The Effect of the Musca domestica Salivary Gland Hypertrophy Virus on Food Consumption in Its Adult Host, the Common House Fly (Diptera: Muscidae).

The Musca domestica salivary gland hypertrophy virus (MdSGHV) substantially enlarges the house fly's salivary glands and prevents or delays ovarian development in its adult host, but the effect that MdSGHV has on the house fly's food consumption is currently unknown. Using house flies from a laboratory-reared colony, we evaluated the effect of MdSGHV infection on food consumption over a 7-d period. Both treatment (virus-infected) and control (saline-injected) flies were provided with a choice of 8% sucrose solution and 4% powdered milk solution to determine food preferences. Quantities of each solution consumed were measured every 24 h for each fly to measure food consumptions. Infected house flies were shown to consume less overall of both solutions than house flies injected with saline. The largest consumption discrepancy was seen between female house flies. Healthy female flies with developing ovaries continued to consume a sugar and protein diet, whereas infected female flies fed predominantly on a sugar diet. Additionally, infected male and female flies consumed significantly lower quantities of protein and sucrose than control flies. This suggests that MdSGHV has a negative consumption effect (e.g., hunger, starvation) on its host. Thus, differences in food consumption of infected and control flies probably represent differences in the nutritional requirements of flies resulting from viral infection.

ICTV Virus Taxonomy Profile: Nudiviridae.

Members of the family Nudiviridae are large dsDNA viruses with distinctive rod-shaped nucleocapsids and circular genomes of 96-232 kbp. Nudiviruses have been identified from a diverse range of insects and crustaceans and are closely related to baculoviruses. This is a summary of the International Committee on Taxonomy of Viruses Report on the taxonomy of the family Nudiviridae, which is available at ictv.global/report/nudiviridae.

Using the SSU, ITS, and Ribosomal DNA Operon Arrangement to Characterize Two Microsporidia Infecting Bruce Spanworm, Operophtera bruceata (Lepidoptera: Geometridae).

Research pertaining to the two closely-related microsporidian genera Nosema and Vairimorpha is hindered by inconsistencies in species differentiation within and between the two clades. One proposal to better delimit these genera is to restructure the Nosema around a "True Nosema" clade, consisting of species that share a characteristic reversed ribosomal DNA operon arrangement and small subunit (SSU) ribosomal DNA sequences similar to that of the Nosema type species, N. bombycis. Using this framework, we assess two distinct microsporidia recovered from the forest insect Bruce spanworm (Operophtera bruceata) by sequencing their SSU and internal transcribed spacer regions. Phylogenetic analyses place one of our isolates within the proposed True Nosema clade close to N. furnacalis and place the other in the broader Nosema/Vairimorpha clade close to N. thomsoni. We found that 25% of Bruce spanworm cadavers collected over the four-year study period were infected with microsporidia, but no infections were detected in cadavers of the Bruce spanworm's invasive congener, the winter moth (O. brumata), collected over the same period. We comment on these findings as they relate to the population dynamics of the Bruce spanworm-winter moth system in this region, and more broadly, on the value of ribosomal DNA operon arrangement in Nosema systematics.

Viral transmission in honey bees and native bees, supported by a global black queen cell virus phylogeny.

In recent decades, we have realized that honey bee viruses are not, in fact, exclusive to honey bees. The potential impact of Apis-affiliated viruses on native pollinators is prompting concern. Our research addresses the issue of virus crossover between honey bees and native bees foraging in the same localities. We measured the presence of black queen cell virus (BQCV), deformed wing virus (DWV) and sacbrood virus (SBV) in managed Apis mellifera (honey bees) and native Andrena spp. (subgenus Melandrena) bee populations in five commercial orchards. We identified viral presence across sites and bees and related these data to measures of bee community diversity. All viruses were found in both managed and native bees, and BQCV was the most common virus in each. To establish evidence for viral crossover between taxa, we undertook an additional examination of BQCV where 74 samples were sequenced and placed in a global phylogenic framework of hundreds of BQCV strains. We demonstrate pathogen sharing across managed honey bees and distantly related wild bees. This phylogenetic analysis contributes to growing evidence for host switching and places local incidence patterns in a worldwide context, revealing multispecies viral transmission.

ICTV Virus Taxonomy Profile: Baculoviridae.

