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Significant progress has been made in the biochemical and genetic characterization of the host-pathogen interaction mediated by insect pathogenic fungi, with the most widely studied being the Ascomycetes (Hypocrealean) fungi, Metarhizium robertsii and Beauveria bassiana. However, few studies have examined the consequences and effects of host (insect) microbes, whether compatible or antagonistic, on the development and survival of entomopathogenic fungi. Host microbes can act on the insect cuticular surface, within the gut, in specialized insect microbe hosting structures, and within cells, and they include a wide array of facultative and/or obligate exosymbionts and endosymbionts. The insect microbiome differs across developmental stages and in response to nutrition (e.g., different plant hosts for herbivores) and environmental conditions, including exposure to chemical insecticides. Here, we review recent advances indicating that insect-pathogenic fungi have evolved a spectrum of strategies for exploiting or suppressing host microbes, including the production of antimicrobial compounds that are expressed at discrete stages of the infection process. Conversely, there is increasing evidence that some insects have acquired microbes that may be specialized in the production of antifungal compounds to combat infection by (entomopathogenic) fungi. Consideration of the insect microbiome in fungal insect pathology represents a new frontier that can help explain previously obscure ecological and pathological aspects of the biology of entomopathogenic fungi. Such information may lead to novel approaches to improving the efficacy of these organisms in pest control efforts.

Background Hytrosaviruses (SGHVs; Hytrosaviridae family) are double-stranded DNA (dsDNA) viruses that cause salivary gland hypertrophy (SGH) syndrome in flies. Two structurally and functionally distinct SGHVs are recognized; Glossina pallidipes SGHV (GpSGHV) and Musca domestica SGHV (MdSGHV), that infect the hematophagous tsetse fly and the filth-feeding housefly, respectively. Genome sizes and gene contents of GpSGHV (~ 190 kb; 160–174 genes) and MdSGHV (~ 124 kb; 108 genes) may reflect an evolution with the SGHV-hosts resulting in differences in pathobiology. Whereas GpSGHV can switch from asymptomatic to symptomatic infections in response to certain unknown cues, MdSGHV solely infects symptomatically. Overt SGH characterizes the symptomatic infections of SGHVs, but whereas MdSGHV induces both nuclear and cellular hypertrophy (enlarged non-replicative cells), GpSGHV induces cellular hyperplasia (enlarged replicative cells). Compared to GpSGHV’s specificity to Glossina species, MdSGHV infects other sympatric muscids. The MdSGHV-induced total shutdown of oogenesis inhibits its vertical transmission, while the GpSGHV’s asymptomatic and symptomatic infections promote vertical and horizontal transmission, respectively. This paper reviews the coevolution of the SGHVs and their hosts (housefly and tsetse fly) based on phylogenetic relatedness of immune gene orthologs/paralogs and compares this with other virus-insect models. Results Whereas MdSGHV is not vertically transmitted, GpSGHV is both vertically and horizontally transmitted, and the balance between the two transmission modes may significantly influence the pathogenesis of tsetse virus. The presence and absence of bacterial symbionts (Wigglesworthia and Sodalis) in tsetse and Wolbachia in the housefly, respectively, potentially contributes to the development of SGH symptoms. Unlike MdSGHV, GpSGHV contains not only host-derived proteins, but also appears to have evolutionarily recruited cellular genes from ancestral host(s) into its genome, which, although may be nonessential for viral replication, potentially contribute to the evasion of host’s immune responses. Whereas MdSGHV has evolved strategies to counteract both the housefly’s RNAi and apoptotic responses, the housefly has expanded its repertoire of immune effector, modulator and melanization genes compared to the tsetse fly. Conclusions The ecologies and life-histories of the housefly and tsetse fly may significantly influence coevolution of MdSGHV and GpSGHV with their hosts. Although there are still many unanswered questions regarding the pathogenesis of SGHVs, and the extent to which microbiota influence expression of overt SGH symptoms, SGHVs are attractive ‘explorers’ to elucidate the immune responses of their hosts, and the transmission modes of other large DNA viruses.

The phloem-feeding Southern chinch bug, Blissus insularis , harbors a high density of the exocellular bacterial symbiont Burkholderia in the lumen of specialized midgut crypts. Here we developed an organ culture method that initially involved incubating the B. insularis crypts in osmotically balanced insect cell culture medium. This approach enabled the crypt-inhabiting Burkholderia spp. to make a transition to an in vitro environment and to be subsequently cultured in standard bacteriological media. Examinations using ribotyping and BOX-PCR fingerprinting techniques demonstrated that most in vitro -produced bacterial cultures were identical to their crypt-inhabiting Burkholderia counterparts. Genomic and physiological analyses of gut-symbiotic Burkholderia spp. that were isolated individually from two separate B. insularis laboratory colonies revealed that the majority of individual insects harbored a single Burkholderia ribotype in their midgut crypts, resulting in a diverse Burkholderia community within each colony. The diversity was also exhibited by the phenotypic and genotypic characteristics of these Burkholderia cultures. Access to cultures of crypt-inhabiting bacteria provides an opportunity to investigate the interaction between symbiotic Burkholderia spp. and the B. insularis host. Furthermore, the culturing method provides an alternative strategy for establishing in vitro cultures of other fastidious insect-associated bacterial symbionts. IMPORTANCE An organ culture method was developed to establish in vitro cultures of a fastidious Burkholderia symbiont associated with the midgut crypts of the Southern chinch bug, Blissus insularis . The identities of the resulting cultures were confirmed using the genomic and physiological features of Burkholderia cultures isolated from B. insularis crypts, showing that host insects maintained the diversity of Burkholderia spp. over multiple generations. The availability of characterized gut-symbiotic Burkholderia cultures provides a resource for genetic manipulation of these bacteria and for examination of the mechanisms underlying insect-bacterium symbiosis.

