Trans-generational transmission of the Glossina pallidipes hytrosavirus depends on the presence of a functional symbiome

The vertically transmitted endosymbionts (Sodalis glossinidius and Wigglesworthia glossinidia) of the tsetse fly (Diptera: Glossinidae) are known to supplement dietary deficiencies and modulate the reproductive fitness and the defense system of the fly. Some tsetse fly species are also infected with the bacterium, Wolbachia and with the Glossina hytrosavirus (GpSGHV). Laboratory-bred G. pallidipes exhibit chronic asymptomatic and acute symptomatic GpSGHV infection, with the former being the most common in these colonies. However, under as yet undefined conditions, the asymptomatic state can convert to the symptomatic state, leading to detectable salivary gland hypertrophy (SGH+) syndrome. In this study, we investigated the interplay between the bacterial symbiome and GpSGHV during development of G. pallidipes by knocking down the symbionts with antibiotic. Intrahaemocoelic injection of GpSGHV led to high virus titre (109 virus copies), but was not accompanied by either the onset of detectable SGH+, or release of detectable virus particles into the blood meals during feeding events. When the F1 generations of GpSGHV-challenged mothers were dissected within 24 h post-eclosion, SGH+ was observed to increase from 4.5% in the first larviposition cycle to >95% in the fourth cycle. Despite being sterile, these F1 SGH+ progeny mated readily. Removal of the tsetse symbiome, however, suppressed transgenerational transfer of the virus via milk secretions and blocked the ability of GpSGHV to infect salivary glands of the F1 progeny. Whereas GpSGHV infects and replicates in salivary glands of developing pupa, the virus is unable to induce SGH+ within fully differentiated adult salivary glands. The F1 SGH+ adults are responsible for the GpSGHV-induced colony collapse in tsetse factories. Our data suggest that GpSGHV has co-evolved with the tsetse symbiome and that the symbionts play key roles in the virus transmission from mother to progeny

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 famil

Improving Sterile Insect Technique (SIT) for tsetse flies through research on their symbiont and pathogens

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

Two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica are closely 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 (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 relate

External development of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in the subterranean termite Heterotermes tenuis Desenvolvimento dos fungos entomopatogênicos Beauveria bassiana E Metarhizium anisopliae no cupim subterrâneo Heterotermes tenuis

The subterranean termite Heterotermes tenuis is one of the main pests of sugarcane and eucalyptus in Brazil, and the use of entomopathogenic fungi, alone or associated to chemicals, is an efficient and environmentally favorable method for its control. Studies related to the fungal development on these insects are important due to the effect of insect behavior on entomopathogens. The objective of this work was to describe the external development of Beauveria bassiana and Metarhizium anisopliae on H. tenuis using Scanning Electron Microscopy (SEM), determining the duration of the different phases of fungal infection. Two fixation techniques for preparing SEM samples were also evaluated. Worker specimens of H. tenuis were inoculated with a 1 x 10(9) conidia mL-1 suspension of the fungi and maintained at 25±1ºC and 70±10% relative humidity. Insects were collected from 0 to 144 hours after inoculation and prepared on SEM stubs for each of the two fixation techniques. The results obtained with the two techniques were compared and duration of the different phases of the infection process were estimated from SEM observations and compared for three fungal isolates. B. bassiana and M. anisopliae have similar development cycles on the termite, but some important differences exist. The penetration, colonization and conidiogenesis phases are relatively faster for M. anisopliae than for B. bassiana, which results in a faster rate of insect mortality. The fixation technique with OsO4 vapor is suitable for preparation of insects to be used in SEM observation of the developmental stages of entomopathogenic fungi.<br>O cupim subterrâneo Heterotermes tenuis , uma das principais pragas da cana-de-açúcar e eucalipto no Brasil, e o uso de fungos entomopatogênicos, isoladamente ou associados a produtos químicos, é um método eficiente e ambientalmente seguro para seu controle. Estudos relacionados ao desenvolvimento fúngico nestes insetos são importantes devido ao efeito do comportamento dos insetos sobre entomopatógenos. O objetivo deste trabalho foi descrever o desenvolvimento de Beauveria bassiana e Metarhizium anisopliae sobre H. tenuis por meio da Microscopia Eletrônica de Varredura (MEV), determinando a duração das fases de infecção fúngica. Também foram avaliadas duas técnicas de fixação para o preparo de amostras para MEV. Operários de H. tenuis foram inoculados com suspensões fúngicas de 1 x 10(9) conídios mL-1 e mantidos a 25 ± 1ºC e umidade relativa de 70 ± 10%. Foram coletados insetos de 0 a 144 horas após a inoculação e preparados pelas duas técnicas de fixação. Foram comparados os resultados obtidos com as duas técnicas e estimadas e comparadas as durações das fases do processo de infecção para três isolados fúngicos por meio de observações em MEV. B. bassiana e M. anisopliae têm ciclos de desenvolvimento semelhantes sobre H. tenuis, mas algumas diferenças importantes existem. As fases de penetração, colonização e conidiogênese são relativamente mais rápidas para M. anisopliae que para B. bassiana, o que resulta em uma taxa mais rápida de mortalidade do inseto. A técnica de fixação com vapor de OsO4 é satisfatória para preparação de insetos para observação do desenvolvimento de fungos entomopatogênicos em MEV