopropylstilbene (ISO) at 0.04 nM in 6 days. The highest IJ recovery percentages was obtained from ISO and ascr#3 for All strain, ascr#5 and ascr#6 for LN2 strain, and ISO and ascr#12 for H06 strain. The ISO detected from Photorhabdus bacteria also induced IJ recovery of S. carpocapsae All. IJ yields was significantly stimulated by all synthesized compounds for S. carpocapsae All, and by most compounds for H. bacteriophora H06. The higher IJ yields varied with ascarosides. Ascr#7 and DMSO was common for the improved IJ yields of both nematode species. The three nematode species showed marked differences in dispersal behavior. In response to the ascarosides or ISO, S. carpocapsae All IJs actively moved with different dispersal rates, H. indica LN2 IJs in very low dispersal rates, and H. bacteriophora H06 IJs in variable and even suppressed rates on the agar plates at least during the assay period. Based on the synthesized standards, ascr#1, ascr#9 and ascr#10 were detected from three nematode species, ascr#5 and ascr#11 also from S. carpocapsae All and H. bacteriophora H06, and ascr#12 also from H. bacteriophora H06 and H. indica LN2. Ascr#9 was most abundant in three nematode species. Compared with the sterile PBS, significantly more ascr#1, ascr#9 and ascr#10 were detected from S. carpocapsae All and H. indica LN2, but less ascr#5 and ascr#11 from S. carpocapsae All, ascr#1, ascr#5, ascr#11 and ascr#12 from H. bacteriophora H06, in the corresponding bacterial supernatant. It seems that the bacterial supernatants could regulate the ascaroside secretion by the three nematode species. These results will provide useful clues for selecting suitable ascarosides to induce the recovery, improve the yield, and enhance the dispersal of the IJs of these nematodes.

A novel volatile deterrent from symbiotic bacteria of entomopathogenic nematodes fortifies field performances of nematodes against fall armyworm larvae.

The core elements of entomopathogenic nematode toxicity towards the fall armyworm Spodoptera frugiperda are associated with symbiotic bacteria. These microbes provide independent control effects and are reported to have repellency to insect pests. However, the ecological background of this nematode-bacteria-insect communication module is elusive. This work aims to identify key chemical cues which drive the trophic interactions through olfactory reception of S. frugiperda, and to inspire implementations with these isolated behavioral regulators in the corn field. A total of 657 volatiles were found within 13 symbiotic bacterial strains, and five of them induced significant electrophysiological responses of S. frugiperda larvae. 2-Hexynoic acid was demonstrated to exhibit a dominant role in deterring S. frugiperda larvae from feeding and localization. Field implementations with this novel volatile deterrent have resulted in fortified nematode applications. 2-Hexynoic acid acts as an excellent novel deterrent and presents remarkable application potential against fall armyworm larvae. Emissions from symbiotic bacteria of entomopathogenic nematodes are key players in chemical communication among insects, nematodes, and microbes. The olfactory perceptions and molecular targets for this volatile are worthy of future research.

Identification of the genes involved in growth characters of medicinal fungus Ophiocordyceps sinensis based on Agrobacterium tumefaciens-mediated transformation.

Ophiocordyceps sinensis, one of the well-known and precious fungal species in the world, parasitizes soil-dwelling larvae of ghost moths on the Tibetan Plateau. The genetic intractability of this extremely psychrophilic and slow-growing O. sinensis fungus is a major limitation on the molecular study. In this study, an Agrobacterium tumefaciens-mediated genetic transformation (ATMT) system for this fungus was established. ATMT procedure was optimized based on the fungal recipient, Agrobacterium strains, and different co-cultivation conditions. Blastospores were ideal recipients for this system. Acetosyringone (AS) was not essential for the transformation of O. sinensis. Sixty to 100 hygromycin B-resistant transformants per 1 × 106 blastospores were obtained. Southern blot analysis indicated the presence of a random single-copy integration of T-DNA into the O. sinensis genome. The insertional transformants with altered growth characters such as colony, blastospore, and fruiting body production were selected to identify the T-DNA flanking sequences by modified hiTAIL-PCR and FPNI-PCR techniques. Eight genes, including genes for an MFS transporter, a 2-oxoglutarate dehydrogenase, a DNA-directed RNA polymerase III complex subunit Rpc37, a cytochrome oxidase subunit I, a mitochondrial import inner membrane translocase subunit tim54, a cytidine deaminase, a phosphoribosylaminoimidazole carboxylase, and a histone H3-like centromeric protein cse-4 were identified. This ATMT system provides a useful tool for gene discovery and characterization of O. sinensis and contributes to the better understanding of the mysterious life cycle of O. sinensis and the molecular interaction between this fungus and its host insects.