This investigation documents the expression of the in vivo dimorphic program exhibited by the insect mycopathogen Metarhizium rileyi. This insect mycopathogen represents the key mortality factor regulating various caterpillar populations in legumes, including subtropical and tropical soybeans. Using two hosts and M. rileyi isolates, we have measured M. rileyi growth rates under in vivo and in vitro conditions and have assessed the pathogen’s impact on host fitness. Significantly, the hyphal bodies-to-mycelia transition that occurs at the late infection stage is regulated by a quorum-sensing molecule(s) (QSM) that triggers hyphal bodies (Hb) to synchronously switch to the tissue-invasive mycelia. Within hours of this transition, the host insect succumbs to mycosis. The production of the QS chemical(s) occurs when a quorum of Hb is produced in the hemolymph (late-stage infection). Furthermore, the QS activity detected in late-stage infected sera is unique and is unrelated to any known fungal QSM. The lack of similar QS activity from conditioned media of M. rileyi suggests that the chemical signal(s) that mediates the dimorphic switch is produced by host tissues in response to a quorum of hyphal bodies produced in the host hemolymph. The serum-based QS activity is retained after lyophilization, mild heat treatment, and proteinase digestion. However, attempts to extract/identify the QSM have been unsuccessful. Results suggest that the observed hyphal body-to-mycelia transition is a multi-step process involving more than one chemical signal.

House flies (Musca domestica L.) throughout the world are infected with the salivary gland hypertrophy virus MdSGHV (Hytrosaviridae). Although the primary route of infection is thought to be via ingestion, flies that are old enough to feed normally are resistant to infection per os, suggesting that the peritrophic matrix (PM) is a barrier to virus transmission. Histological examination of the peritrophic matrix of healthy flies revealed a multilaminate structure produced by midgut cells located near the proventriculus. SEM revealed the PM to form a confluent sheet surrounding the food bolus with pores/openings less than 10 nm in diameter. TEM revealed the PM to be multilayered, varying in width from 350 to 900 nm, and generally thinner in male than in female flies. When flies were fed on the reducing agents dithiothetriol (DTT) or tris (2-caboxyethyl)phosphine hydrochloride (TCEP) for 48 h before per os exposure to the virus, infection rates increased 10- to 20-fold compared with flies that did not receive the reducing agent treatments. PM’s from flies treated with DTT and TCEP displayed varying degrees of disruption, particularly in the outer layer, and lacked the electron-dense inner layer facing the ectoperitrophic space. Both drugs were somewhat toxic to the flies, resulting in > 40% mortality at doses greater than 10 mM (DTT) or 5 mM (TCEP). DTT increased male fly susceptibility (55.1% infected) more than that of females (7.8%), whereas TCEP increased susceptibility of females (42.9%) more than that of males (26.2%). The cause for the sex differences in response to oral exposure the reducing agents is unclear. Exposing flies to food treated with virus and the reducing agents at the same time, rather than pretreating flies with the drugs, had no effect on susceptibility to the virus. Presumably, the reducing agent disrupted the enveloped virus and acted as a viricidal agent. In summary, it is proposed that the reducing agents influence integrity of the PM barrier and increase the susceptibility of flies to infection by MdSGHV.

Hytrosaviridae family members replicate in the salivary glands (SGs) of their adult dipteran hosts and are transmitted to uninfected hosts via saliva during feeding. Despite inducing similar gross symptoms (SG hypertrophy; SGH), hytrosaviruses (SGHVs) have distinct pathobiologies, including sex-ratio distortions in tsetse flies and refusal of infected housefly females to copulate. Via unknown mechanism(s), SGHV replication in other tissues results in reduced fecundity in tsetse flies and total shutdown of vitellogenesis and sterility in housefly females. We hypothesized that vitellogenesis shutdown was caused by virus-induced modulation of hormonal titers. Here, we used RNA-Seq to investigate virus-induced modulation of host genes/pathways in healthy and virus-infected houseflies, and we validated expression of modulated genes (n=23) by RT-qPCR. We also evaluated the levels and activities of hemolymph AMPs, levels of endogenous sesquiterpenoids, and impacts of exogenous hormones on ovarian development in viremic females. Of the 973 housefly unigenes that were significantly modulated (padj ≤0.01, log2FC ≤−2.0 or ≥2.0), 446 and 527 genes were downregulated and upregulated, respectively. While the most downregulated genes were related to reproduction (embryogenesis/oogenesis), the repertoire of upregulated genes was overrepresented by genes related to non-self recognition, ubiquitin-protease system, cytoskeletal traffic, cellular proliferation, development and movement, and snRNA processing. Overall, the virus, Musca domestica salivary gland hytrosavirus (MdSGHV), induced the upregulation of various components of the siRNA, innate antimicrobial immune, and autophagy pathways. We show that MdSGHV undergo limited morphogenesis in the corpora allata/corpora cardiaca (CA/CC) complex of M. domestica. MdSGHV replication in CA/CC potentially explains the significant reduction of hemolymph sesquiterpenoids levels, the refusal to mate, and the complete shutdown of egg development in viremic females. Notably, hormonal rescue of vitellogenesis did not result in egg production. The mechanism underlying MdSGHV-induced sterility has yet to be resolved.

