Culture-based analysis of Pristionchus-associated microbiota from beetles and figs for studying nematode-bacterial interactions

Abstract

The interplay with bacteria is of crucial importance for the interaction of multicellular organisms with their environments. Studying the associations between the nematode model organisms Caenorhabditis elegans and Pristionchus pacificus with bacteria constitutes a powerful system to investigate these interactions at a mechanistic level. P. pacificus is found in association with scarab beetles in nature and recent studies revealed the succession and dynamics of this nematode and its microbiome during the decomposition of one particular host species, the rhinoceros beetle Oryctes borbonicus on La Réunion Island. However, these studies were performed using culture-free methods, with no attempt made to establish bacterial cultures from the beetle-nematode ecosystem and to investigate the effects of these microbes on life history traits in P. pacificus. Here, we establish and characterize a collection of 136 bacterial strains that have been isolated from scarab beetles and figs, another Pristionchus-associated environment, as a resource for studying their effect on various nematode traits. Classification based on 16S sequencing identified members of four bacterial phyla with the class of Gammaproteobacteria representing the majority with 81 strains. Assessing the survival of P. pacificus on individual bacteria allowed us to propose candidate groups of pathogens such as Bacillaceae, Actinobacteria, and Serratia. In combination with chemoattraction data, it was revealed that P. pacificus is able to recognize and avoid certain groups of pathogens, but not others. Our collection of bacterial strains forms a natural resource to study the effects of bacterial diet on development and other traits. Furthermore, these results will form the basis of future studies to elucidate the molecular mechanisms of recognition and pathogenicity.

Fig 1. Regional maps of beetle and fig sampling sites.

(A) Map of La Réunion Island showing the approximate beetle and fig sampling sites. O. borbonicus, Adoretus sp., Hoplia sp. and A. godefroyi beetles were collected to isolate Pristionchus-associated bacteria. Similarly, F. mauritiana figs were sampled and processed for bacteria isolation. (B) and (C) F. sycomorus (Brummeria, Pretoria, South Africa) and F. racemosa (Hanoi, Vietnam) figs were dissected under sterile conditions from Pristionchus-positive specimens to isolate bacteria.

Fig 2. Taxonomic distribution of bacterial isolates.

(A) Circles show the distribution of bacterial strains at the level of phyla (innermost circle), class (middle circle), and family (outermost circle) based on classification of the 16S ribosomal RNA gene [27]. Proteobacteria are by far the largest group (N = 104 strains). (B) Distribution of bacterial strains that were isolated from beetles on La Réunion Island.

Fig 3. Survival of P. pacificus nematodes on different bacteria.

(A) Bars show median and interquartile ranges of survival of P. pacificus worms in response to various bacterial classes. All pairwise tests for significantly different survival were done by a Wilcoxon rank-sum test. Results that remained significant after Bonferroni correction are highlighted. (B) Distribution of survival rate for deeply sampled and highly variable bacterial classes (Bacilli and Gammaproteobacteria) at higher phylogenetic resolution (family level). Within the class Bacilli, decreased survival is mostly due to strains of the family Bacillaceae. Within the class Gammaproteobacteria, the largest variability in nematode survival is observed in the family Enterobacteriaceae. (C) Distribution of survival within Enterobacteriaceae at genus level.

Fig 4. Chemoattraction towards different bacteria.

(A) While most bacterial strains seem to be preferred over control spots, the class Bacilli shows a significantly repulsive effect (Wilcoxon test, Bonferroni corrected p-value<0.05) in comparison to three other bacterial classes. (B) Within the class Bacilli, the repulsive effect is largely due to strains of the family Bacillaceae.

Fig 5. Correlation between survival and chemotaxis.

(A) Testing for the correlation between survival and chemotaxis data we found a weak trend (Spearman’s rho = 0.154, P = 0.075) for bacteria resulting in higher survival rates to also have higher chemotaxis indices, compared to strains that do not support growth or are pathogenic. (B) Similar correlation tests for those bacterial strains isolated from beetles where again, no strong signal was observed (Spearman’s rho = 0.17, P = 0.134).