A viral mutualist employs posthatch transmission for vertical and horizontal spread among parasitoid wasps.

Heritable symbionts display a wide variety of transmission strategies to travel from one insect generation to the next. Parasitoid wasps, one of the most diverse insect groups, maintain several heritable associations with viruses that are beneficial for wasp survival during their development as parasites of other insects. Most of these beneficial viral entities are strictly transmitted through the wasp germline as endogenous viral elements within wasp genomes. However, a beneficial poxvirus inherited by Diachasmimorpha longicaudata wasps, known as Diachasmimorpha longicaudata entomopoxvirus (DlEPV), is not integrated into the wasp genome and therefore may employ different tactics to infect future wasp generations. Here, we demonstrated that transmission of DlEPV is primarily dependent on parasitoid wasps, since viral transmission within fruit fly hosts of the wasps was limited to injection of the virus directly into the larval fly body cavity. Additionally, we uncovered a previously undocumented form of posthatch transmission for a mutualistic virus that entails external acquisition and localization of the virus within the adult wasp venom gland. We showed that this route is extremely effective for vertical and horizontal transmission of the virus within D. longicaudata wasps. Furthermore, the beneficial phenotype provided by DlEPV during parasitism was also transmitted with perfect efficiency, indicating an effective mode of symbiont spread to the advantage of infected wasps. These results provide insight into the transmission of beneficial viruses among insects and indicate that viruses can share features with cellular microbes during their evolutionary transitions into symbionts.

Establishment and characterization of the Bactrocera dorsalis (Diptera: Tephritidae) embryonic cell line QAU-Bd-E-2.

In this study, we successfully established a Bactrocera dorsalis (Diptera: Tephritidae) embryonic cell line, i.e., QAU-Bd-E-2, from the insect eggs. The cells have been stably passaged for more than 60 times in TNM-FH medium with 10% fetal bovine serum (FBS). QAU-Bd-E-2 cells are adherent cells. Most of the cells were round, spindle-shaped, and rod-shaped. Round cells accounted for 82.3%, with a diameter of 13.9 ± 2.6 µm; spindle-shaped cells accounted for 9.8%, with the size of 51.2 ± 11.2 µm × 10.3 ± 3.1 µm; the rod-shaped cells accounted for 7.9%, with the size of 35.2 ± 9.4 µm × 12.0 ± 2.5 µm. The mitochondrial cytochrome oxidase I subunit (CoI) gene from QAU-Bd-E-2 cells was amplified, and the 657 bp fragment had a 100% similarity with the CoI gene of B. dorsalis, suggesting that the cell line was derived from B. dorsalis. The chromosome number of QAU-Bd-E-2 cells was mostly 12, which is the same as the B. dorsalis chromosome number. The cell density of QAU-Bd-E-2 cells reached the maximum (3.4 × 106 cells/mL) at 192 h, and the population doubling time was 31.9 h. Bactrocera dorsalis cripavirus (BdCV) could replicate in QAU-Bd-E-2 cells, suggesting that this cell line could be used for in-depth study of the relationship between virus and host.

Tephritid fruit flies have a large diversity of co-occurring RNA viruses.

Tephritid fruit flies are amongst the most devastating pests of horticulture, and Sterile Insect Technique (SIT) programs have been developed for their control. Their interactions with viruses are still mostly unexplored, yet, viruses may negatively affect tephritid health and performance in SIT programs, and, conversely, constitute potential biological control agents. Here we analysed ten transcriptome libraries obtained from laboratory populations of nine tephritid species from Australia (six species of Bactrocera, and Zeugodacus cucumis), Asia (Bactrocera dorsalis) and Europe (Ceratitis capitata). We detected new viral diversity, including near-complete (>99%) and partially complete (>80%) genomes of 34 putative viruses belonging to eight RNA virus families. On average, transcriptome libraries included 3.7 viruses, ranging from 0 (Z. cucumis) to 9 (B. dorsalis). Most viruses belonged to the Picornavirales, represented by fourteen Dicistroviridae (DV), nine Iflaviridae (IV) and two picorna-like viruses. Others were a virus from Rhabdoviridae (RV), one from Xinmoviridae (both Mononegavirales), several unclassified Negev- and toti-like viruses, and one from Metaviridae (Ortervirales). Using diagnostic PCR primers for four viruses found in the transcriptome of the Bactrocera tryoni strain bent wings (BtDV1, BtDV2, BtIV1, and BtRV1), we tested nine Australian laboratory populations of five species (B. tryoni, Bactrocera neohumeralis, Bactrocera jarvisi, Bactrocera cacuminata, C. capitata), and one field population each of B. tryoni, B. cacuminata and Dirioxa pornia. Viruses were present in most laboratory and field populations yet their incidence differed for each virus. Prevalence and co-occurrence of viruses in B. tryoni and B. cacuminata were higher in laboratory than field populations. This raises concerns about the potential accumulation of viruses and their potential health effects in laboratory and mass-rearing environments which might affect flies used in research and control programs such as SIT.