The termite gut accomplishes key physiologic functions that underlie termite symbiosis and sociality. However, potential candidate functions of the host-symbiont holobiome have not yet been explored across seemingly divergent processes such as digestion, immunity, caste differentiation, and xenobiotic tolerance. This study took a meta-analysis approach for concurrently studying host and symbiont gut metatranscriptome responses of the lower termite Reticulitermes flavipes, which has ancestral characteristics and hosts a diverse mix of eukaryotic and bacterial symbionts. Thirteen treatments were compared from 5 categories (dietary, social, hormonal, immunological, and xenobiotic), revealing 3 main insights. First, each of the 5 tested colonies had distinct magnitudes of transcriptome response, likely as a result of unique symbiont profiles, which highlights the uniqueness of individual termite colonies. Second, after normalization to standardize colony response magnitudes, unique treatment-linked metatranscriptome topologies became apparent. Third, despite colony and topology differences, 4 co-opted master genes emerged that were universally responsive across diverse treatments. These master genes encode host functions related to protein translation and symbiont functions related to protein degradation and pore formation in microbial cell walls. Three of the 4 master genes were from co-evolved protist symbionts, highlighting potentially co-evolved roles for gut symbiota in coordinating functional responses of the collective host-symbiont holobiome. Lastly, for host genes identified, these results provide annotations of recent termite genome sequences. By revealing conserved domain genes, as well as apparent roles for gut symbiota in holobiome regulation, this study provides new insights into co-opted eusocial genes and symbiont roles in termite sociobiology.

Salivary gland hytrosaviruses (SGHVs) are entomopathogenic dsDNA, enveloped viruses that replicate in the salivary glands (SGs) of the adult dipterans, Glossina spp (GpSGHV) and Musca domestica (MdSGHV). Although belonging to the same virus family (Hytrosaviridae), SGHVs have distinct morphologies and pathobiologies. Two GpSGHV strains potentially account for the differential pathologies in lab-bred tsetse. New data suggest incorporation of host-derived cellular proteins and lipids into mature SGHVs. In addition to within the SGs, MdSGHV undergoes limited replication in the corpora allata, potentially disrupting hormone biosynthesis, and GpSGHV replicates in the milk glands providing a transmission conduit to progeny tsetse. Whereas MdSGHV is a potential biocontrol agent, the vertically transmitted GpSGHV is unsuitable for tsetse vector control but does jeopardize tsetse mass rearing.

In this paper it is proposed that one potential component by which the Musca domestica salivary gland hypertrophy virus (MdSGHV) infects individual flies is through cuticular damage. Breaks in the cuticle allow entry of the virus into the hemocoel causing the infection. Male flies typically have a higher rate of infection and a higher rate of cuticular damage than females. A model for the transmission of MdSGHV was formulated assuming several potential and recognized means of transmission. The model yields results that are in agreement with field data that measured the infection rate in house flies on dairy farms in Florida. The results from this model indicate that MdSGHV will be maintained at a stable rate within house fly populations and support the future use of MdSGHV as a birth control agent in house fly management.

Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of the trypanosomes, which cause human African trypanosomosis (HAT) or sleeping sickness in humans and African animal trypanosomosis (AAT) or nagana in animals. Due to the lack of effective vaccines and inexpensive drugs for HAT, and the development of resistance of the trypanosomes against the available trypanocidal drugs, vector control remains the most efficient strategy for sustainable management of these diseases. Among the control methods used for tsetse flies, Sterile Insect Technique (SIT), in the frame of area-wide integrated pest management (AW-IPM), represents an effective tactic to suppress and/or eradicate tsetse flies. One constraint in implementing SIT is the mass production of target species. Tsetse flies harbor obligate bacterial symbionts and salivary gland hypertrophy virus which modulate the fecundity of the infected flies. In support of the future expansion of the SIT for tsetse fly control, the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture implemented a six year Coordinated Research Project (CRP) entitled “Improving SIT for Tsetse Flies through Research on their Symbionts and Pathogens”. The consortium focused on the prevalence and the interaction between the bacterial symbionts and the virus, the development of strategies to manage virus infections in tsetse colonies, the use of entomopathogenic fungi to control tsetse flies in combination with SIT, and the development of symbiont-based strategies to control tsetse flies and trypanosomosis. The results of the CRP and the solutions envisaged to alleviate the constraints of the mass rearing of tsetse flies for SIT are presented in this special issue.

Termites are highly eusocial insects that thrive on recalcitrant materials like wood and soil and thus play important roles in global carbon recycling and also in damaging wooden structures. Termites, such as Reticulitermes flavipes (Rhinotermitidae), owe their success to their ability to extract nutrients from lignocellulose (a major component of wood) with the help of gut-dwelling symbionts. With the aim to gain new insights into this enzymatic process we provided R. flavipes with a complex lignocellulose (wood) or pure cellulose (paper) diet and followed the resulting differential gene expression on a custom oligonucleotide-microarray platform. We identified a set of expressed sequence tags (ESTs) with differential abundance between the two diet treatments and demonstrated the source (host/symbiont) of these genes, providing novel information on termite nutritional symbiosis. Our results reveal: (1) the majority of responsive wood- and paper-abundant ESTs are from host and symbionts, respectively; (2) distinct pathways are associated with lignocellulose and cellulose feeding in both host and symbionts; and (3) sets of diet-responsive ESTs encode putative digestive and wood-related detoxification enzymes. Thus, this study illuminates the dynamics of termite nutritional symbiosis and reveals a pool of genes as potential targets for termite control and functional studies of termite-symbiont interactions.

