Uncovering Active Bacterial Symbionts in Pollen-Feeding Beetles

Microbial symbionts enable many phytophagous insects to specialize on plant-based diets through a range of metabolic services. Pollen comprises one plant tissue consumed by such herbivores. While rich in lipids and protein, its nutrient content is often imbalanced and dicult-to-access due to a digestibly recalcitrant cell wall. Pollen quality can be further degraded by harmful allelochemicals. To identify microbes that may aid in palynivory, we performed cDNA-based 16S rRNA metabarcoding on three related pollen beetles (Nitidulidae: Meligethinae) exhibiting different dietary breadths: Brassicogethes aeneus, B. matronalis, and Meligethes atratus. Nine bacterial symbionts (i.e. 97% OTUs) exhibited high metabolic activity during active feeding. Subsequent PCR surveys revealed varying prevalence of those from three Rickettsialles genera - Lariskella, Rickettsia and Wolbachia - within beetle populations. Our ndings lay the groundwork for future studies on the inuence of phylogeny and diet on palynivorous insect microbiomes, and roles of symbionts in the use of challenging diets.


Main Text
Many phytophagous insects harbor symbiotic microorganisms that play fundamental roles in their hostplant interactions [1]. Symbiotic bacteria can synthesize essential diet-limited metabolites, break down defensive or recalcitrant plant compounds, or recycle/upgrade metabolites that are metabolically inaccessible to insects [1,2]. Through such activities, they can have a large impact on the adaptation of insects with varying degrees of dietary specialization.
The beetle subfamily Meligethinae includes ~700 species that develop in budding owers [3,4] of Dicots and Monocots (palms), with both larvae and adults consuming pollen as their primary food. Evolution within this subfamily has been characterized by repeated host shifts towards plants of different tribes or genera within the same botanical family, and more rarely towards members of unrelated families [5,6,7,8]. Overall, Meligethinae are remarkably variable in their degrees of host-plant specialization within several genera [3]. These factors position this subfamily as a suitable group for studies on how phylogeny and diet shape symbiont communities [9,10]. Given the precedent for microbial digestion of pollen [11], this group holds further promise for exploring symbionts' impacts on diet use and diversi cation.
To initiate such investigation, we studied microbiomes of three Meligethinae species varying in host-plant specialization. Brassicogethes aeneus is a renowned pest of oilseed rape crops [12] and feed on various Brassicacae (e.g. Brassica, Sinapis, Cakile, and Biscutella). Its congener, B. matronalis, is monophagous, feeding exclusively on Hesperis matronalis (Brassicaceae). Meligethes atratus is a more distantly related oligophagous beetle, associated with genera from the Rosaceae (i.e. Rosa, Crataegus and Prunus) [5,7]. Highlighting active bacterial communities in these beetles, we performed 16S rRNA metabarcoding on cDNA libraries from a total of 66 dissected individuals collected while feeding on their respective host plants (Supplementary Methods). cDNA was synthesized from two pooled RNA extractions per species -one from dissected head tissue and one from abdomens -aiding in preliminary ascertainment of whether bacteria are con ned to the midgut and hindgut, or more broadly distributed throughout tissues beyond. After quality control and ltering of our six Illumina V4 sequencing libraries, we obtained 69,382 reads (B. aeneus = 21,337; B. matronalis = 19,549; M. atratus = 28,496; Supplementary data). Sequences were clustered into Operational Taxonomic Units (OTUs) at 97% sequence similarity, enabling us to compute the relative 'activity' [13] of distinct symbionts.
Among these Rickettsiales we detected differences in abundance/activity across the three pollen-beetle species: Wolbachia, Rickettsia and Lariskella, dominated in B. aeneus, M. atratus and B. matronalis, respectively, and were rare or inactive in the other species (Fig. 1). Since Rickettsiales are common insect symbionts and since some may assist phytophagy [28], we further explored their presence in DNA from 71 separately collected Meligethinae specimens through diagnostic PCR (Supplementary Methods). The generated data largely validated initial ndings (Table 1), supporting the con nement of Lariskella to B. matronalis, and the occurrence of Rickettsia in the two generalist species, most notably M. atratus. Wolbachia was more widespread than expected from our initial study, suggesting uctuating prevalence or environmental modulation of Wolbachia activity.  Fig. 1; Supplementary data) -four were Enterobacteriales (32.32%), while the fth belonged to the Pseudomonadales (4.08%). Like the Rickettsiales, Enterobacteriales exhibited differential activity patterns across the three Meligethinae. For instance, Sodalis, a common facultative symbiont [29], was only active in the two generalist species. In other hosts, Sodalis provision nutrients such as tyrosine, lysine, biotin and ribo avin [30], suggesting the potential for bene cial impacts. Another bacterium, Morganella -found in other insects and beetles [31,32] -was also active in the two generalist species (Fig. 1). While several functions, and even commensalism, are possible, Sodalis and Morganella, like other Enterobacteriales, could aid the breakdown of plant cell wall compounds, including pectin [33].
Based on common plant-association for their closest relatives, we speculate that two other Enterobacteriales OTUs (i.e. 6 and 46), Pantoea/Erwinia and Pseudomonas, represent transiently acquired bacteria, noting that they accordingly showed higher activity/abundance in mid-and hind-gut containing tissues of B. matronalis (Fig. 1). Related bacteria are, however, known symbionts of insects. Based on prior ndings, these microbes could shape pollen cell wall breakdown [34,35], nutrient provisioning or recycling [36,37], or diet detoxi cation [38].
In conclusion, three pollen-feeding beetles from the Meligethinae harbor variable active microbiomes. Close relatives of abundantly active bacteria encode functions of possible use in a plant-based diet, with potential to in uence utilization of a sometimes nitrogen-rich [39], yet often nutrient-imbalanced [40], welldefended tissue [41]. Yet belonging to two different genera, the two generalist species shared more symbionts ( Fig. 1; Table 1). Future studies on other pollen-beetles are needed to weigh the combined in uence of host-plant use and phylogeny in structuring symbiotic communities.
Declarations Figure 1 Relative abundance (= 'activity') of bacterial genera across the three Meligethinae species (polyphagous or oligophagous = B. aeneus and M. atratus; monophagous = B. matronalis) on V4 amplicon sequencing of 16S rRNA from 2015-collected specimens. Bar graphs for each library (one column = pooled tissues from all eld-caught beetles; AB = abdomen, HE = head) show the percentage of denoised and qualitycontrolled Illumina sequence reads classi ed to genera. Rare bacteria (never exceeding a relative