Isolation, culture and characterization of Arsenophonus symbionts from two insect species reveal loss of infectious transmission and extended host range

Abstract

Vertically transmitted “Heritable” microbial symbionts represent an important component of the biology and ecology of invertebrates. These symbioses evolved originally from ones where infection/acquisition processes occurred within the environment (horizontal transmission). However, the pattern of evolution that follows transition from horizontal to vertical transmission is commonly obscured by the distant relationship between microbes with differing transmission modes. In contrast, the genus Arsenophonus provides an opportunity to investigate these processes with clarity, as it includes members that are obligate vertically transmitted symbionts, facultative vertically transmitted symbionts, strains with mixed modes of transmission and ones that are purely horizontally transmitted. Significantly, some of the strains are culturable and amenable to genetic analysis. We first report the isolation of Arsenophonus nasoniae strain aPv into culture from the ectoparasitic wasp Pachycrepoideus vindemmiae and characterize the symbiosis. We demonstrate maternal vertical transmission and find no evidence for paternal inheritance, horizontal transmission or reproductive parasitism phenotypes. This leads us to conclude this strain, in contrast to related strains, is a facultative heritable symbiont which is likely to be beneficial. We then report the serendipitous discovery and onward culture of a strain of Arsenophonus (strain aPb) from the blue butterfly, Polyommatus bellargus. This association extends the range of host species carrying Arsenophonus nasoniae/Arsenophonus apicola symbionts beyond the Hymenoptera for the first time. We perform basic metabolic analysis of the isolated strains using Biolog plates. This analysis indicates all strains utilize a restricted range of carbon sources, but these restrictions are particularly pronounced in the A. nasoniae aPv strain that is solely vertically transmitted. Finally, we demonstrate the Arsenophonus sp. strain aPb from the blue butterfly can infect Galleria waxworms, providing a model system for investigating the functional genetics of Arsenophonus-insect interactions. These results are consistent with a model of reduced metabolic competence in strains evolving under vertical transmission only. The data also broadens the range of host species infected with nasoniae/apicola clade strains beyond the Hymenoptera, and indicate the potential utility of the Galleria model for investigation of symbiosis mechanism.

Genome dynamics across the evolutionary transition to endosymbiosis

“…To gain a precise view of the tempo and mode of evolution during the transition to persistent endosymbiosis, we leveraged the wide diversity of host-associated lifestyles in the gammaproteobacterial genus Arsenophonus. Within this clade, there are environmentally acquired extracellular pathogens, extracellular endosymbionts with mixed modes of transmission, intracellular facultative endosymbionts with vertical transmission, and obligate vertically transmitted endosymbionts where the partners are co-dependent [6][7][8][9][10]. Importantly, extracellular pathogens and endosymbionts with mixed modes of transmission are very closely related.…”

“…Seven Arsenophonus isolates were isolated to pure culture and sequenced within this project: A. nasoniae isolates aNv_UK, aNv_CH and aNv_CAN derived from Nasonia vitripennis from the UK, Switzerland and Canada respectively, A. nasoniae aPv from Pachycrepoideus vindemmiae and the Arsenophonus strain aPb from the butterfly Polyommatus bellargus [9], Arsenophonus nasoniae strain aIh previously identified in the parasitoid wasp Ixodiphagus hookeri from a questing Ixodes ricinus tick in The Netherlands [28] and Arsenophonus apicola strain aApi_AU from Australian honey bees described in [29]. A further strain, Ca.…”

“…However, metabolic capabilities are not markedly distinct between vertically transmitted endosymbionts and environmentally-acquired strains. The completeness of metabolic pathways broadly reflects the abilities of strains to grow in in vitro cell-free culture [9]. One notable exception is beta oxidation of fatty acids, which was only intact in environmentally acquired strains; this result was sustained when draft genomes for the triatominarum group were additionally examined (Supplementary Figure S4).…”

16S rRNA Gene Sequencing of Six Psyllid Species of the Family Carsidaridae Identified Various Bacteria Including <i>Symbiopectobacterium</i>

“…Although Arsenophonus and Sodalis are among the symbionts most frequently observed in arthropod hosts including psyllid lineages ( Thao et al , 2000a ; Spaulding and von Dohlen, 2001 ; Hansen et al , 2007 ; Moran et al , 2008 ; Sloan and Moran, 2012 ; Arp et al , 2014 ; Hall et al , 2016 ; Morrow et al , 2017 ; Nakabachi et al , 2022a , 2022b ), Symbiopectobacterium is the lineage identified for the first time in Psylloidea. Arsenophonus have been found in diverse insect groups, including wasps ( Gherna et al , 1991 ; Nadal-Jimenez et al , 2023 ), bees (Hymenoptera: Apoidea) ( Nadal-Jimenez et al , 2022a ), aphids ( Russell et al , 2003 ; Wulff and White, 2015 ; Tian et al , 2019 ; Zhang et al , 2021 ; Yorimoto et al , 2022 ), psyllids ( Spaulding and von Dohlen, 1998 , 2001 ; Subandiyah et al , 2000 ; Thao et al , 2000a ; Hansen et al , 2007 ; Hall et al , 2016 ; Morrow et al , 2017 ; Nakabachi et al , 2022a , 2022b ), whiteflies ( Thao and Baumann, 2004 ; Chiel et al , 2007 ; El Hamss et al , 2021 ), triatomine bugs (Hemiptera: Heteroptera) ( Hypsa and Dale, 1997 ), lice (Psocodea: Anoplura) ( Sasaki-Fukatsu et al , 2006 ; Allen et al , 2007 ; Kirkness et al , 2010 ), louse flies ( Dale et al , 2006 ; Nováková et al , 2009 , 2016 ), and ticks (Arachnida: Ixodida) ( Grindle et al , 2003 ). The types of symbiotic relationships with hosts exhibit a wide diversity from parasitism ( e.g.…”