Distinct isolates of the invertebrate pathogenic alga Helicosporidium sp., collected from different insect hosts and different geographic locations, were processed to sequence the 18S rDNA and β-tubulin genes. The sequences were analyzed to assess genetic variation within the genus Helicosporidium and to design Helicosporidium-specific 18S rDNA primers. The specificity of these primers was demonstrated by testing not only on the Helicosporidium sp. isolates, but also on two trebouxiophyte algae known to be close Helicosporidium relatives, Prototheca wickerhamii and Prototheca zopfii. The genus-specific primers were used to develop a culture-independent assay aimed at detecting the presence of Helicosporidium spp. in environmental waters. The assay was based on the PCR amplification of 18SrDNA gene fragments from metagenomic DNA preparations, and it resulted in the amplification of detectable products for all sampled sites. Phylogenetic analyses that included the environmental sequences demonstrated that all amplification products clustered in a strongly supported, monophyletic Helicosporidium clade, thereby validating the metagenomic approach and the taxonomic origin of the produced environmental sequences. In addition, the phylogenetic analyses established that Helicosporidium spp. isolated from coleopteran hosts are more closely related to each other than they are to the isolate collected from a dipteran host. Finally, the phylogenetic trees depicted intergeneric relationships that supported a Helicosporidium-Prototheca cluster but did not support a Helicosporidium-Coccomyxa grouping, suggesting that pathogenicity to invertebrates evolved at least twice independently within the trebouxiophyte green algae.

Dense populations of extracellular bacteria were detected in midgut crypts of the southern chinch bug, Blissus insularis Barber (Hemiptera: Blissidae). Examination by epifluorescent and transmission electron microscopy revealed that the bacteria covered the luminal surface of the crypts and filled the entire lumen. Attempts to culture the extracellular endosymbionts in various media failed. Sequencing and phylogenetic analyses of 16S rRNA gene clones obtained from insects of five Florida populations showed high nucleotide homology to either betaproteobacterial Burkholderia spp. (243 clones from five populations) or gammaproteobacterial Pseudomonas spp. (58 clones from one population). Using Burkholderia-specific primers, bacteria were detected in the egg, nymph, and adult stages. Fluorescent in situ hybridization with genus-specific oligonucleotide probes confirmed the localization of Burkholderia in the crypts. Quantitative real-time PCR showed that antibiotic treatments of nymphs significantly reduced the amount of Burkholderia 16S rRNA gene copies in chinch bugs sampled 11 days after the treatment. Furthermore, these treatments resulted in retarded development and high mortality of B. insularis, indicating a beneficial impact of Burkholderia on its host.

A two-year field study was conducted in an orange grove in the United States (Florida) to characterize the phenology of the entomopathogen Hirsutella citriformis Speare infecting adults of the Asian citrus psyllid, Diaphorina citri Kuwayama. On the average, 23% of adults observed on mature leaves were killed by H. citriformis. These dead psyllids were characterized as being mummified and covered to various extents by synnemata produced by the fungus. Mummified cadavers were most abundant on citrus leaves during the fall and winter months, with the maximal percentage of mummified psyllids sometimes exceeding 75% of the total number of adults observed. Mummified cadavers were nearly absent each spring, presumably because relative humidity levels were suboptimal for the fungus at this time. Based on dispersion analyses, a monitoring plan for mummified cadavers would best include multiple samples in individual trees as well as multiple tree samples throughout a grove. Mummified cadavers with synnemata, which serve as point sources for new infections of the fungus in psyllids, were observed to remain on leaves for a mean of 68 days (one cadaver remained on a leaf for 168 days). Rainfall was positively correlated with the number of days mummies remained on leaves while mean daily air temperature was negatively correlated. Mummified cadavers were abundant in the summer during 2006 but not during 2007. This may have been a density-dependent consequence of low psyllid host populations in the grove in 2007. Alternatively, combination sprays of oil and copper applied during 2007 may have suppressed the fungus. This latter possibility prompted a laboratory investigation into the toxicity to H. citriformis of six chemicals commonly used in citrus. Copper hydroxide, petroleum oil, and elemental sulfur at maximum label rates each significantly reduced the infectivity of a laboratory culture of H. citriformis while copper sulfate pentahydrate, aluminum tris and alpha-keto/humic acids did not. This finding indicates that citrus growers interested in capitalizing on H. citriformis as a biological control agent of D. citri should avoid applying high rates of copper hydroxide, oil or sulfur.

Past surveys of feral house fly populations have shown that Musca domestica salivary gland hypertrophy virus (MdSGHV) has a worldwide distribution, with an average prevalence varying between 0.5% and 10%. How this adult-specific virus persists in nature is unknown. In the present study, experiments were conducted to examine short-term transmission efficiency and long-term persistence of symptomatic MdSGHV infections in confined house fly populations. Average rates of disease transmission from virus-infected to healthy flies in small populations of 50 or 100 flies ranged from 3% to 24% and did not vary between three tested geographical strains that originated from different continents. Introduction of an initial proportion of 40% infected flies into fly populations did not result in epizootics. Instead, long-term observations demonstrated that MdSGHV infection levels declined over time, resulting in a 10% infection rate after passing through 10 filial generations. In all experiments, induced disease rates were significantly higher in male flies than in female flies and might be explained by male-specific behaviors that increased contact with viremic flies and/or virus-contaminated surfaces.

SummaryIn this study, eight commercial and three feral bumble bee (Bombus impatiens Cresson and Bombus pensyivanicus DeGeer respectively, Hymenoptera: Apidae) colonies were tested for the presence of Kodamaea ohmeri (Ascomycota: Saccharomycotina), a yeast known to attract small hive beetles (SHB) (Aethina tumida Murray, Coleoptera: Nitidulidae) to honey bee (Apis mellifera L., Hymenoptera: Apidae) colonies. Swabs of commercial bumble bee colonies and homogenates of bumble bee colony components (adults, brood, honey, pollen and wax) were plated on selective media. The resulting yeast isolates were compared to K ohmeri previously isolated from SHB. Yeasts were detected in all of the commercial bumble bee colony swab samples (n = 56) and a selected subsample was shown through molecular, chemical, and microbiological evidence to be K. ohmeri. For the second part of the study, feral bumble bee colonies were excavated and evaluated for the presence of any SHB life stage (none was found). Adult bees and swabs fr...

cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma – mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.

