To B or not to B: Arsenophonus as a source of B-vitamins in whiteflies

Insect lineages feeding on nutritionally restricted diets such as phloem, xylem, or blood, were able to diversify by acquiring bacterial species that complemented the missing nutrients. These bacteria, considered obligate/primary endosymbionts, share a long evolutionary history with their hosts. In some cases, however, these endosymbionts are not able to fulfill all the nutritional requirements of their host, driving the acquisition of additional symbiotic species. Whiteflies, which feed on phloem, established an obligate relationship with Candidatus Portiera aleyrodidarum, who provides essential amino acids and carotenoids to the host. As many Whiteflies species harbor additional endosymbionts, they could provide their hosts with missing nutrients. To test this hypothesis, genomes of several endosymbionts from the whiteflies Aleurodicus dispersus, A. floccissimus and Trialeurodes vaporariorum were sequenced and analyzed. All three species were found to harbor an endosymbiont from the genus Arsenophonus, and the two former also host Wolbachia. A comparative analysis of the three Arsenophonus genomes revealed that although all of them are capable of synthesizing B-vitamins and cofactors, such as pyridoxal, riboflavin, or folate, their genomes and phylogenetic relationship vary greatly. Arsenophonus of A. floccissimus and T. vaporariorum belong to the same clade and display characteristics of facultative endosymbionts, such as large genomes (3 Mb) with thousands of genes, many pseudogenes, intermediate GC content, and mobile genetic elements (MGEs). In contrast, Arsenophonus of A. dispersus belongs to a different lineage and displays the characteristics of a primary endosymbiont, such as a reduced genome (670 kb) with 400 genes, 32% GC content, and no MGEs. However, the presence of 274 pseudogenes suggests that this symbiotic association is more recent than other reported hemipteran’s primary endosymbionts. Arsenophonus of A. dispersus gene repertoire is completely integrated in the symbiotic consortia, and the biosynthesis of most vitamins occurs in shared pathways with its host. In addition, Wolbachia have also retained the ability to produce riboflavin, FAD, and folate, and may have a nutritional contribution. Taken together, our results show that Arsenophonus have a pivotal place in whiteflies nutrition by their ability to produce B-vitamins, even if they diverge and/or go through a genome reduction process.


180
The small number of shared protein clusters results from the reduced proteome of ARAD, which 181 is mainly a subset of the larger ARTV and ARAF proteomes. Only 4 out of the 10 ARAD  (Table S5). (C) Synteny between the draft genomes of ARTV and ARAF. Contig edges are displayed as dashed lines. Contigs of ARTV were ordered with Mauve using ARAF as reference for a better visualization.
Wolbachia ARAD and ARAF showed a core genome of 686 clusters, and 253 and 169 species 193 8 specific clusters, respectively ( Figure 3A, Table S6). It should be noted, however, that the  (Table S6).
(B) Synteny between the draft genomes (see Table 1) WBAD and WBAF. Contigs of WBAF were ordered with Mauve using WBAD as reference for a better visualization.
The proteomes of the five endosymbionts were functionally classified according to the Cluster     (Table S2). ARAF and ARTV also require complementation by their host in order to produce 251 folate. ARTV lost most of the pathway to produce thiamine, and the missing steps are not 252 complemented by its host. Additionally, B. tabaci seems to be able to produce biotin using some  tabaci genome. This suggests that these whiteflies are not able to produce pantothenate and 274 they acquire it from the diet or other sources ( Figure 5B and Table S2). However, failure on 275 horizontal transferred genes detection could be an artifact from the methods used, since they 276 are unable to discover new horizontally transferred genes in the whiteflies screened.

277
The third endosymbiont of A. dispersus and A. floccissimus, Wolbachia WBAD and WBAF, 278 may be also contributing to the vitamin/cofactor synthesis of the system, as they have the 279 complete pathway for riboflavin-FMN-FAD synthesis and potentially are able to produce folate 280 from some intermediate precursors ( Figure 5B).