Introduction
Aphids (Hemiptera: Aphididae) are plant sap-feeding insects that interact with a range of microbial mutualists and pathogens. Almost all aphid species carry the obligate nutritional symbiont, Buchnera aphidicola, which is maternally transmitted and upgrades the nutritional profile of plant phloem (Douglas 2009). Nine facultative, heritable symbionts have also been identified in different species that can confer conditional benefits including defense against parasitic wasps and fungal pathogens (Oliver et al. 2010, Oliver and Martinez 2014, Vorburger 2014, Guo et al. 2017). Aphids frequently encounter viruses in the environment, including economically-important plant viruses that they vector (Brault et al. 2010) and pathogenic viruses specialized on aphids. The latter include single-stranded DNA viruses in the family Parvoviridae (Piccovirales) and positive-sense ssRNA viruses in the families Picornaviridae and Dicistroviridae (Picornavirales) (Van der Wilk et al. 1997, Moon et al. 1998, van Munster et al. 2003, Ryabov 2007, Ryabov et al. 2009, Asgari and Johnson 2010, Liu et al. 2016). However, little is known about the interactions among the many protective symbionts in aphids and pathogenic viruses.
Acyrthosiphon pisum virus (APV), is a picorna-like virus that persistently infects the pea aphid, Acyrthosiphon pisum (Van den Heuvel et al. 1997). The 10 kb APV genome contains two open reading frames (P1, P2) encoding a protease, helicase, RNA-dependent RNA polymerase and capsid protein (Van der Wilk et al. 1997). While primarily detected in epithelial cells of the gut and salivary glands, APV has also been weakly detected in the ovaries of pea aphids (Van den Heuvel et al. 1997, Lu et al. 2020). APV is vertically transmitted at moderate rates, and can be horizontally transferred from aphids to plants (Van den Heuvel et al. 1997, Lu et al. 2020), but no studies have shown aphid acquisition of APV from plants. APV exerts variable effects on aphid growth, survival and reproduction (Van den Heuvel et al. 1997, Lu et al. 2020), but how pea aphid genotype and facultative symbionts influence APV infection has not previously been investigated.
Hamiltonella defensa (Yersiniaceae: γ-Proteobacteria) is one of the most studied facultative symbionts in pea aphids because certain strains confer high levels of resistance against parasitoid wasps likeAphidius ervi (Hymenoptera: Braconidae) (Oliver and Higashi 2019). Protective strains of H. defensa further host specific variants of a bacteriophage named APSE which as a provirus expresses virulence genes that have been implicated in disabling parasitoid development (Oliver et al. 2009, Brandt et al. 2017, Lynn-Bell et al. 2019, Rouïl et al. 2020, Boyd et al. 2021). H. defensa strains infected by APSE-3 confer high levels of protection (> 85% of parasitized aphids survive) while strains infected by APSE-2 or APSE-8 provide moderate protection (40-60%) (Weldon et al. 2013, Oliver and Higashi 2019). Aphid genotype also contributes to resistance to parasitoids (Martinez et al. 2018). Another facultative symbiont associated with pea aphids, Regiella insecticola , is closely related to H. defensa (Patel et al. 2019) and confers protection against specialist entomopathogenic fungi like Pandora neoaphidis(Entomophthorales) (Scarborough et al. 2005, Parker et al. 2013). Levels of protection conferred by R. insecticola also vary with symbiont strain and host genotype (Parker et al. 2017).
We recently discovered an APV in certain pea aphid lines maintained in our laboratory. In this study, we assembled the genome of this APV and conducted assays that examined transmission and fitness effects on aphids by generating genetically homogeneous lines that controlled aphid and Buchnera genotypes while manipulating facultative symbiont presence and APV infection. We report that aphid fitness was adversely affected in aphids without facultative symbionts, effects which were worsened in aphid carrying H. defensa . In contrast, negative fitness impacts were ameliorated in aphids hosting R. insecticola . Overall, our results identify heretofore unknown interactions between facultative symbionts and a pathogenic virus that strongly influence host fitness.