Cell-1 is a host-derived beta-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell-1 using eukaryotic (Baculovirus Expression Vector System; BEVS) and prokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily obtained in solubilized form and more active than the E. coli–expressed enzyme. Km and Vmax values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993% w/v and 1.056 µmol/min/mg. Additional characterization studies on the BEVS-expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5–7.5 and 50–60°C, is inhibited by EDTA, and displays enhanced activity up to 70°C in the presence of CaCl2. These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol-oxidizing laccases, and others. © 2010 Wiley Periodicals, Inc.

Frankliniellafusca (Hinds) (Thysanoptera: Thripidae) is the predominant thrips species found inhabiting and reproducing in peanut, Arachis hypogaea L. (Fabaceae), and is one of at least seven thrips species reported to transmit Tomato spotted wilt virus (TSWV). The entomogenous nematode Thripinema fuscum Tipping & Nguyen (Tylenchida: Allantonematidae), a natural enemy of F. fusca, parasitizes larval and adult populations under field conditions. All known Thripinema species render the host female thrips sterile and have the potential to suppress pest populations to near extinction. As a result, secondary spread of TSWV in peanut is reduced. Reduction of the virus under field conditions may also be due to lower transmission rates caused by parasite-induced alterations in host feeding behavior. Therefore, the feeding rates of healthy and parasitized F. fusca male and female cohorts on leaf discs were recorded daily for 10 days and digital images were subjected to image analysis and viral transmission rates were compared daily using double antibody sandwich enzyme-linked immunosorbent assay. Thripinema fuscum reduced the feeding of female F. fusca by nearly 65%, and the ability of females to transmit TSWV by 50%. Potential mechanisms underlying the parasite-induced alterations in feeding behavior and transmission are discussed. Parasitism by T. fuscum significantly reduced male longevity, but female longevity was not affected. These results provide further evidence that T. fuscum aids in regulating viruliferous F. fusca pest populations and suggests its potential as a biological control agent for inoculative release in peanut.

TheMusca domesticasalivary gland hypertrophy virus (MdSGHV) is a large dsDNA virus that infects and sterilizes adult houseflies. The transcriptome of this newly described virus was analysed by rapid amplification of cDNA 3′-ends (3′-RACE) and RT-PCR. Direct sequencing of 3′-RACE products revealed 78 poly(A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs not amplified by 3′-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either 3′-RACE or RT-PCR. A series of 5′-RACE reactions were conducted on selected ORFs that were identified by 3′-RACE to be transcribed in tandem (tandem transcripts). In the majority of cases, the downstream ORFs were detected as single transcripts as well as components of the tandem transcripts, whereas the upstream ORFs were found only in tandem transcripts. The only exception was the upstream ORF MdSGHV084, which was differentially transcribed as a single transcript at 1 and 2 days post-infection (days p.i.) and as a tandem transcript (MdSGHV084/085) at 2 days p.i. Transcriptome analysis of MdSGHV detected splicing in the 3′ untranslated region (3′-UTR) and extensive heterogeneity in the polyadenylation signals and cleavage sites. In addition, 23 overlapping antisense transcripts were found. In conclusion, sequencing the 3′-RACE products without cloning served as an alternative approach to detect both 3′-UTRs and transcript variants of this large DNA virus.

The MdSGHV is a double-stranded DNA virus that replicates in the salivary glands of infected adult house flies. Transmission of this non-occluded, enveloped virus is believed to be mediated orally via deposition and consumption of oral secretions composed of salivary gland secretions and crop contents. In this study, transmission electron micrographs of crops from infected flies showed numerous enveloped virions in the crop lumen adjacent to the cuticular intima, as well as on the hemocoel side in close vicinity to muscle cells. Oral treatments of newly emerged flies with viremic salivary gland homogenates, crop homogenates, or gradient-purified virus resulted in an average 44% infection. Virus released via oral secretion was infectious when ingested by newly emerged adult flies, resulting in an average 66% infection. Using quantitative real-time PCR, MdSGHV DNA was quantified in oral secretions and excreta obtained from viremic flies. Between 2 and 4 days post-infection (dpi), viral copy numbers in oral secretions increased exponentially and from 5 to 21 dpi each infected fly released an average 106 MdSGHV copies per feeding event. Excreta samples collected overnight from individual infected flies at 5 dpi contained an average 6.5 × 105 viral copies. Low but detectable infection rates were produced when newly emerged flies were challenged with excreta samples. In summary, evaluation of the quantity and infectivity of MdSGHV released by individual infected house flies clearly showed that deposition of oral secretions and excreta onto a shared food substrate is the main route of natural MdSGHV transmission among adult house flies.

A fungal pathogen that killed adult Diaphorina citri Kuwayama (Asian citrus psyllid) (Hemiptera: Psyllidae) in Florida citrus groves during the fall of 2005 was identified and characterized. Investigation of this pathogen is important because D. citri vectors citrus greening disease (Huanglongbing), which was reported in Florida in 2005. The morphological and genetic data generated herein support identification of the fungus as Isaria fumosorosea Wize (Ifr) (=Paecilomyces fumosoroseus) (Hypocreales: Cordycipitaceae) from the Asian citrus psyllid (Ifr AsCP). Koch's postulates were fulfilled after the fungus was isolated in vitro and transmitted to healthy psyllids, which then exhibited a diseased-phenotype similar to that observed in the field. Both in vitro growth characteristics and two Ifr AsCP-specific molecular markers discriminated the psyllid pathogen from another local Ifr isolate, Ifr 97 Apopka. These molecular markers will be useful to track the dynamics of this disease in D. citri populations. The potential for utilizing Ifr to complement existing psyllid pest management strategies is discussed.