Analysis of Volatile Components in Different Ophiocordyceps sinensis and Insect Host Products.

The artificial production of Ophiocordyceps sinensis mycelia and fruiting bodies and the Chinese cordyceps has been established. However, the volatile components from these O. sinensis products are not fully identified. An efficient, convenient, and widely used approach based on headspace solid-phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography and quadrupole time-of-flight mass spectrometry (GC×GC-QTOFMS) was developed for the extraction and the analysis of volatile compounds from three categories of 16 products, including O. sinensis fungus, Thitarodes hosts of O. sinensis, and the Chinese cordyceps. A total of 120 volatile components including 36 alkanes, 25 terpenes, 17 aromatic hydrocarbons, 10 ketones, 5 olefines, 5 alcohols, 3 phenols, and 19 other compounds were identified. The contents of these components varied greatly among the products but alkanes, especially 2,5,6-trimethyldecane, 2,3-dimethylundecane and 2,2,4,4-tetramethyloctane, are the dominant compounds in general. Three categories of volatile compounds were confirmed by partial least squares-discriminant analysis (PLS-DA). This study provided an ideal method for characterizing and distinguishing different O. sinensis and insect hosts-based products.

The high-throughput production of dsRNA against sacbrood virus for use in the honey bee Apis cerana (Hymenoptera: Apidae).

Sacbrood virus (SBV) is a serious threat to honey bees. Currently, there is no specific drug available for the treatment of SBV that does not affect the quality of the bee product. RNA interference (RNAi) is an important antiviral strategy for disease control. To effectively utilize this technology, the large-scale production and purification of double-stranded RNA (dsRNA) is necessary. Here, a dsRNA-expressing plasmid targeting the VP1 gene of Chinese sacbrood virus (CSBV) was constructed and expressed in Escherichia coli (E. coli) HT115 (DE3). After lysing and ethanol precipitation from E. coli, dsRNA VP1 was purified with an anion exchange chromatography column. Second instar larvae of Apis cerana were fed the purified dsRNA VP1. A significant decrease in larval mortality and the level of expression of the VP1 gene after CSBV infection was demonstrated after the ingestion of dsRNA VP1. This result provides a potential method for the large-scale production of dsRNA to protect A. cerana from CSBV infection.

Laboratory Rearing of Thitarodes armoricanus and Thitarodes jianchuanensis (Lepidoptera: Hepialidae), Hosts of the Chinese Medicinal Fungus Ophiocordyceps sinensis (Hypocreales: Ophiocordycipitaceae).

Ophiocordyceps sinensis (Ophiocordycipitaceae) is an entomopathogenic fungus endemic to the Tibetan Plateau, at elevations ranging between 3,000 and 5,000 m. The fungus-insect complex is useful in healthcare but limited in the field, so there is an urgent need to develop an artificial rearing system of both the fungus and its insect hosts. Large-scale artificial rearing of the Thitarodes insect hosts is crucial. This paper reports results of the artificial cultivation and complete life tables of two host species of O. sinensis, Thitarodes armoricanus and Thitarodes jianchuanensis (Lepidoptera: Hepialidae), under low-altitude laboratory conditions. The larvae were reared on carrots in plastic containers at 9­13°C and 50­80% RH. Both experimental insect species had long and unusual life cycle; it took 263­494 and 443­780 d for T. jianchuanensis and T. armoricanus, respectively, to complete a developmental cycle, including egg, larval instars L1-L9, pupa, and adult. The larvae did develop into pupae from the L7, L8, or L9 instar larvae. Although the total survival rates of both insect species were low (12.0% for T. jianchuanensis and 1.6% for T. armoricanus), the experimental populations successfully developed into the next generation owing to high egg production by fertilized females (averages of 703 and 355 eggs per female in the Yunnan and Sichuan species, respectively). Successful artificial rearing of host insect species for O. sinensis under low temperature conditions will allow the cultivation of this important fungus-insect complex to ensure its protection as a bio-resource and for commercial supply.

Rhf1 gene is involved in the fruiting body production of Cordyceps militaris fungus.