The family Baculoviridae comprises large viruses with circular dsDNA genomes ranging from 80 to 180 kbp. The virions consist of enveloped, rod-shaped nucleocapsids and are embedded in distinctive occlusion bodies measuring 0.15-5 µm. The occlusion bodies consist of a matrix composed of a single viral protein expressed at high levels during infection. Members of this family infect exclusively larvae of the insect orders Lepidoptera, Hymenoptera and Diptera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Baculoviridae, which is available at www.ictv.global/report/baculoviridae.

Effect of Diet on Adult House Fly (Diptera: Muscidae) Injected With the Salivary Gland Hypertrophy Virus (MdSGHV).

Research to date on the salivary gland hypertrophy virus (SGHV) in three species of flies has focused on adult flies having access to and taking a proteinaceous diet. Since many studies have shown that diet affects viral infection in numerous organisms, this study examined the effect of a protein-free diet on the effect of the SGHV virus in adult house flies, Musca domestica. L. Adults infected with the virus, and maintained on a sugar diet only, showed salivary glands with a blue rather than a grayish color and mild hypertrophy compared with protein-fed flies. It was possible to retrieve the virus from these glands and successfully infect noninfected flies. When injected at various ages, female flies fed only sugar showed that regardless of age, sugar-fed flies still became infected and showed the pathology of the glands. In addition, electron microscope studies revealed at the ultrastructural level that there was no difference between viral replication in cells from salivary glands of adults fed a proteinaceous-free diet and those feeding on protein.

The Operophtera brumata Nucleopolyhedrovirus (OpbuNPV) Represents an Early, Divergent Lineage within Genus Alphabaculovirus.

Operophtera brumata nucleopolyhedrovirus (OpbuNPV) infects the larvae of the winter moth, Operophtera brumata. As part of an effort to explore the pesticidal potential of OpbuNPV, an isolate of this virus from Massachusetts (USA)-OpbuNPV-MA-was characterized by electron microscopy of OpbuNPV occlusion bodies (OBs) and by sequencing of the viral genome. The OBs of OpbuNPV-MA consisted of irregular polyhedra and contained virions consisting of a single rod-shaped nucleocapsid within each envelope. Presumptive cypovirus OBs were also detected in sections of the OB preparation. The OpbuNPV-MA genome assembly yielded a circular contig of 119,054 bp and was found to contain little genetic variation, with most polymorphisms occurring at a frequency of < 6%. A total of 130 open reading frames (ORFs) were annotated, including the 38 core genes of Baculoviridae, along with five homologous repeat (hr) regions. The results of BLASTp and phylogenetic analysis with selected ORFs indicated that OpbuNPV-MA is not closely related to other alphabaculoviruses. Phylogenies based on concatenated core gene amino acid sequence alignments placed OpbuNPV-MA on a basal branch lying outside other alphabaculovirus clades. These results indicate that OpbuNPV-MA represents a divergent baculovirus lineage that appeared early during the diversification of genus Alphabaculovirus.

The phylogenetic relationship and cross-infection of nucleopolyhedroviruses between the invasive winter moth (Operophtera brumata) and its native congener, Bruce spanworm (O. bruceata).

Disease can affect biological invasions by acting as either a synergist or antagonist. Disease-mediated invasions have important implications for understanding the spread of invasive insects, which cost billions of dollars in damages annually. One such non-native, destructive insect is the winter moth, Operophtera brumata L. (Lepidoptera: Geometridae), which causes defoliation and mortality of deciduous trees in its introduced range. In the northeastern United States, winter moth populations overlap with a native congener, Bruce spanworm, Operophtera bruceata Hulst. Nucleopolyhedrovirus (NPV), appears to be an important natural enemy in Bruce spanworm and there is some evidence that the NPV infection found in winter moth in the northeastern U.S. may originate from Bruce spanworm. By sequencing two viral genes (the polyhedrin and p74 genes) from field-collected larvae of both species, we found that the winter moth virus (OpbuNPV) is distinct from the virus from Bruce spanworm (OpbrNPV). However, the two viruses do constitute a clade within the Alphabaculovirus Group 2 NPVs, indicating that they are more similar to each other than they are to other lepidopteran viruses, even other geometrid-derived NPVs. As far as we know, this is the first report of sequences from an NPV from Bruce spanworm. Results from cross infection trials suggest that cross infection is uncommon if it occurs at all. Our results show that these two closely related species have distinct viruses and, unlike previous suggestions, Bruce spanworm virus is not mediating the winter moth invasion.

RNAi: future in insect management.