In the search for potential cytotoxic substances produced by Nomuraea rileyi, an active compound was isolated from mycosed insects through an activity guided fractionation process. The compound, cytotoxic against the Sf9 insect cell line, was identified to be ergosterol peroxide (5α, 8α-epidioxy-24(R)-methylcholesta-6, 22-dien-3β-ol) using nuclear magnetic resonance techniques, infrared spectrometry, and mass spectroscopy. Anticancer screens demonstrated that ergosterol peroxide at micromolar concentrations inhibited the growth of hormone-dependent breast cancer cell line (T47D), hormone-independent breast cancer cell line (MDA-MB-231), human epidermoid carcinoma in mouth cell line (KB), human cervical carcinoma cell line (HeLa), lung cancer cell line (H69AR) and human cholangiocarcinoma cell line (HuCCA-1). Ergosterol peroxide showed moderate effects against Spodoptera litura larvae; 46.7% mortality via topical application after 7 day post-treatment whereas the insect’s death was not found in per os application. The amounts of ergosterol peroxide produced by N. rileyi cultures under in vitro and in vivo were determined. The physiological levels of ergosterol peroxide detected in mycosed and mummified cadavers were very low (0.011 and 0.386 μg/larva) less then levels that either inhibited insect cell proliferation or caused insecticidal activity.

The genome of the virus that causes salivary gland hypertrophy in Musca domestica (MdSGHV) was sequenced. This non-classified, enveloped, double stranded, circular DNA virus had a 124,279bp genome. The G + C content was 43.5% with 108 putative methionine-initiated open reading frames (ORFs). Thirty ORFs had homology to database proteins: eleven to proteins coded by both baculoviruses and nudiviruses (p74, pif-1, pif-2, pif-3, odv-e66, rr1, rr2, iap, dUTPase, MMP, and Ac81-like), seven to nudiviruses (mcp, dhfr, ts, tk and three unknown proteins), one to baculovirus (Ac150-like), one to herpesvirus (dna pol), and ten to cellular proteins. Mass spectrum analysis of the viral particles' protein components identified 29 structural ORFs, with only p74 and odv-e66 previously characterized as baculovirus structural proteins. Although most of the homology observed was to nudiviruses, phylogenetic analysis showed that MdSGHV was not closely related to them or to the baculoviruses.

The mite fungal pathogen Hirsutella thompsonii produces a single polypeptide chain, insecticidal protein named hirsutellin A (HtA) that is composed of 130 amino acid residues. This protein has been purified from its natural source and produced as a recombinant protein in Escherichia coli. Spectroscopic analysis has determined that the two protein forms are indistinguishable. HtA specifically inactivates ribosomes and produces the α-fragment characteristic of ribotoxin activity on rRNA. Behaving as a cyclizing ribonuclease, HtA specifically cleaves oligonucleotides that mimick the sarcin/ricin loop of the ribosome, as well as selected polynucleotides and dinucleosides. HtA interacts with phospholipid membranes as do other ribotoxins. As a consequence of its ribonuclease activity and its ability to interact with cell membranes, HtA exhibits cytotoxic activity on human tumor cells. On the basis of these results, HtA is considered to be a member of the ribotoxin group of proteins, although it is significantly smaller (130 aa) than all known ribotoxins that are composed of 149/150 amino acids. Ribotoxins are members of a larger family of fungal ribonucleases whose members of smaller size (100/110 aa) are not cytotoxic. Thus, the characterization of the fungal ribotoxin HtA represents an important milestone in the study of the diversity and the function of fungal ribonucleases. Proteins 2008. © 2008 Wiley-Liss, Inc.

SummaryHoney bee colony infestation by the small hive beetle (SHB) is associated with fermentation of hive materials. Pollen, beetles, and robbing bees (ten of each) were collected from hives infested with SHB in both Florida and Kenya. Plating of homogenized bodies of beetles and bees and comb swabs resulted in smooth cream-colored yeast colonies that formed pseudomycelial cells as they aged. Fatty acid profiles of yeast isolates from Florida and Kenya most closely matched the profiles of Candida krusei and C. sake, respectively. However, the DNA sequence of the 28S and 5.8S-ITS2 of both the Florida and Kenya isolates were, 99–100 % homologous to Kodamaea ohmeri. The ITSI region differed between the two geographic strains. The two strains produced similar volatile profiles which were attractive to SHB and contained compounds also found in honey bee alarm pheromone.

The effectiveness of two lures for trapping the small hive beetle, Aethina tumida, by means of in-hive traps was tested by field trials in apiaries located in Florida, Delaware, and Pennsylvania during 2003-2005. Both lures included a mixture (pollen dough) consisting of bee pollen and commercial pollen substitute formulated with or without glycerol and honey. Before it was used in the traps, the dough was conditioned either by the feeding of adult small hive beetles or by inoculation with the yeast Kodamaea ohmeri (NRRL Y-30722). Traps baited with conditioned dough captured significantly more beetles than unbaited traps, and traps positioned under the bottom board of a hive captured significantly more beetles than traps located at the top of a hive. In fact, baited in-hive bottom board traps nearly eliminated the beetles from colonies at a pollination site in Florida. However, when these honey bee colonies were moved to an apiary, trap catch increased markedly over time, indicating a resurgence of the beetle population produced by immigration of beetles from nearby hives or emerging from the soil. In tests at three Florida apiaries during 2006, yeast-inoculated dough baited bottom board traps captured significantly more beetles than unbaited traps, showing the effectiveness of yeast-inoculated dough as a lure and its potential as a tool in managing the small hive beetle.

