Lactic Acid Bacteria Are Prevalent in the Infrabuccal Pockets and Crops of Ants That Prefer Aphid Honeydew

Abstract

Ants are evolutionarily successful species and occupy diverse trophic and habitat niches on the earth. To fulfill dietary requirements, ants have established commensalism with both sap-feeding insects and bacteria. In this study, we used high-throughput sequencing of the bacterial 16S rRNA gene to characterize the bacterial composition and structure of the digestive tracts in three species of Formica ants and Lasius niger (Linnaeus)-species that predominantly feed on honeydew secreted by aphids. We found that bacterial communities displayed species- and colony-level signatures, and that bacterial communities in the infrabuccal pockets and crops were different from those in the midguts and hindguts. Lactobacillus and Wolbachia were dominant in the infrabuccal pockets and crops of workers, whereas Wolbachia was dominant in the midguts, hindguts and brood (larvae, pupae and cocoons). To learn more about the dominant Lactobacillus in ants, we assessed its prevalence in a wide range of aphid-tending ants using diagnostic PCR. We found that Lactobacillus was more prevalent in Formicinae than in Myrmicinae species. We also isolated four strains of lactic acid bacteria (Lactobacillus sanfranciscensis, Lactobacillus lindneri, Weissella cibaria and Fructobacillus sp.) from the infrabuccal pockets and crops of aphid-tending ants using a culture-dependent method. Two predominant lactic acid bacterial isolates, Lactobacillus sanfranciscensis (La2) and Weissella cibaria (La3), exhibited abilities in catabolizing sugars (sucrose, trehalose, melezitose and raffinose) known to be constituents of hemipteran honeydew. These findings contribute to further understanding the association between ants, aphids and bacteria, and provide additional information on the function of lactic acid bacteria in ants.

Keywords: ants; aphids; bacterial communities; digestive tract; lactic acid bacteria.

FIGURE 1

Relative abundance of bacteria (phyla and genera) found in four ant species sequenced by Illumina HiSeq 2500 platform, and significant different bacteria across samples. (A) Abundance of bacteria showing phyla with more than 1% relative abundance in all samples. (B) Abundance of bacteria showing genera with more than 1% relative abundance in all samples. (C) Abundance of bacterial genera across each ant species. (D) Abundance of bacterial genera across different digestive tract regions and broods of three Formica ant species, and in heads and gasters of L. niger. (E) Bacterial genera that significantly differ (FDR adjusted p-value < 0.05) in abundance across different digestive tract regions and broods of three Formica ant species, and heads and gasters of L. niger. Significant bacterial genera were ranked in decreasing order based on their LDA score (x-axis). The mini heatmap to the right of the plot indicated whether the relative abundance of bacterial genera was higher (red) or lower (blue) in each group.

FIGURE 2

Non-metric Multidimensional scaling (NMDS) analysis (Bray-Curtis distance) showing differences in bacterial structures. (A) NMDS analysis of bacterial structures across four ant species. (B) NMDS analysis of bacterial structures across three colonies (Fj1, Fj2, Fj3) of F. japonica. (C) NMDS analysis of bacterial structures across three colonies (Fs1, Fs2, Fs3) of F. sanguinea. (D) NMDS analysis of bacterial structures across two colonies (Fg1, Fg2) of F. gagatoides. (E) NMDS analysis of bacterial structures across two colonies (Ln1, Ln2) of L. niger. (F) NMDS analysis of bacterial structures across different digestive tract regions and brood (larvae, pupae, and cocoons) of three Formica ant species. Dots in different color represented different groups. PERMANOVA tests showed statistical analysis between groups.

FIGURE 3

Relative abundance of bacteria found in the IBPs and crops of Formica ants, heads and gasters of L. niger sequenced by PacBio SMRT platform. (A) Abundance of dominant bacterial genera. (B) Abundance of bacterial species.

FIGURE 4

Phylogenetic circle tree based on a distance matrix of 16S rRNA genes from 15 isolated strains of lactic acid bacteria, representative ASVs of Lactobacillus, and their most closely related bacterial sequences in NCBI. The phylogenetic tree was constructed by ClustalW using the maximum-likelihood method within the MEGA 7.0 package. Closely related sequences were shown together with accession numbers (in parentheses) from GenBank. Bootstrap value based on 2,000 replication displayed the significance of the interior nodes, and are shown at branch points. Solid black dots, isolated lactic acid bacteria strains; solid black triangle, respective Lactobacillus ASVs. Branches in different color represents groups of lactic acid bacteria.

FIGURE 5

Catabolism of sugars (sucrose, trehalose, melezitose, and raffinose) by two strains of lactic acid bacteria. (A) Lactobacillus sanfranciscensis (La2). (B) Weissella cibaria (La3). Different letters indicate significant differences among different lines (Duncan’s multiple range test). Values are means ± SEM; n = 3.