The RNA Virome and Its Dynamics in an Invasive Fruit Fly, Bactrocera dorsalis, Imply Interactions Between Host and Viruses.

The oriental fruit fly, Bactrocera dorsalis (Hendel), is an important invasive agricultural insect pest with a wide host range, and has spread around the world over the last century. This evolutionary trait may have arisen primarily from interactions between B. dorsalis and other invertebrates that share the same ecological niches. The invasive behavior of B. dorsalis also frequently exposes them to diverse species of viruses. Thereby, RNA viromes may be useful microbial markers to understand the ecological evolution of B. dorsalis as well as to investigate virus-host interactions. Here, we reported eight novel RNA viruses in B. dorsalis of a lab colony, including four positive-strand RNA viruses, two negative-strand RNA viruses, and two double-stranded RNA viruses using high-throughput sequencing technology. Analysis of the virus-derived small RNAs suggested that most of these viruses may be active and trigger the host antiviral RNAi responses. The viruses were also detected in various geographical populations of B. dorsalis, implying that there is a strong association between the viromes and host. In addition, these viruses infected specific fly tissues, predominately the central nervous system and gut. Furthermore, we explored the dynamics of the viruses when hosts were exposed to short- or long-term stressors, which showed that titers of some viruses were responsively altered in the stressed B. dorsalis. The discovery of these viruses may enrich our understanding of the species diversity of RNA viruses and also provide information on viruses in association with host adaptation in insects.

Morphogenesis and cytopathic effects of the Diachasmimorpha longicaudata entomopoxvirus in host haemocytes.

The Diachasmimorpha longicaudata entomopoxvirus (DlEPV), the first reported symbiotic entomopoxvirus, occurs in the venom apparatus of D. longicaudata female wasps and is introduced into Anastrepha suspensa larvae during parasitism. The DlEPV 250-300 kb double stranded DNA genome encodes putative proteins having 30 to >60% amino acid identity with poxvirus homologs such as DNA helicase, DNA-dependent RNA polymerase, and the poxvirus-specific rifampicin resistance protein. Although the molecular characterization of DlEPV is progressing, little is known about its morphogenesis in and effects on host haemocytes. This paper describes (1) haemocytes of third instar A. suspensa, (2) DlEPV infection and morphogenesis, and (3) DlEPV-induced changes in haemocytes. A. suspensa third instars have 3-4 haemocyte morphotypes. Dot blots of DNA from infected haemocytes hybridized with a digoxigenin-labeled DlEPV genomic probe as early as 4 h post-parasitism (hpp) and the intensity of the signal increased with time through 40 hpp. Immunofluorescence microscopy localized DlEPV proteins in cytoplasmic (but not nuclear) sites of infected haemocytes, within 24-36 hpp. Electron microscopy confirmed the presence of viral envelopes, immature spheroids with centric nucleoids, budding virus, and extracellular enveloped virus in three haemocyte types, 24-84 hpp and later. Infected haemocytes exhibited blebbing, DNA concatenation, and inability to encapsulate sephadex beads in vitro. These data indicate that DlEPV disrupts the normal function of host haemocytes, thereby insuring the successful development of D. longicaudata offspring and as such should be regarded as a symbiont of the wasp.

Transmission of the Diachasmimorpha longicaudata rhabdovirus (DlRhV) to wasp offspring: an ultrastructural analysis.

During oviposition, the parasitic wasp Diachasmimorpha longicaudata introduces an entomopoxvirus (DlEPV) and a rhabdovirus (DlRhV) into larvae of its tephritid fruit fly host Anastrepha suspensa. DlEPV and DlRhV replicate, respectively, in host hemocytes and epidermal cells. Both viruses, like many beneficial viruses of parasitic wasps, are retained in all wasp generations but their avenue(s) of transmission are unknown. This study tests the hypothesis that DlRhV is transmitted transovarially or through larval feeding on infected host hemolymph. Transmission electron microscopy (TEM) revealed no virions in pre-vitellogenic or vitellogenic ova, or in the lateral oviduct of D. longicaudata females. However, numerous virions occurred in subchorionic regions of 33-36-h-old oviposited eggs. This suggests that DlRhV is introduced into the egg either as (a) intact virions after chorionogenesis but prior to oviposition and/or as (b) unencapsidated RNA molecules, undetectable by TEM in pre-vitellogenic ova, that subsequently replicate and assemble into mature virions. DlRhV particles also occurred in the midgut lumen of 20-24-h-old wasp first instars, suggesting that they were ingested. These virions may have been released from the egg into the hemolymph during hatching or may have come from virions introduced by the female wasp directly into the host, separate from the egg. DlRhV particles were also evident in the intracellular vesicles and intercellular spaces of the larval midgut. Taken together, these data support the hypothesis that DlRhV is transovarially transmitted as virions and/or as unencapsidated RNA. Further studies are needed to determine whether the DlRhV that ultimately resides within the female wasp's accessory gland filaments is the progeny of the virus from the egg and/or larval midgut cells.