The response of the small hive beetle (SHB), Aethina tumida Murray, to volatiles from a pollen-based diet conditioned by the feeding of 100 adult virgin female or male SHBs (4–8 weeks old) for 1, 3, 7 or 14 days is described and compared to that of the same diet inoculated with the yeast Kodamaea ohmeri (NRRL Y-30722), isolated from the beetle. In a wind tunnel, volatiles from pollen dough conditioned by beetles of either sex for 3 or 7 days lured significantly more beetles into traps than volatiles from unconditioned dough. In contrast, trap captures with volatiles from dough conditioned for 1 and 14 days were weakly attractive. In cage bioassays, when naive, unfed, virgin, SHBs (3–4 days old) were given a choice between yeast-inoculated pollen dough and non-inoculated dough, the responses were similar to those obtained in the wind tunnel with dough conditioned by SHBs for 3 and 7 days. Chemical analysis revealed high levels of fermentation-related products in volatiles that attracted the beetle.

The glassy-winged sharpshooter (GWSS), Homalodisca coagulata (Say), is an efficient vector of the phytopathogenic bacterium Xyllela fastidiosa. Native to the Southeast US, it was introduced to California, where its presence has caused a sharp increase in X. fastidiosa-based disease. Here are described three fungi associated with GWSS in its native range, including a Hirsutella species that is the predominant mycopathogen detected in sampled GWSS populations. Also described are potentially novel species of Pseudogibellula and Sporothrix. Data garnered from examination of in␣situ and in vitro morphological characters were compared to published keys and type specimen descriptions to achieve morphology-based identification. ITS1-5.8S-ITS2 and partial sequences of the large subunit (LSU)-rDNA, actin, and β-tubulin genes of several GWSS Hirsutella isolates were sequenced and analyzed. Phylogenetic analysis of the LSU-rDNA and tubulin of the GWSS Hirsutella in combination with sequence data generated from other Hirsutella species defined the GWSS isolates to belong to subgroup II within this genus. The combination of phenotypic and genotypic data demonstrated GWSS Hirsutella to be a new species and as such, has been designated Hirsutella homalodiscae nom. prov. Species-specific PCR primers, designed from intron and exon regions of the actin and tubulin genes, were used in a multiplex reaction for the in situ identification of H. homalodiscae. A total of 246 mummified GWSS collected from sites in the southeastern US were subjected to multiplex PCR analysis. More than 70% of these samples tested positive for the presence of H. homalodiscae demonstrating it to be the major mycopathogen distributed throughout the native range of GWSS.

In the accompanying paper [ Cho, E.-M., Liu, L., Farmerie, W. & Keyhani, N. O. (2006) . Microbiology 152, 2843–2854], the analysis of expressed sequence tag (EST) libraries derived from homogeneous single-cell populations of aerial conidia, in vitro blastospores and submerged conidia of the entomopathogenic fungus Beauveria (Cordyceps) bassiana has been reported. Here an extended EST analysis is presented of complex cell mixtures derived from fungal cells sporulating on chitin or grown under culture conditions inducing the production of the B. bassiana secondary metabolite, oosporein. Fungal material used for the construction of the libraries included germinating conidia and blastospores, mycelia, as well as cells in various developmental stages. Approximately 2500 5′ end sequences were determined from random sequencing of clones from each library, and were clustered into 277 contigs with 1069 singlets, and 306 contigs with 1064 singlets, for the chitin and oosporein libraries, respectively. Almost half (45–50 %) of the sequences in each library displayed either no significant similarity (e value >10−4) or similarity to hypothetical proteins found in the NCBI database. Approximately 20–25 % of the sequences in each library could be annotated by gene ontology terms. A comparative analysis between the two libraries, as well as the libraries in the accompanying paper, is presented. A set of 4360 clustered and unique sequences was characterized. The data are indicative of a highly plastic gene expression repertoire being available to B. bassiana for growth during different environmental and developmental conditions, and provides a dataset for gene discovery and genome annotation.

An entomopathogenic fungus (IndGH 96), identified as Paecilomyces reniformis, was isolated from long-horned grasshoppers (Orthoptera: Tettigoniidae) in Sulawesi, Indonesia. The phenotypic and molecular data identified the IndGH 96 as a P. reniformis. We present the first comprehensive characterization of this species using morphological features, sequencing of the ITS1-5.8s-ITS2 region, D1/D2 region of 28S of rDNA, and a portion of the tubulin gene, and laboratory bioassays. Distinguishing features include a hyphal body stage during vegetative growth and the production of distinctly curved, light-green conidia. High dosage bioassays showed that IndGH 96 was infectious to both long-horned and short-horned grasshoppers but not to the house cricket, Acheta domestica, or to the lepidopterans velvetbean caterpillar, Anticarsia gemmatalis or fall armyworm, Spodoptera frugiperda. Phenotypic and genetic analyses suggest that IndGH 96 and other isolates of P. reniformis are more closely related to Nomuraea rileyi than to other species of Paecilomyces.

Amplified fragment length polymorphism (AFLP) was used to investigate polymorphism among 43 Hirsutella thompsonii isolates (33 from Thailand) obtained from various mite species. The outgroups were an unidentified Hirsutella isolate along with Hirsutella nodulosa and Hirsutella kirchneri. Phylogenetic analyses of the AFLP data showed significant variation among isolates and the existence of three H. thompsonii clades. We also investigated the isolates using PCR with specific primers for the Hirsutella exotoxin gene Hirsutelin A (HtA) and 18 of these isolates were used for sequencing of the partial beta-tubulin gene. Phylogenetic analyses of beta-tubulin sequences showed two distinct H. thompsonii clades, one of which included AFLP clades I and II. For both markers grouping of the H. thompsonii isolates was not related to either host mite species or geographical origin, although for the HtA gene one clade contained only isolates with no detectable HtA band. These results confirm the high intraspecific polymorphism of H. thompsonii, and maximum likelihood analysis showed no monophyletic group within this species. To refine the taxonomy of this genus other studies should be undertaken using additional molecular markers and several other Hirsutella isolates.

