A novel intracellular mutualistic bacterium in the invasive ant Cardiocondyla obscurior

Abstract

The evolution of eukaryotic organisms is often strongly influenced by microbial symbionts that confer novel traits to their hosts. Here we describe the intracellular Enterobacteriaceae symbiont of the invasive ant Cardiocondyla obscurior, 'Candidatus Westeberhardia cardiocondylae'. Upon metamorphosis, Westeberhardia is found in gut-associated bacteriomes that deteriorate following eclosion. Only queens maintain Westeberhardia in the ovarian nurse cells from where the symbionts are transmitted to late-stage oocytes during nurse cell depletion. Functional analyses of the streamlined genome of Westeberhardia (533 kb, 23.41% GC content) indicate that neither vitamins nor essential amino acids are provided for the host. However, the genome encodes for an almost complete shikimate pathway leading to 4-hydroxyphenylpyruvate, which could be converted into tyrosine by the host. Taken together with increasing titers of Westeberhardia during pupal stage, this suggests a contribution of Westeberhardia to cuticle formation. Despite a widespread occurrence of Westeberhardia across host populations, one ant lineage was found to be naturally symbiont-free, pointing to the loss of an otherwise prevalent endosymbiont. This study yields insights into a novel intracellular mutualist that could play a role in the invasive success of C. obscurior.

Figure 1

Genomic structure of Westeberhardia representational Circos plot (Krzywinski et al., 2009) illustrating genomic properties of Westeberhardia. Tile plots show the distribution of protein-coding genes (black bars) and ribosomal binding sites (red bars). The histogram in the inner circle shows GC content in percent for 1 kb windows.

Figure 2

Westeberhardia metabolic reconstruction. Intact pathways are shown in black lines and unclear pathways (missing a specific gene or having it pseudogenized) are shown in gray lines. Exporters are represented using green ovals, whereas exporters/importers are represented using blue ovals with the name of the family/superfamily they belong to, otherwise the protein or complex name is used. ATP synthase is shown twice to represent an additional metabolic capability. Essential and non-essential amino acids are shown in red and purple lettering, respectively. Cofactors and vitamins are represented in blue. Blurred compounds represent those for which biosynthesis or import cannot be accounted for based on the genomic data, according to MetaCyc. Relevant genes involved in the biosynthesis of nucleotides and peptidoglycan are indicated. A single frameshift is found in adk and murA; therefore, they might be young pseudogenes, or an RNA polymerase or ribosomal slippage would be required to produce a functional protein.

Figure 3

Intraspecific and temporal dynamics of Westeberhardia infection. (a) In genomic coverage data for pooled haploid males mapped against the Westeberhardia reference, Westeberhardia reads (We) were exclusively present in the Brazil, Ilhéus (2009) sample (BR, blue) and no reads could be detected in the OypB, Japan (JP, red) sample, whereas coverage of C. obscurior reads (Cobs) mapped against the C. obscurior reference is similar. (b) Real time-quantitative PCRs on DNA level for the nrdB gene confirm the absence of Westeberhardia in larvae (L), prepupae (PP) and female (queen and worker) pupae (PW=pupa white, PB=pupa brown) of the JP OypB population, whereas all those developmental stages are infected in the BR Una (2012) (letters indicate significances for within-population comparisons for BR). (c and d) Prevalence of Westeberhardia in queens (c) and workers (d) across different populations of C. obscurior from BR (blue), JP (red) and Tenerife, SP (gray), as revealed by qPCR (c) and diagnostic PCR (d), of the nrdB gene. For each lineage, 6–8 colonies and per colony 9–10 young workers and 6–10 queens were tested. Bars represent medians and whiskers denote quartiles. Note that while Westeberhardia infection status of workers varies between and within populations of C. obscurior, it is almost fixed in queens across all lineages except OypB. (e and f) Morph (e) and age (f) dependency of relative amounts of Westeberhardia in C. obscurior individuals from BR (Una, 2012) determined by real-time quantitative PCR. Normalized nrdB copy numbers are elevated in queens compared with all other morphs (Q=queens; W=workers; M=males winged; MW=males wingless) (e), increase with age in queens, but decrease with age in workers (numbers after Q/W show age in days, V=virgin queens, letters indicate significant differences for within-caste comparisons) (f). Sample sizes are given within parentheses.

Figure 4

Localization of Westeberhardia in adults and pupae of C. obscurior (from Brazil, Ilhéus, 2009). Symbionts were stained in longitudinal sections through the abdomen with the Westeberhardia-specific probe Wcard2-Cy5 (green) and host cell nuclei were counterstained with DAPI (blue). (a–c) Localization of Westeberhardia in gut-associated bacteriomes (bac) in the pupae of a queen (a), a worker (b) and a male (c). