Comparative metatranscriptomic signatures of wood and paper feeding in the gut of the termite Reticulitermes flavipes (Isoptera: Rhinotermitidae).

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.

Analysis of the structural proteins from the Musca domestica hytrosavirus with an emphasis on the major envelope protein.

The Musca domestica hytrosavirus (MdHV), a member of the family Hyrosaviridae, is a large, dsDNA, enveloped virus that infects adult house flies and causes a diagnostic hypertrophy of the salivary gland. Herein, studies were directed at identifying key structural components of the viral envelope and nucleocapsid. SDS-PAGE of detergent-treated virus fractions identified protein bands unique to the envelope and nucleocapsid components. Using prior LC-MSMS data we identified the viral ORF associated with the major envelope band, cloned and expressed recombinant viral antigens, and prepared a series of polyclonal sera. Western blots confirmed that antibodies recognized the target viral antigen and provided evidence that the viral protein MdHV96 underwent post-translational processing; antibodies bound to the target high molecular weight parent molecule as well as distinct sets of smaller bands. Immuno gold electron microscopy demonstrated that the anti-MdHV96 sera recognized target antigens associated with the envelope. The nucleocapsids migrated from the virogenic stroma in the nucleus through the nuclear membrane into the cytoplasm, where they acquired an initial envelope that contained MdHV96. This major envelope protein, appeared to incorporate into intracellular membranes of both the caniculi and rough endoplasmic reticulum membranes and mediate binding to the nucleocapsids. Oral infection bioassays demonstrated that the anti-HV96 polyclonal sera acted as neutralizing agents in suppressing the levels of orally acquired infections.

Salivary gland hypertrophy virus of house flies in Denmark: prevalence, host range, and comparison with a Florida isolate.

House flies (Musca domestica) infected with Musca domestica salivary gland hypertrophy virus (MdSGHV) were found in fly populations collected from 12 out of 18 Danish livestock farms that were surveyed in 2007 and 2008. Infection rates ranged from 0.5% to 5% and averaged 1.2%. None of the stable flies (Stomoxys calcitrans), rat-tail maggot flies (Eristalis tenax) or yellow dung flies (Scathophaga stercoraria) collected from MdSGHV-positive farms displayed characteristic salivary gland hypertrophy (SGH). In laboratory transmission tests, SGH symptoms were not observed in stable flies, flesh flies (Sarcophaga bullata), black dump flies (Hydrotaea aenescens), or face flies (Musca autumnalis) that were injected with MdSGHV from Danish house flies. However, in two species (stable fly and black dump fly), virus injection resulted in suppression of ovarian development similar to that observed in infected house flies, and injection of house flies with homogenates prepared from the salivary glands or ovaries of these species resulted in MdSGHV infection of the challenged house flies. Mortality of virus-injected stable flies was the highest among the five species tested. Virulence of Danish and Florida isolates of MdSGHV was similar with three virus delivery protocols, as a liquid food bait (in sucrose, milk, or blood), sprayed onto the flies in a Potter spray tower, or by immersiion in a crude homogenate of infected house flies. The most effective delivery system was immersion in a homogenate of ten infected flies/ml of water, resulting in 56.2% and 49.6% infection of the house flies challenged with the Danish and Florida strains, respectively.

Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics.

The European house cricket, Acheta domesticus L., is highly susceptible to A. domesticus densovirus (AdDNV). Commercial rearings of crickets in Europe are frequently decimated by this pathogen. Mortality was predominant in the last larval stage and young adults. Infected A. domesticus were smaller, less active, did not jump as high, and the adult females seldom lived more than 10-14 days. The most obvious pathological change was the completely empty digestive caecae. Infected tissues included adipose tissue, midgut, epidermis, and Malpighian tubules. Sudden AdDNV epizootics have decimated commercial mass rearings in widely separated parts of North America since the autumn of 2009. Facilities that are producing disease-free crickets have avoided the importation of crickets and other non-cricket species (or nonliving material). Five isolates from different areas in North America contained identical sequences as did AdDNV present in non-cricket species collected from these facilities. The North American AdDNVs differed slightly from sequences of European AdDNV isolates obtained in 1977, 2004, 2006, 2007 and 2009 and an American isolate from 1988. The substitution rate of the 1977 AdDNV 5kb genome was about two nucleotides per year, about half of the substitutions being synonymous. The American and European AdDNV strains are estimated to have diverged in 2006. The lepidopterans Spodoptera littoralis and Galleria mellonella could not be infected with AdDNV. The Jamaican cricket, Gryllus assimilis, and the European field cricket, Gryllus bimaculatus, were also found to be resistant to AdDNV.

Impact of house fly salivary gland hypertrophy virus (MdSGHV) on a heterologous host, Stomoxys calcitrans.

The effect of Musca domestica salivary gland hypertrophy virus (MdSGHV) on selected fitness parameters of stable flies, Stomoxys calcitrans (L.), was examined in the laboratory. Virus-injected stable flies of both genders suffered substantially higher mortality than control flies. By day 9, female mortality was 59.3 +/- 10.1% in the virus group compared with 23.7 +/- 3.7% in the controls; mortality in virus-injected males was 78.1 +/- 3.1% compared with 33.3 +/- 9.3% for controls. Fecundity of control flies on days 6-9 was 49-54 eggs deposited per live female per day (total, 8,996 eggs deposited), whereas virus-injected flies produced four to five eggs per female on days 6-7 and less then one egg per female per day thereafter (total, 251 eggs). Fecal spot deposition by virus-injected flies was comparable to controls initially but decreased to approximately 50% of control levels by day 4 after injection; infected flies produced only 26% as many fecal spots as healthy flies on days 6 and 7. None of the virus-injected stable flies developed symptoms of salivary gland hypertrophy. Quantitative real-time polymerase chain reaction demonstrated virus replication in injected stable flies, with increasing titers of virus genome copies from one to four days after injection. MdSGHV in stable flies displayed tissue tropism similar to that observed in house fly hosts, with higher viral copy numbers in fat body and salivary glands compared with ovaries. Virus titers were approximately 2 orders of magnitude higher in house fly than in stable fly hosts, and this difference was probably due to the absence of salivary gland hypertrophy in the latter species.

Phenol-oxidizing laccases from the termite gut.

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.

Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family.

Salivary gland hypertrophy viruses (SGHVs) have been identified from different dipteran species, such as the tsetse fly Glossina pallidipes (GpSGHV), the housefly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). These viruses share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 500-1,000 nm in length and 50-100 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120 to 190 kbp and having G + C ratios ranging from 28 to 44%; (iii) they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility. The available information on the complete genome sequence of GpSGHV and MdSGHV indicates significant co-linearity between the two viral genomes, whereas no co-linearity was observed with baculoviruses, ascoviruses, entomopoxviruses, iridoviruses and nudiviruses, other large invertebrate DNA viruses. The DNA polymerases encoded by the SGHVs are of the type B and closely related, but they are phylogenetically distant from DNA polymerases encoded by other large dsDNA viruses. The great majority of SGHV ORFs could not be assigned by sequence comparison. Phylogenetic analysis of conserved genes clustered both SGHVs, but distantly from the nudiviruses and baculoviruses. On the basis of the available morphological, (patho)biological, genomic and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This proposed family currently comprises two unassigned species, G. pallidipes salivary gland hypertrophy virus and M. domestica salivary gland hypertrophy virus, and a tentative unassigned species, M. equestris salivary gland hypertrophy virus. Here, we present the characteristics and the justification for establishing this new virus family.

Serine proteases identified from a Costelytra zealandica (White) (Coleoptera: Scarabaeidae) midgut EST library and their expression through insect development.

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.

Characterization of an Indonesian isolate of Paecilomyces reniformis.

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.

Hirsutella thompsonii and Metarhizium anisopliae as potential microbial control agents of Varroa destructor, a honey bee parasite.

The potential of Hirsutella thompsonii Fisher and Metarhizium anisopliae (Metschinkoff) as biological control agents of the parasitic mite, Varroa destructor Anderson and Trueman was evaluated in the laboratory and in observation hives. In the laboratory, time required for 90% cumulative mortality of mites (LT(90)) was 4.16 (3.98-4.42) days for H. thompsonii and 5.85 (5.48-7.43) days for M. anisopliae at 1.1 x 10(3) conidia mm(-2). At a temperature (34+/-1 degrees C) similar to that of the broodnest in a honey bee colony, Apis mellifera L., H. thompsonii [LC(90)=9.90 x 10(1) (5.86-19.35) conidia mm(-2) at Day 7] and M. anisopliae [LC(90)=7.13 x 10(3) (2.80-23.45) conidia mm(-2) at Day 7] both showed significant virulence against V. destructor. The applications of H. thompsonii to observation hives resulted in significant mortality of mites, and reduction of the number of mites per bee 21 and 42 days post-treatments. The treatments did not significantly affect the mite population in sealed brood. However, the fungus must have persisted because infected mites were still observed [82.97+/-(0.6)%] 42 days post-treatment. In addition, the fungus was found to sporulate on the host. A small percentage [2.86+/-(0.2)%] of dead mites found in the control hives also showed fungal infection, suggesting that adult bees drifted between hives and disseminated the fungus. H. thompsonii was harmless to the honey bees at the concentrations applied and did not have any deleterious effects on the fecundity of the queens. Microbial control with fungal pathogens provides promising new avenues for control of V. destructor and could be a useful component of an integrated pest management program for the honey bee industry.

In vivo and in vitro development of the protist Helicosporidium sp.

We describe the discovery and developmental features of a Helicosporidium sp. isolated from the black fly Simulium jonesi. Morphologically, the helicosporidia are characterized by a distinct cyst stage that encloses three ovoid cells and a single elongate filamentous cell. Bioassays have demonstrated that the cysts of this isolate infect various insect species, including the lepidopterans, Helicoverpa zea, Galleria mellonella, and Manduca sexta, and the dipterans, Musca domestica, Aedes taeniorhynchus, Anopheles albimanus, and An. quadrimaculatus. The cysts attach to the insect peritrophic matrix prior to dehiscence, which releases the filamentous cell and the three ovoid cells. The ovoid cells are short-lived in the insect gut with infection mediated by the penetration of the filamentous cell into the host. Furthermore, these filamentous cells are covered with projections that anchor them to the midgut lining. Unlike most entomopathogenic protozoa, this Helicosporidium sp. can be propagated in simple nutritional media under defined in vitro conditions, providing a system to conduct detailed analysis of the developmental biology of this poorly known taxon. The morphology and development of the in vitro produced cells are similar to that reported for the achorophyllic algae belonging to the genus Prototheca.

Pathobiology of amber disease, caused by Serratia Spp., in the New Zealand grass grub, Costelytra zealandica.

Amber disease in the New Zealand grass grub (Costelytra zealandica) is caused by some strains of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae). When treated with pathogenic isolates, larvae ceased feeding within 48 h, developed an amber coloration after 72 h, and entered a long chronic phase without feeding. An acute dose of 2-4 x 10(4) pathogenic bacteria was sufficient to produce disease in 50% of treated larvae. Time to death was directly related to temperature. At 15 degrees C, infected larvae remained in a chronic, nonfeeding state for more than 4 months prior to death. Nonpathogenic isolates, lacking the disease-causing plasmid (pADAP), had no effect on either feeding or disease. Twenty-four hours after ingestion, bacteria were found predominantly in the hindgut and growth occurred primarily within the fermentation chamber and in the head section of the larvae. Nonpathogenic strains did not multiply in treated larvae. Treatment of diseased larvae with antibiotic eliminated Serratia cells from the insects but did not result in restoration of feeding or the dark gut characteristic of the healthy larva.

Comparative analysis of the binding of antibodies prepared against the insect Spodoptera exigua and against the mycopathogen Nomuraea rileyi.

Polyclonal antibodies were produced in mice against Spodoptera exigua (beet armyworm) larval hemolymph and hemocytes and against cell wall surfaces of hyphal bodies and hyphae of the entomopathogenic hyphomycete Nomuraea rileyi. In addition to exhibiting strong activity against their original antigenic substrates, all of the antibodies cross-react extensively with other substrates. The hemolymph antibody binds to hemocytes and vice versa, and both antibodies cross-react to the insect fat body basement membrane (extracellular matrix (ECM) and to N. rileyi and Beauveria bassiana (another entomopathogenic fungus) cell wall surfaces (ECM). Likewise, the anti-fungal antibodies cross-react with S. exigua hemolymph and hemocytes, especially the granules that may contain ECM components, and with fat body basement membrane. These cross-reactivities are specific as indicated by negative controls in the microscopy and Western blotting assays. Parallel labeling experiments using Con A suggest that the reactive epitopes contain mannose; however, none of the antibodies bind to mannose residues of nonentomopathogenic Candida albicans or Saccharomyces cerevisiae yeast cells. Thus, these cross-reactivities suggest that the host mimicry expressed by surface components of entomopathogenic fungi represents an important pathogenic determinant.

Characterization of monoclonal antibodies against cell wall epitopes of the insect pathogenic fungus, Nomuraea rileyi: differential binding to fungal surfaces and cross-reactivity with host hemocytes and basement membrane components.

Monoclonal antibodies (MAbs) were generated against epitopes on yeast-like hyphal bodies and hyphae of the entomopathogenic hyphomycete, Nomuraea rileyi. Two MAbs (4C10, 2H4) bind to epitopes common to both hyphal bodies and hyphae, whereas MAb 4E9 binds only to hyphal surfaces. 4C10 and 2H4 appear to be directed towards carbohydrate portions of cell surface mannoproteins, as evidenced by similarities in staining patterns between these MAbs and Concanavalin A on Western blots of N. rileyi cell wall extracts. These MAbs cross-react with antigens on blastospore and hyphal surfaces of two other entomopathogenic fungi, Beauveria bassiana and Paecilomyces farinosus in fluorescence microscopy assays, but do not cross-react with a non-entomopathogenic strain of Candida albicans or with Saccharomyces cerevisiae yeasts. MAb 4C10 also cross-reacts with immunocompetent granular hemocytes from Spodoptera exigua (beet armyworm) and Trichoplusia ni (cabbage looper) larvae and with S. exigua plasmatocytes. Electron microscopy revealed that this MAb binds to a component in cytoplasmic granules in the hemocytes, and that surface labeling may be due to the release of this MAb-positive component upon degranulation. MAb 2H4 does not cross-react with granular hemocytes, but does bind to plasmatocytes and hemocytes that tightly adhere to the substrate in monolayer assays. Additionally, MAb 4C10 specifically labels a basement membrane epitope on S. exigua fat body, suggesting that this antibody binds to mannose residues on extracellular matrix glycoproteins. Cross-reactivity of these N. rileyi MAbs with insect hemocyte and tissue components indicates that fungal surface epitopes can mimic host surface molecules, which could explain why N. rileyi hyphal bodies are not recognized by granulocytes and are able to circulate freely in the hemolymph without binding to basement membranes lining the hemocoel.

Attachment of Metarhizium anisopliae to the southern green stink bug Nezara viridula cuticle and fungistatic effect of cuticular lipids and aldehydes.

In this paper we examined the conidial attachment of Metarhizium anisopliae on the southern green stink bug, Nezara viridula, using the exuvia and nymphal stage of the host as a substrate for M. anisopliae conidiospores. Initial studies using fluorescein isothiocyanate (FITC)-labeled conidia examined the differential binding of conidia to various sites on the cuticle. Both the topography and the chemistry of the cuticle affected conidial adhesion. Conidia were trapped in areas containing large numbers of setae (e.g., antennal tips, apical portions of tibia and tarsi). Chemical treatments to remove the cuticle proteins did not affect conidial adhesion, but solvent extraction of cuticular lipids significantly reduced the adhesion of M. anisopliae spores. Germination of M. anisopliae conidia attached to N. viridula cuticle was much less than conidia attached to other insect cuticle substrates. After a 24-hr incubation, only 5-20% of the conidia produced detectable germ tubes. The aldehyde (E)-2-decenal, a primary component of the stink bug scent gland, was detected in cuticle extracts and found to be selectively fungistatic to certain entomopathogenic fungi, including M. anisopliae. The hydrocarbon fraction (nC13 and nC21 to nC31 hydrocarbon series) served as a binding substrate for M. anisopliae, but conidia did not degrade these hydrocarbons and did not use them as a carbon source.

The mode of action of hirsutellin A on eukaryotic cells.

A 16-kDa protein toxin was purified from Hirsutella thompsonii var thompsonii and named hirsutellin A (HtA). At 0.5 and 5.0 microM concentrations, HtA caused detectable cytopathic effects on Spodoptera frugiperda cells (Sf-9) within 2-4 hr and completely inhibited Sf-9 cell growth at 4 days posttreatment. Electron microscope data showed that the HtA treated Sf-9 cells became hypotrophied and internal organelles and cell membranes were disrupted. At the same concentration, HtA effectively inhibited Brome mosaic virus protein synthesis of both rabbit reticulocyte and wheat germ in vitro translation system. The ribosomal RNA extracted from HtA treated Sf-9 cells produced a smaller RNA (approximately 528 bases) than untreated Sf-9 cells. In summary, HtA is the first mycotoxin of a invertebrate mycopathogen determined to possess ribosomal inhibiting activity and appears to possess some specificity to invertebrate cells.

Extraction and characterization of the insecticidal toxin hirsutellin A produced by Hirsutella thompsonii var. thompsonii.

Hirsutellin A (HtA) produced by Hirsutella thompsonii var. thompsonii (strain JAB-04) was extracted and purified using a combination of ion-exchange, gel-permeation, and immunoaffinity chromatography. The identity of the purified HtA was confirmed by amino acid analysis and N-terminal sequencing. Monoclonal antibodies prepared against HtA were capable of detecting 25-50 ng of HtA by direct sandwich ELISA. In addition, utilizing Western blot methods, the antibodies were shown to be specific to HtA. The production of HtA was monitored during submerged fermentation. The peak level of exocellular HtA (13-14 micrograms/ml) was during the late exponential growth phase (39-45 h), determined by utilizing a combination of densitometric analysis of the 16.3-kDa bands on SDS-PAGE gels and ELISA. HtA production was directly correlated with mycelial growth. Twenty-one-hour culture filtrates were highly toxic to larvae of the greater wax moth. Pure HtA at a final concentration of 40 pmol was highly toxic to Galleria mellonella larvae.

Antigens of Aspergillus fumigatus produced in vivo.

Immunoblots of extracts from kidneys of mice infected intravenously with Aspergillus fumigatus were probed with human sera from patients with aspergilloma. A limited number of antigens were detected with molecular masses of 31, 36, 56, 84, 88 and 200 kDa. These antigens can be clustered into two classes: (i) galactomannan and/or galactofuran-containing glycoproteins; and (ii) antigenic proteins (31, 36 and 88 kDa) exempt of the galactofuran epitope. Antigens belonging to this second class were also produced in vitro but were not the major proteins expressed by A. fumigatus in a glucose-asparagin medium.