Bacterial vitamin B12 production enhances nematode predatory behavior

Abstract

Although the microbiota is known to affect host development, metabolism, and immunity, its impact on host behavior is only beginning to be understood. In order to better characterize behavior modulation by host-associated microorganisms, we investigated how bacteria modulate complex behaviors in the nematode model organism Pristionchus pacificus. This nematode is a predator that feeds on the larvae of other nematodes, including Caenorhabditis elegans. By growing P. pacificus on different bacteria and testing their ability to kill C. elegans, we reveal large differences in killing efficiencies, with a Novosphingobium species showing the strongest enhancement. This enhanced killing was not accompanied by an increase in feeding, which is a phenomenon known as surplus killing, whereby predators kill more prey than necessary for sustenance. Our RNA-seq data demonstrate widespread metabolic rewiring upon exposure to Novosphingobium, which facilitated screening of bacterial mutants with altered transcriptional responses. We identified bacterial production of vitamin B₁₂ as an important cause of such enhanced predatory behavior. Although vitamin B₁₂ is an essential cofactor for detoxification and metabolite biosynthesis, shown previously to accelerate development in C. elegans, supplementation with this enzyme cofactor amplified surplus killing in P. pacificus, whereas mutants in vitamin B₁₂-dependent pathways reduced surplus killing. By demonstrating that production of vitamin B₁₂ by host-associated microbiota can affect complex host behaviors, we reveal new connections between animal diet, microbiota, and nervous system.

Fig. 1. Bacterial diet modulates killing behavior in P. pacificus.

a Eurystomatous (Eu) and stenostomatous (St) mouth forms. Eu worms are capable of predation and have a wide mouth with two teeth, whereas St worms feed on bacteria and have a narrower mouth with one tooth. b A predatory P. pacificus adult biting a C. elegans larvae. c Corpse assay of P. pacificus predators fed upon C. elegans larvae following growth on a variety of bacteria from Pristionchus-associated environments; five predators are fed prey for 2 h for each assay. N = 5 replicates for each assay. d Bite assay after growth on either an E. coli OP50 or Novosphingobium L76 diet to assess the effect on P. pacificus surplus-killing behavior. Numbers of bites, successful bites and feeding was quantified during a 10 min interval while fed upon C. elegans larvae. e Corpse assay of P. pacificus fed with E. coli OP50, Novosphingobium L76 or of E. coli OP50 with Novosphingobium L76 supernatant. N = 10 replicates for each assay for d and e. f Corpse assays of P. pacificus previously fed with a mixture of Novosphingobium L76 and E. coli OP50 at 1/10, 1/100, and 1/1000 concentrations. Low concentrations of Novosphingobium L76 in the diet is sufficient to influence killing behavior. Bacteria were spotted to NGM without peptone to prevent bacterial growth. N = 10 replicates for each assay.

Fig. 2. Bacterial diet influences gene expression in P. pacificus.

a RNA-seq analysis of P. pacificus in response to a diet of Novosphingobium L76 compared with E. coli OP50. The pathways with most significant enrichment (FDR-corrected P < 0⁻⁵) in downregulated and (b) upregulated genes are shown. c The dietary sensor Ppa-acs-19.1::RFP is highly expressed in ventral gland, hypodermal and intestinal cells following an E. coli OP50 diet, whereas a N. lin. LE124 diet induces expression only in ventral gland cells. The co-injection marker Ppa-egl-20::RFP is expressed in the tail. d Ppa-stdh-1::RFP is expressed in the intestinal and hypodermal cells with expression strongly upregulated on N. lin. LE124 compared with an E. coli OP50 diet. e Expression of the Ppa-acs-19.1::RFP dietary sensor after feeding on N. lin. LE124 transposon mutants with mutations in vitamin B₁₂ (N. lin. LE124 CbiQ::Tn5), purine (N. lin. LE124 PurH::Tn5), pyrimidine biosynthesis (N. lin. LE124 PryD::Tn5) and nitrogen metabolism (N. lin. LE124 GlnD::Tn5). Mutants increase the expression of the dietary sensor in comparison to a N. lin. LE124 wild-type diet. f Corpse assay of P. pacificus after feeding on various N. lin. LE124 mutants. There is decreased killing efficiency compared to a N. lin. LE124 wild-type diet. N = 10 replicates for each assay.

Fig. 3. Vitamin B₁₂ containing diet regulates surplus-killing behavior and development.

a Corpse assays showing effects of vitamin B₁₂ supplementation on P. pacificus predation efficiency with P. pacificus fed on either E. coli OP50, N. lin. LE124, N. lin. LE124 CbiQ::Tn5, 500 nM vitamin B₁₂ supplemented E. coli OP50 or 500 nM vitamin B₁₂ supplemented N. lin. LE124 CbiQ::Tn5 prior to assays. b Bite assays showing effects of vitamin B₁₂ supplementation on P. pacificus killing behavior with P. pacificus fed on either E. coli OP50, N. lin. LE124, N. lin. LE124 CbiQ::Tn5, 500 nM vitamin B₁₂ supplemented E. coli OP50 or 500 nM vitamin B₁₂ supplemented N. lin. LE124 CbiQ::Tn5 prior to assays. c Developmental staging of C. elegans and P. pacificus showing percentage of third larval stage (L3), early fourth larval stage (L4), mid fourth larval stage (L4), late fourth larval stage (L4) and young adults on plates after feeding with E. coli OP50, Comamonas DA18877 and N. lin. LE124 for either 45 h (C. elegans) or 56 h (P. pacificus). d Bite assays of P. pacificus fed with E. coli OP50, Comamonas DA18877 and N. lin. LE124. N = 10 replicates for each assay in figure.

Fig. 4. Vitamin B₁₂ influence on development is conserved in various nematodes.

a Corpse assays of P. pacificus wild-type (PS312) and mutant animals defective in vitamin B₁₂-dependent pathways Ppa-metr-1 and Ppa-mce-1 fed with E. coli OP50 supplemented with/without 500 nM vitamin B₁₂. b Corpse assays of PS312 and Ppa-metr-1 fed with E. coli OP50 supplemented with/without 10 mM methionine. N = 10 replicates for each assay. c, d Comparative volume measurement of C. elegans, P. pacificus, Parastrongyloides trichosuri, Rhabditophanes sp., Steinernema carpocapsae and Allodiplogaster sudhausi after growing on bacterial plates supplemented or not-supplemented with vitamin B₁₂. N = 60 for each assay. The entomopathogenic nematode Steinernema carpocapsae can only be cultured using the specific symbiontic bacterium Xenorhabdus nematophila.