Members of the genus Helicosporidium are the first described algal insect pathogens. They have a close affinity to the non-photosynthetic algae of the genus Prototheca, and have a wide host range, infecting many species of aquatic and terrestrial insects. In this study the infectivity of two Helicosporidium spp. isolates, originating from a black fly (SjHe) and an aquatic weevil (CsHe), was tested against a weevil Diaprepes abbreviatus (L.) and three mosquito species Anopheles quadrimaculatus Say, Culex quinquefasciatus Say, and Aedes aegypti (L.). The weevil constitutes a new experimental host record for helicosporidia. The CsHe isolate was more virulent than the SjHe isolate in D. abbreviatus. Anopheles quadrimaculatus was the most susceptible mosquito species measured by infection rate and mortality. The infectivity and virulence of SjHe and CsHe isolates did not differ in any of the mosquito species.

During infection (in vivo), the entomopathogenic fungus Beauveria bassiana produces yeast-like cells that are surrounded by modified cell walls. These modifications have been related to the fungus ability to limit recognition by the host defense system. The composition of the in vivo cell wall was analyzed using a combination of cytochemical and molecular techniques. The in vivo cell walls still contained both chitin and 1,3-beta-glucan, but they were significantly thinner than in vitro cell walls (50-60 nm versus 100-160 nm, respectively). The difference in cell wall thickness was correlated with transcriptional regulation of cell wall-related genes: quantitative RT-PCR reactions demonstrated that B. bassiana chitin synthase (CHS) and glucan synthase (FKS) genes are down regulated in vivo. These analyses indicate that in vivo-triggered phenotypic modifications, including cell wall adjustments, are controlled by molecular mechanisms that include regulation of gene expression at the transcriptional level.

The genetic diversity of 79 Nomuraea rileyi isolates from various lepidopteran hosts in Asia, North America, and South America was evaluated using amplified fragment length polymorphism (AFLP) analysis. Cluster analysis separated the N. rileyi isolates into two major groups and seven subgroups. The resulting dendrogram generally classified the N. rileyi isolates based on insect host and geographical region. The haplotypic diversity index of N. rileyi subpopulations from each location and host origin was ranging from 0.8788 to 1.000. However, analysis of molecular variance (AMOVA) demonstrated no significant differences (p = 0.3421) among N. rileyi isolates from different continents. Whereas the genetic variation among the N. rileyi populations from the different host insects within each continent was significantly different (p

Helicosporidia are obligate invertebrate pathogens with a unique and highly adapted mode of infection. The evolutionary history of Helicosporidia has been uncertain, but several recent molecular phylogenetic studies have shown an unexpectedly close relationship to green algae, and specifically to the opportunistic pathogen Prototheca. To date, molecular sequences from Helicosporidia are restricted to those genes used for phylogenetic reconstruction and genes related to the existence and function of its cryptic plastid. We have therefore conducted a small expressed sequence tag (EST) project on Helicosporidium sp., yielding about 700 unique sequences. We have examined the functional distribution of known genes, the distribution of EST abundance, and the prevalence of previously unknown gene sequences. To demonstrate the potential utility of large amounts of data, we have used ribosomal proteins to test whether the phylogenetic position of Helicosporidium inferred from a small number of genes is broadly supported by a large number of genes. We conducted phylogenetic analyses on 69 ribosomal proteins and found that 98% supported the green algal origin of Helicosporidia and 80% support a specific relationship with Prototheca. Overall, these data multiply the available molecular information from Helicosporidium 100-fold, which should provide the basis for new insights into these unusual but interesting parasites.

A fragment of the Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) plastid genome has been sequenced. The genome architecture was compared to that of both a non-photosynthetic relative (Prototheca wickerhamii) and a photosynthetic relative (Chlorella vulgaris). Comparative genomic analysis indicated that Helicosporidium and Prototheca are closely related genera. The analyses also revealed that the Helicosporidium sp. plastid genome has been rearranged. In particular, two ribosomal protein-encoding genes (rpl19 and rps23) appeared to have been transposed, or lost from the Helicosporidium sp. plastid genome. RT-PCR reactions demonstrated that the retained plastid genes were transcribed, suggesting that, despite rearrangement(s), the Helicosporidium sp. plastid genome has remained functional. The modified plastid genome architecture is a novel apomorphy that indicates that the Helicosporidia are highly derived green algae, more so than Prototheca spp. As such, they represent a promising model to study organellar genome reorganizations in parasitic protists.

A new Helicosporidium spp. isolate recently purified from an aquatic weevil, Cyrtobagus salviniae Calder & Sands, was capable of infecting and reproducing in three heterologous noctuid hosts. Regardless of host species, oral treatment of Heliocoverpa zea (Boddie), Spodoptera exigua (Hubner), or Trichoplusia ni (Hubner) early instars with cyst preparations of Helicosporidium spp. resulted in ≈50% infection of the challenged larvae. The sex ratio did not differ between infected and control groups, suggesting the existence of a natural, nonsex-related resistance to the disease. Injection of Helicosporidium spp. into the hemocoels of late instars resulted in virtually 100% infection, indicating that resistance is related to the ingestion of the pathogen and therefore affiliated with midgut-mediated barriers. The pathogen’s development was not interrupted by metamorphosis; likewise, the infection did not necessarily interrupt the insects’ development. When treated as early instars, 50–90% of the infected larvae formed pupae, of which 20–30% emerged as adults. However, a high proportion of the infected adults (62–86%) had malformed wings, and their longevity was reduced compared with that of healthy adults. Infected S. exigua adults that seemed to be morphologically healthy were able to mate and produce viable offspring. The disease was detected in five of 12 groups of progeny produced by infected adults. However, the relative infection rate in the filial generation was low (2–5%). To our knowledge, this is the first evidence for a vertical transmission of helicosporidial infection.