Cordyceps militaris is an important medicinal fungus. Commercialization of this fungus needs to improve the fruiting body production by molecular engineering. An improved Agrobacterium tumefaciens-mediated transformation (ATMT) method was used to select an insertional mutant (g38) which exhibited fast stromatal differentiation and increased yield. The Rhf1 gene encoding filamentation protein was destroyed by a single T-DNA and no Rhf1 transcription was detected in mutant g38. To verify the function of the Rhf1 gene, RNA interference plasmid and overexpression vector of the Rhf1 gene were constructed and transferred to the wild-type JM4 by ATMT. Fast stromatal differentiation and larger fruiting bodies were found in the RNAi-Rhf1 mutants (JM-iRhf1). In the overexpression mutants (JM-OERhf1), neither stromata nor fruiting bodies appeared. The rescued strain (38-OERhf1) showed similar growth characteristics as JM4. These results indicated that the Rhf1 gene was involved in the stromatal differentiation and the shape formation of fruiting bodies.

Regulation of morphogenesis and pathogenicity by OsMep2, OsCph1, and OsPes1 in dimorphic entomopathogenic fungus Ophiocordyceps sinensis (Hypocreales: Ophiocordycipitaceae).

Polarized growth plays a key role in all domains of their biology, including morphogenesis and pathogenicity of filamentous fungi. However, little information is available about the determinants of polarized growth. The fungal Mep2, Pes1, and Cph1 proteins were identified to be involved in the dimorphic transition between yeast and hyphal forms in Candida albicans. In this study, evidence that the dimorphic fungal entomopathogen Ophiocordyceps sinensis Mep2, Pes1, and Cph1 proteins are involved in polarized growth is presented. OsMep2 was significantly upregulated at aerial hyphae and conidia germination stages. OsCph1 was significantly upregulated at aerial hyphae, conidia initiation, and conidia germination stages, and OsPes1 was significantly upregulated at the conidia germination stage. Deletions of OsMep2, OsCph1, and OsPes1 provoked defects in the polarized growth. The abilities of hyphal formation and the yields of blastospores and conidia for the ∆ OsMep2, ∆OsCph1, and ∆ OsPes1 mutants were significantly reduced. The conidia yields of the ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were decreased by 69.17%, 60.90%, and 75.82%, respectively. Moreover, the pathogenicity of the ∆ OsMep2, ∆OsCph1, and ∆ OsPes1 mutants against Thitarodes xiaojinensis was significantly reduced. The mummification rate caused by wide type and ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were 36.98% ± 8.52%, 0.31% ± 0.63%, 1.15% ± 1.57%, and 19.69% ± 5.6%, respectively. These results indicated that OsMep2, OsCph1, and OsPes1 are involved in the regulation of hyphal formation, sporulation, and pathogenicity of O. sinensis. This study provided a basis for the understanding of the fungal dimorphic development and improving the efficiency of artificial cultivation of O. sinensis.

Ascarosides and Symbiotic Bacteria of Entomopathogenic Nematodes Regulate Host Immune Response in Galleria mellonella Larvae.

Insects protect themselves through their immune systems. Entomopathogenic nematodes and their bacterial symbionts are widely used for the biocontrol of economically important pests. Ascarosides are pheromones that regulate nematode behaviors, such as aggregation, avoidance, mating, dispersal, and dauer recovery and formation. However, whether ascarosides influence the immune response of insects remains unexplored. In this study, we co-injected ascarosides and symbiotic Photorhabdus luminescens subsp. kayaii H06 bacteria derived from Heterorhabditis bacteriophora H06 into the last instar larvae of Galleria mellonella. We recorded larval mortality and analyzed the expressions of AMPs, ROS/RNS, and LPSs. Our results revealed a process in which ascarosides, acting as enhancers of the symbiotic bacteria, co-induced G. mellonella immunity by significantly increasing oxidative stress responses and secreting AMPs (gallerimycin, gloverin, and cecropin). This led to a reduction in color intensity and the symbiotic bacteria load, ultimately resulting in delayed host mortality compared to either ascarosides or symbiotic bacteria. These findings demonstrate the cross-kingdom regulation of insects and symbiotic bacteria by nematode pheromones. Furthermore, our results suggest that G. mellonella larvae may employ nematode pheromones secreted by IJs to modulate insect immunity during early infection, particularly in the presence of symbiotic bacteria, for enhancing resistance to invasive bacteria in the hemolymph.

The Effect of Comb Cell Size on the Development of Apis mellifera Drones.

