The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa.

Wolbachia infections can present different phenotypes in hosts, including different forms of reproductive manipulation and antiviral protection, which may influence infection dynamics within host populations. In populations of Drosophila pandora two distinct Wolbachia strains coexist, each manipulating host reproduction: strain wPanCI causes cytoplasmic incompatibility (CI), whereas strain wPanMK causes male killing (MK). CI occurs when a Wolbachia-infected male mates with a female not infected with a compatible type of Wolbachia, leading to nonviable offspring. wPanMK can rescue wPanCI-induced CI but is unable to induce CI. The antiviral protection phenotypes provided by the wPanCI and wPanMK infections were characterized; the strains showed differential protection phenotypes, whereby cricket paralysis virus (CrPV)-induced mortality was delayed in flies infected with wPanMK but enhanced in flies infected with wPanCI compared to their respective Wolbachia-cured counterparts. Homologs of the cifA and cifB genes involved in CI identified in wPanMK and wPanCI showed a high degree of conservation; however, the CifB protein in wPanMK is truncated and is likely nonfunctional. The presence of a likely functional CifA in wPanMK and wPanMK’s ability to rescue wPanCI-induced CI are consistent with the recent confirmation of CifA’s involvement in CI rescue, and the absence of a functional CifB protein further supports its involvement as a CI modification factor. Taken together, these findings indicate that wPanCI and wPanMK have different relationships with their hosts in terms of their protective and CI phenotypes. It is therefore likely that different factors influence the prevalence and dynamics of these coinfections in natural Drosophila pandora hosts. IMPORTANCE Wolbachia strains are common endosymbionts in insects, with multiple strains often coexisting in the same species. The coexistence of multiple strains is poorly understood but may rely on Wolbachia organisms having diverse phenotypic effects on their hosts. As Wolbachia is increasingly being developed as a tool to control disease transmission and suppress pest populations, it is important to understand the ways in which multiple Wolbachia strains persist in natural populations and how these might then be manipulated. We have therefore investigated viral protection and the molecular basis of cytoplasmic incompatibility in two coexisting Wolbachia strains with contrasting effects on host reproduction.

The native Australian soldier flies, Inopus spp. (Diptera: Stratiomyidae), are agricultural pests of economic importance to the sugarcane industry. While adult soldier flies do not feed on sugarcane, larvae spend one to two-years underground feeding on roots, causing mechanical and systemic damage to crops (Saccharum officinarum L.) that impacts yield. Current measures of pest control commonly target above ground pests and are ineffective against solider fly larvae, highlighting the importance of novel control methods. A screen of the salivary gland transcriptome of Inopus flavus (James) revealed the presence of viral RNA belonging to a potentially novel member of the Dicistroviridae family. Viruses from this family have been found naturally infecting insects from a range of taxonomic groups and they often cause pathogenesis in their hosts. To characterise the genetic and physical properties of the new virus, the positive RNA genome was analysed using a combination of sequencing approaches. The virus genome is organised similarly to members of the Dicistroviridae with two open reading frames (ORF) the first encoding non-structural proteins and the second encoding structural proteins. The genome includes two potential internal ribosomal entry sites (IRES) one within the 5' UTR and the other in the intergenic region (IGR). Based on the amino acid sequences of the non-structural and structural polyproteins encoded by the two ORF soldier fly virus groups within the dicistrovirus family. Virus particles purified from infected larvae and visualised by electron microscopy are icosahedral, non-enveloped, and 30 nm in diameter. The genetic and physical characteristics of this novel soldier fly virus are consistent with it being a member of the Dicistroviridae.

The native Australian soldier flies, Inopus spp. (Diptera: Stratiomyidae), are agricultural pests of economic importance to the sugarcane industry. While adult soldier flies do not feed on sugarcane, larvae spend one to two-years underground feeding on roots, causing mechanical and systemic damage to crops (Saccharum officinarum L.) that impacts yield. Current measures of pest control commonly target above ground pests and are ineffective against solider fly larvae, highlighting the importance of novel control methods. A screen of the salivary gland transcriptome of Inopus flavus (James) revealed the presence of viral RNA belonging to a potentially novel member of the Dicistroviridae family. Viruses from this family have been found naturally infecting insects from a range of taxonomic groups and they often cause pathogenesis in their hosts. To characterise the genetic and physical properties of the new virus, the positive RNA genome was analysed using a combination of sequencing approaches. The virus genome is organised similarly to members of the Dicistroviridae with two open reading frames (ORF) the first encoding non-structural proteins and the second encoding structural proteins. The genome includes two potential internal ribosomal entry sites (IRES) one within the 5’ UTR and the other in the intergenic region (IGR). Based on the amino acid sequences of the non-structural and structural polyproteins encoded by the two ORF soldier fly virus groups within the dicistrovirus family. Virus particles purified from infected larvae and visualised by electron microscopy are icosahedral, non-enveloped, and 30 nm in diameter. The genetic and physical characteristics of this novel soldier fly virus are consistent with it being a member of the Dicistroviridae.