The bacterial community of entomophilic nematodes and host beetles

Abstract

Insects form the most species-rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode-insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle-nematode-bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five-year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high-throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate-reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect-associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate-reducing bacteria suggests a possible link between beetle-bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.

Keywords: bacteria; beetles; entomophilic nematodes; microbiome; next-generation sequencing.

Figure 1. Five-year sampling summary of southern California scarab beetles and their associated nematodes

Only nematode infested beetles are represented. Proportion of nematode infestations on three major species of beetles. Diplogasteriodes sp. 1 shows the strongest host preference for Amblonoxia palpalis beetles while the closely related species Diplogasteriodes sp. 2 was isolated only from Serica alternata. P. pacificus was the most abundant nematode species isolated from the most commonly captured Cyclocephala pasadenae and Cyclocephala hirta beetles. The three beetle species are shown in relative size proportions, with the Cyclocephala spp. body length representing ~1 cm.

Figure 2. Most P. pacificus strains from Los Angeles show neutral or negative chemotaxis towards the oriental beetle pheromone

All but one out of twelve P. pacificus strains obtained from Cyclocephala beetles in 2012 show neutral (rlh54-rlh56, ***P<0.001) or repulsive (rlh59-rlh71, ****P<0.0001) chemotaxis response to the oriental beetle pheromone (z)-7-tetradece-2-one (Zhang et al. 1994; Herrmann et al. 2007). 10–21 trials of greater than 10 nematodes each were assayed for each strain. Dunnett’s multiple comparisons test was performed against the reference Washington strain, PS1843.

Figure 3. The beetle bacterial microbiome is driven by differences between beetle genera

(A) Principle coordinates plot of pair-wise Bray-Curtis similarities shows that the bacterial microbiomes differ according to beetle species (P=0.001). (B) A heat map of the average relative abundance of bacterial taxa occurring at ≥1% in the whole beetle microbiome, as arranged by abundance in the Amblonoxia microbiome. Amblonoxia palpalis and Serica alternata had higher abundance of anaerobes from the family Synergistaceae, Desulfovibrionaceae and Desulfobacteraceae. Taxon assignment is at the family level unless otherwise indicated and darker shades denote higher abundance. Significant difference in corrected P values for FDR *<0.05, **<0.01 (Kruskal-Wallis). ^Includes Cyclocephala hirta and hybrids from Los Angeles.

Figure 4. Entomophilic nematode infestation does not alter the diversity of the beetle bacterial microbiome

(A) Globally, diversity differed across beetle species. Results of a Nemenyi post-hoc test in the PMCMR package of R found that diversity was different between C. hirta from Ohio and C. pasadenae/hirta from California (P<0.01) and between C. hirta and S. alternata. (P<0.05). (B) However, within each beetle species, there was no difference in bacterial diversity in beetles infested with nematodes(+) compared to those not infested(−). The comparison was not possible in C. longula due to only a single case of nematode infestation. (P>0.05, Kruskal-Wallis nonparametric test).

Figure 5. The cultivable nematode bacterial microbiome does not differ by nematode species or beetle host

(A) The beetle-derived nematode microbiome do not differ according to nematode species (P=0.61), nor (B) by which beetles the nematodes were isolated from (P=0.18).