The growth and development of honeybees are influenced by many factors, one of which is the cell size of the brood comb. Larger worker bees can be obtained by being raised in bigger cells. However, whether cell size has the same effect on drone development is still unknown. Here, using 3D-printed foundations, we observed the development of drones kept in comb cells of different sizes from the late larval stage through eclosion. The results showed that drones in larger cell-size combs had heavier body weights, longer body lengths, and larger head widths, thorax widths, and abdomen widths compared to those in smaller cell-size combs. Furthermore, regardless of developmental stages, the drones' body weights increased linearly with the comb's cell size. However, the other morphological changes of drones in different developmental stages were out of proportion to the cell-size changes, resulting in smaller cells with a higher fill factor (thorax width/cell width). Our findings confirm that comb cell size affects the development of honeybees; drones become bigger when raised in large cells.

Tandemly expanded OR17b in Himalaya ghost moth facilitates larval food allocation via olfactory reception of plant-derived tricosane.

Herbivorous insects utilize intricate olfactory mechanisms to locate food plants. The chemical communication of insect-plant in primitive lineage offers insights into evolutionary milestones of divergent olfactory modalities. Here, we focus on a system endemic to the Qinghai-Tibetan Plateau to unravel the chemical and molecular basis of food preference in ancestral Lepidoptera. We conducted volatile profiling, neural electrophysiology, and chemotaxis assays with a panel of host plant organs to identify attractants for Himalaya ghost moth Thitarodes xiaojinensis larvae, the primitive host of medicinal Ophiocordyceps sinensis fungus. Using a DREAM approach based on odorant induced transcriptomes and subsequent deorphanization tests, we elucidated the odorant receptors responsible for coding bioactive volatiles. Contrary to allocation signals in most plant-feeding insects, T. xiaojinensis larvae utilize tricosane from the bulbil as the main attractant for locating native host plant. We deorphanized a TxiaOR17b, an indispensable odorant receptor resulting from tandem duplication of OR17, for transducing olfactory signals in response to tricosane. The discovery of this ligand-receptor pair suggests a survival strategy based on food location via olfaction in ancestral Lepidoptera, which synchronizes both plant asexual reproduction and peak hatch periods of insect larvae.

Large cells suppress the reproduction of Varroa destructor.

BACKGROUND: The parasitic mite, Varroa destructor has posed a threat to the health and survival of European honey bees, Apis mellifera worldwide. There is a prevailing belief that small comb cells could provide a management tool against Varroa mites. However, the hypothesis that smaller cells can impede Varroa reproduction has not been fully tested. Here, we tested this hypothesis under laboratory conditions by using two distinct Varroa in vitro rearing systems: one involved gelatin capsules of different sizes, specifically size 00 (0.95 mL) versus size 1 (0.48 mL), and the second consisted of brood comb cells drawn on 3D printed foundations with varying cell sizes, ranging from 5.0 mm to 7.0 mm at 0.5 mm intervals. RESULTS: The results showed that mother mites in size 00 cells had significantly lower fecundity and fertility compared to those in size 1 cells. Interestingly, the reproductive suppression in larger cells could be reversed by adding an extra worker larva. Similarly, gonopore size of mother mites was smaller in size 00 cells, but restored with another host larva. Furthermore, both the fecundity and fertility of mother mites decreased linearly with the size of brood comb cells. CONCLUSIONS: Our results suggest that the reproduction of V. destructor is hindered by larger cells, possibly because larger brood cells disperse or weaken host volatile chemical cues that are crucial for Varroa reproduction. The insights derived from this study are expected to hold significant implications for the implementation of Varroa management programs. © 2024 Society of Chemical Industry.

A ghost moth olfactory prototype of the lepidopteran sex communication.

Sex role differentiation is a widespread phenomenon. Sex pheromones are often associated with sex roles and convey sex-specific information. In Lepidoptera, females release sex pheromones to attract males, which evolve sophisticated olfactory structures to relay pheromone signals. However, in some primitive moths, sex role differentiation becomes diverged. Here, we introduce the chromosome-level genome assembly from ancestral Himalaya ghost moths, revealing a unique olfactory evolution pattern and sex role parity among Lepidoptera. These olfactory structures of the ghost moths are characterized by a dense population of trichoid sensilla, both larger male and female antennal entry parts of brains, compared to the evolutionary later Lepidoptera. Furthermore, a unique tandem of 34 odorant receptor 19 homologs in Thitarodes xiaojinensis (TxiaOr19) has been identified, which presents overlapped motifs with pheromone receptors (PRs). Interestingly, the expanded TxiaOr19 was predicted to have unconventional tuning patterns compared to canonical PRs, with nonsexual dimorphic olfactory neuropils discovered, which contributes to the observed equal sex roles in Thitarodes adults. Additionally, transposable element activity bursts have provided traceable loci landscapes where parallel diversifications occurred between TxiaOr19 and PRs, indicating that the Or19 homolog expansions were diversified to PRs during evolution and thus established the classic sex roles in higher moths. This study elucidates an olfactory prototype of intermediate sex communication from Himalaya ghost moths.

Characterization in biological traits of entomopathogenic nematodes isolated from North China.

In a series of bioassays, thirty-one isolates that were collected from diverse locations in northern China and the laboratory kept isolate Steinernema carpocapsae All, were compared in order to select superior isolates for biological control of Bradysia odoriphaga. Virulence of the isolates against B. odoriphaga was significantly different among nematode isolates. Tolerance of infective juveniles (IJs) to heat, cold, and desiccation differed significantly among and within species. Strains from S. carpocapsae, S. ceratophorum, S. longicaudum, Heterorhabditis indica, and H. bacteriophora were more heat tolerant than strains from S. feltiae, S. hebeiense, S. monticolum, and H. megidis. Heterorhabditis megidis, H. bacteriophora, and S. carpocapsae showed better cold tolerance than the other species. High desiccation tolerance was recorded for S. carpocapsae, S. hebeiense, and S. ceratophorum. The infectivity of IJ of these species against Galleria mellonella larvae was not significantly different between the treated and non-treated IJ after the nematodes had been exposed to 40°C for 2 h, -5°C for 8 h or 25% glycerin for 72 h. Nematode survival was significantly affected by exposure time and IJ concentration when exposed to 40°C or -5°C. All nematode isolates lost their infectivity against G. mellonella after exposure to -5°C for 16 h, except for H. megidis LFS10, which had a low infectivity of 3.3%. A hierarchical classification analysis classified the isolates in four main clusters. The fourth cluster, composed of 13 isolates, grouped the isolates that scored well for most traits.

Differentially expressed genes of Coptotermes formosanus (Isoptera: Rhinotermitidae) challenged by chemical insecticides.

Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) termites are harmful social insects to wood constructions. The current control methods heavily depend on the chemical insecticides with increasing resistance. Analysis of the differentially expressed genes mediated by chemical insecticides will contribute to the understanding of the termite resistance to chemicals and to the establishment of alternative control measures. In the present article, a full-length cDNA library was constructed from the termites induced by a mixture of commonly used insecticides (0.01% sulfluramid and 0.01% triflumuron) for 24 h, by using the RNA ligase-mediated Rapid Amplification cDNA End method. Fifty-eight differentially expressed clones were obtained by polymerase chain reaction and confirmed by dot-blot hybridization. Forty-six known sequences were obtained, which clustered into 33 unique sequences grouped in 6 contigs and 27 singlets. Sixty-seven percent (22) of the sequences had counterpart genes from other organisms, whereas 33% (11) were undescribed. A Gene Ontology analysis classified 33 unique sequences into different functional categories. In general, most of the differential expression genes were involved in binding and catalytic activity.

Microbial landscapes of the rhizosphere soils and roots of Luffa cylindrica plant associated with Meloidogyne incognita.

