Gut bacteria of weevils developing on plant roots under extreme desert conditions

Abstract

Background: Many phytophagous insects, whose diet is generally nitrogen-poor, rely on gut bacteria to compensate for nutritional deficits. Accordingly, we hypothesized that insects in desert environments may evolve associations with gut bacteria to adapt to the extremely low nutrient availability. For this, we conducted a systematic survey of bacterial communities in the guts of weevils developing inside mud chambers affixed to plant roots in the Negev Desert of Israel, based on 16S rRNA gene amplicon sequencing.

Results: Our analyses revealed that gut bacterial communities in weevil larvae were similar across a wide geographical range, but differed significantly from those of the mud chambers and of the surrounding soils. Nevertheless, a high proportion of bacteria (including all of the core bacteria) found in the weevils were also detected in the mud chambers and soils at low relative abundances. The genus Citrobacter (of the Enterobacteriaceae family) was the predominant group in the guts of all individual weevils. The relative abundance of Citrobacter significantly decreased at the pupal and adult stages, while bacterial diversity increased. A mini literature survey revealed that members of the genus Citrobacter are associated with nitrogen fixation, recycling of uric acid nitrogen, and cellulose degradation in different insects.

Conclusions: The results suggest that although weevils could potentially acquire their gut bacteria from the soil, weevil host internal factors, rather than external environmental factors, were more important in shaping their gut bacterial communities, and suggest a major role for Citrobacter in weevil nutrition in this challenging environment. This study highlights the potential involvement of gut bacteria in the adaptation of insects to nutritional deficiencies under extreme desert conditions.

Keywords: Beetle; Citrobacter; Desert ecosystem; Nutrient; Symbiont; Weevil.

Fig. 1

Mud chamber affixed to the root of Salsola inermis, and larva, pupa, and adult (Menecleonus virgatus) in the mud chamber

Fig. 2

Bacterial community composition in the guts of Conorhynchus palumbus (N = 25), mud chamber (N = 25) and surrounding soil (N = 25). (a) PCoA plot displaying unweighted UniFrac distance. The percent variation explained by each principle coordinate is shown. (b) Venn diagram representing the number of OTUs that are unique to each of the sample type and shared between them. (c) Mean relative abundance of bacterial genera. For weevil, only those genera with > 1% mean relative abundance across all weevil samples are shown; for mud chamber and soil, only the top five genera are shown, whereas all remaining sequences are represented as others. S24–7, Actinomycetales, Rhodobacteraceae, and Frankineae are provided because the phylotypes were not classified to lower taxonomic levels. (d) Phylogenetic diversity. Columns with different letters are different at P < 0.05 based on Tukey’s post hoc tests

Fig. 3

Mean relative abundance of bacterial genera in the guts of Conorhynchus palumbus larvae across 11 different sites (Ashalim: N = 6; Abu Haduba, Dimona2, Dimona1, Mamshit, Havat MaShash, Tlalim, Yeruham, and Neot Hovav: N = 5; Mitzpe Ramon: N = 4; Revivim: N = 3). S24–7 was provided because the phylotype was not classified to lower taxonomic level. Only those genera with > 1% mean relative abundance across all weevil samples are shown, whereas all remaining sequences are represented as others

Fig. 4

Bacterial community composition in the guts of Conorhynchus palumbus at different developmental stages (larva: N = 44, pupa: N = 9, adult: N = 7). (a) PCoA plot displaying unweighted UniFrac distance. The percent variation explained by each principle coordinate is shown. (b) Venn diagram representing number of OTUs that are unique to each of developmental stage and shared between them. (c) Mean relative abundance of bacterial genera. Only those genera with > 1% mean relative abundance across all samples are shown, whereas all remaining sequences are represented as others. S24–7 was provided because the phylotype was not classified to lower taxonomic level. (d) Phylogenetic diversity. Columns with different letters are different at P < 0.05 based on Tukey’s post hoc tests

Fig. 5

(a) Mean relative abundance of bacterial genera in the guts of weevil larvae Conorhynchus palumbus (N = 88) and Menecleonus virgatus (N = 8). (b) Neighbour-Joining phylogenetic tree constructed from MUSCLE alignment of the V4 region of the 16S rRNA gene of two dominant representative Citrobacter sequences obtained in this study, different Citrobacter sequences obtained from a range of insects, and representative Enterobacter and Klebsiella sequences (of the Enterobacteriaceae family) obtained from the other weevil species in MEGA v10.0. Pseudomonas sp. (of the Pseudomonadaceae family) was used as the out group. Genbank accession No. is shown for each sequence obtained from Genbank, while the OTU No. (the same as in the deposited datasets in Genbank) is shown for the sequence obtained in this study. Numbers on the branches are bootstrap values. Only bootstrap values greater than 70 are shown. Sequences in bold were obtained in this study