RNA interference is a post- transcriptional, gene regulation mechanism found in virtually all plants and animals including insects. The demonstration of RNAi in insects and its successful use as a tool in the study of functional genomics opened the door to the development of a variety of novel, environmentally sound approaches for insect pest management. Here the current understanding of the biogenesis of the two RNAi classes in insects is reviewed. These are microRNAs (miRNAs) and short interfering RNAs (siRNAs). Several other key approaches in RNAi -based for insect control, as well as for the prevention of diseases in insects are also reviewed. The problems and prospects for the future use of RNAi in insects are presented.

Analysis of the genome of the sexually transmitted insect virus Helicoverpa zea nudivirus 2.

The sexually transmitted insect virus Helicoverpa zea nudivirus 2 (HzNV-2) was determined to have a circular double-stranded DNA genome of 231,621 bp coding for an estimated 113 open reading frames (ORFs). HzNV-2 is most closely related to the nudiviruses, a sister group of the insect baculoviruses. Several putative ORFs that share homology with the baculovirus core genes were identified in the viral genome. However, HzNV-2 lacks several key genetic features of baculoviruses including the late transcriptional regulation factor, LEF-1 and the palindromic hrs, which serve as origins of replication. The HzNV-2 genome was found to code for three ORFs that had significant sequence homology to cellular genes which are not generally found in viral genomes. These included a presumed juvenile hormone esterase gene, a gene coding for a putative zinc-dependent matrix metalloprotease, and a major facilitator superfamily protein gene; all of which are believed to play a role in the cellular proliferation and the tissue hypertrophy observed in the malformation of reproductive organs observed in HzNV-2 infected corn earworm moths, Helicoverpa zea.

Identification of a nucleopolyhedrovirus in winter moth populations from Massachusetts.

Winter moth, Operophtera brumata, originally from Europe, has invaded eastern Massachusetts causing widespread defoliation and damage to many deciduous tree species and a variety of crop plants in the infested area. We identified O. brumata nucleopolyhedrovirus (OpbuNPV) in winter moth larvae collected from field sites in Massachusetts by using PCR to amplify a 482 bp region of the baculovirus polyhedrin gene. Viral sequences were also detected in winter moth pupae that failed to emerge, suggesting that these insects may have died as a result of viral infection. This represents the first report of OpbuNPV in winter moth populations in the US.

Presence and prevalence of viruses in local and migratory honeybees (Apis mellifera) in Massachusetts.

Migratory and local bees in Massachusetts were analyzed for seven viruses. Three were detected: black queen cell virus (BQCV), deformed wing virus (DWV), and sacbrood virus (SBV). DWV was most common, followed closely by BQCV and then by SBV. BQCV and SBV were present at significantly higher rates in the migratory bees assayed, bringing into question the impact that these bees have on the health of local bee populations.

Two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica are related and form a distinct phylogenetic clade.

Glossina pallidipes and Musca domestica salivary gland hypertrophy viruses (GpSGHV and MdSGHV) replicate in the nucleus of salivary gland cells causing distinct tissue hypertrophy and reduction of host fertility. They share general characteristics with the non-occluded insect nudiviruses, such as being insect-pathogenic, having enveloped, rod-shaped virions, and large circular double-stranded DNA genomes. MdSGHV measures 65x550 nm and contains a 124 279 bp genome (approximately 44 mol% G+C content) that codes for 108 putative open reading frames (ORFs). GpSGHV, measuring 50x1000 nm, contains a 190 032 bp genome (28 mol% G+C content) with 160 putative ORFs. Comparative genomic analysis demonstrates that 37 MdSGHV ORFs have homology to 42 GpSGHV ORFs, as some MdSGHV ORFs have homology to two different GpSGHV ORFs. Nine genes with known functions (dnapol, ts, pif-1, pif-2, pif-3, mmp, p74, odv-e66 and helicase-2), a homologue of the conserved baculovirus gene Ac81 and at least 13 virion proteins are present in both SGHVs. The amino acid identity ranged from 19 to 39 % among ORFs. An (A/T/G)TAAG motif, similar to the baculovirus late promoter motif, was enriched 100 bp upstream of the ORF transcription initiation sites of both viruses. Six and seven putative microRNA sequences were found in MdSGHV and GpSGHV genomes, respectively. There was genome. Collinearity between the two SGHVs, but not between the SGHVs and the nudiviruses. Phylogenetic analysis of conserved genes clustered both SGHVs in a single clade separated from the nudiviruses and baculoviruses. Although MdSGHV and GpSGHV are different viruses, their pathology, host range and genome composition indicate that they are related.