Introduction: The root-knot nematodes (RKN), especially Meloidogyne spp., are globally emerging harmful animals for many agricultural crops. Methods: To explore microbial agents for biological control of these nematodes, the microbial communities of the rhizosphere soils and roots of sponge gourd (Luffa cylindrica) infected and non-infected by M. incognita nematodes, were investigated using culture-dependent and -independent methods. Results: Thirty-two culturable bacterial and eight fungal species, along with 10,561 bacterial and 2,427 fungal operational taxonomic units (OTUs), were identified. Nine culturable bacterial species, 955 bacterial and 701 fungal OTUs were shared in both four groups. More culturable bacterial and fungal isolates were detected from the uninfected soils and roots than from the infected soils and roots (except no fungi detected from the uninfected roots), and among all samples, nine bacterial species (Arthrobacter sp., Bacillus sp., Burkholderia ambifaria, Enterobacteriaceae sp., Fictibacillus barbaricus, Microbacterium sp., Micrococcaceae sp., Rhizobiaceae sp., and Serratia sp.) were shared, with Arthrobacter sp. and Bacillus sp. being dominant. Pseudomonas nitroreducens was exclusively present in the infested soils, while Mammaliicoccus sciuri, Microbacterium azadirachtae, and Priestia sp., together with Mucor irregularis, Penicillium sp., P. commune, and Sordariomycetes sp. were found only in the uninfected soils. Cupriavidus metallidurans, Gordonia sp., Streptomyces viridobrunneus, and Terribacillus sp. were only in the uninfected roots while Aspergillus sp. only in infected roots. After M. incognita infestation, 319 bacterial OTUs (such as Chryseobacterium) and 171 fungal OTUs (such as Spizellomyces) were increased in rhizosphere soils, while 181 bacterial OTUs (such as Pasteuria) and 166 fungal OTUs (such as Exophiala) rose their abundance in plant roots. Meanwhile, much more decreased bacterial or fungal OTUs were identified from rhizosphere soils rather than from plant roots, exhibiting the protective effects of host plant on endophytes. Among the detected bacterial isolates, Streptomyces sp. TR27 was discovered to exhibit nematocidal activity, and B. amyloliquefaciens, Bacillus sp. P35, and M. azadirachtae to show repellent potentials for the second stage M. incognita juveniles, which can be used to develop RKN bio-control agents. Discussion: These findings provided insights into the interactions among root-knot nematodes, host plants, and microorganisms, which will inspire explorations of novel nematicides.

Occurrence and prevalence of seven bee viruses in Apis mellifera and Apis cerana apiaries in China.

Populations of Apis mellifera and Apis cerana in China were surveyed for seven bee viruses: acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), sacbrood virus (SBV), and Isreal acute paralysis virus (IAPV). No KBV was detected from any samples of the two species. In A. mellifera, DWV was the most prevalent virus, but in A. cerana, SBV was the dominant. Simultaneous multiple infections of viruses were common in both species. This is the first report of detection of IAPV and CBPV in A. cerana.

Entomopathogenic nematode production and application technology.

Production and application technology is critical for the success of entomopathogenic nematodes (EPNs) in biological control. Production approaches include in vivo, and in vitro methods (solid or liquid fermentation). For laboratory use and small scale field experiments, in vivo production of EPNs appears to be the appropriate method. In vivo production is also appropriate for niche markets and small growers where a lack of capital, scientific expertise or infrastructure cannot justify large investments into in vitro culture technology. In vitro technology is used when large scale production is needed at reasonable quality and cost. Infective juveniles of entomopathogenic nematodes are usually applied using various spray equipment and standard irrigation systems. Enhanced efficacy in EPN applications can be facilitated through improved delivery mechanisms (e.g., cadaver application) or optimization of spray equipment. Substantial progress has been made in recent years in developing EPN formulations, particularly for above ground applications, e.g., mixing EPNs with surfactants or polymers or with sprayable gels. Bait formulations and insect host cadavers can enhance EPN persistence and reduce the quantity of nematodes required per unit area. This review provides a summary and analysis of factors that affect production and application of EPNs and offers insights for their future in biological insect suppression.

Cordyceps militaris (Hypocreales: Cordycipitaceae): transcriptional analysis and molecular characterization of cox1 and group I intron with putative LAGLIDADG endonuclease.

The full-length cytochrome c oxidase subunit I gene (cox1) containing a group I intron was isolated from an important medical fungus Cordyceps militaris (Cordycipitaceae). The open reading frame (ORF) of 1,593 nucleotides encoded a predicted protein COX1 of 530 amino acids. The group I intron encoded a putative homing endonuclease (HE) with two LAGLIDADG motifs. RT-PCR and Northern analysis showed a mature transcript of spliced cox1. Both 5'exon-intron and intron-3'exon junctions were also found by RT-PCR, suggesting the possible presence of unspliced cox1 RNA in total RNA. Sequence comparison by BLASTn showed that the coding region of cox1 (CRcox1) of C. militaris had significant similarities to those of related species (such as Cordyceps bassiana and C. brongniartii), while the intron had no significant homologous sequences of Cordycipitaceae fungi in NCBI database. The phylogenetic tree based on the CRcox1 confirmed the present taxonomic status of related species, but the cox1 introns were phylogenetically distinct. Compared to C. bassiana and C. brongniartii, the cox1 intron of C. militaris exhibited specific splicing site and different intronic ORF. The analysis of the folding RNA structures of the known cox1 introns from Cordyceps species showed different base pairs and conserved regions (P1-P10) in their structures. The present results provide useful information on the studies of cox1 intron splicing and Cordyceps evolution.