Infection with the insect virus Hz-2v alters mating behavior and pheromone production in female Helicoverpa zea moths.

The effect of Hz-2V virus infection on the reproductive physiology and behavior of infected Helicoverpa zea female moths was examined. In the absence of males, infected females exhibited calling behavior and called as often but for shorter periods on average than control females. As expected, control females mated with males for extend periods when they were present and did not call after mating, while virus-infected females made many frequent contacts with males and continued to call even after these contacts. Virus-infected females were found to produce five to seven times more pheromone than control females and attracted twice as many males as did control females in flight tunnel experiments. The ability of Hz-2V to alter the physiology and behavior of infected females observed here may serve to facilitate the transmission of virus in insect populations.

In vivo dose-response of insects to Hz-2V infection.

BACKGROUND: Hz-2V infection of female Helicoverpa zea moths is manifested as insects that are either sterile "agonadal" individuals with malformed reproductive tissues or fertile asymptomatic carriers which are capable of transmitting virus on to their progeny. Virus infected progeny arising from eggs laid by asymptomatic carrier females may themselves be either sterile agonadals or asymptomatic carriers. RESULTS: By injecting virus into female moths, a correlation was established between virus doses administered to the females and the levels of resulting asymptomatic and sterile progeny. CONCLUSIONS: The results of these experiments indicate that high virus doses produced a higher level of agonadal progeny and lower doses produced higher levels of asymptomatic carriers.

Horizontal transmission of Hz-2V by virus infected Helicoverpa zea moths.

Helicoverpa zea female moths productively infected with Hz-2V have malformed reproductive tissues and are sterile. Virus replication in infected females occurs primarily in the reproductive tissues and culminates with the accumulation of virus-filled vesicles, which form plugs of virus covering the reproductive openings of these insects. The location of this large concentration of virus particles at the terminal abdominal segment of infected females suggests that it may serve as a source of virus that can be transmitted horizontally between moths during mating. In mating experiments it was found that healthy males are attracted to and attempt to mate with infected females, and that these males are able transmit Hz-2V to healthy females during subsequent matings, giving rise to virus infected progeny.

Pathology and ultrastructure of Hz-2V infection in the agonadal female corn earworm, Helicoverpa zea.

The pathology and ultrastructure of the reproductive tract of Hz-2V-infected female corn earworm moths, Helicoverpa zea, were studied. The identity of malformed reproductive tissues found in virus-infected moths was determined by examining these tissues in moths that were infected with the virus at different life stages. Malformation of reproductive tissues in the progeny of virus-infected female moths was first observed by 3 days post-pupation (dpp), indicating that virus replication had altered the differentiation of these tissues very early on in their development. The ultrastructure of the grossly malformed agonadal reproductive tissues from insects aged 3-10dpp revealed the absence of the cuticular lining found in the oviducts of normal moths, and the proliferation of epithelial cells in these infected oviduct tissues. In addition, large quantities of virus were found aggregated into a large mass in the lumen of the malformed cervix bursa of 10dpp agonadal female pharate adult moths. Prior to eclosion, the virus in the cervix bursa was observed separated into spherical masses, which are thought to exude through the ductus bursa and collect over the vulva, forming a viral "waxy plug" that is likely to play an important role in virus transmission.

Pathology and ultrastructure of the insect virus, Hz-2V, infecting agonadal male corn earworms, Helicoverpa zea.

The pathology of the reproductive tract of Hz-2V-infected agonadal male corn earworm moths, Helicoverpa zea, was studied. The examination of the reproductive tissues of adult agonadal males infected with Hz-2V during different lifestages allowed us to positively correlate the grossly malformed tissues of typical agonadal male moths to the corresponding normal tissues in uninfected males. The reproductive tissues responsible for producing sperm, a pheromonostatic peptide (PSP), and the spermatophore in normal male moths were absent or grossly malformed in the agonadal male moths. Hz-2V was observed replicating in one area of these malformed reproductive tissues in pharate adult males as early as 7 days post-pupation. Interestingly, reproductive tissues essential for initiation of copulation and transfer of reproductive fluids into a female moth during mating appear to be intact and may be functional. These data suggest that agonadal adult males are able to mate with healthy female moths and transfer Hz-2V particles, without fertilizing female moths or altering their sexual receptivity to further mating with other male moths.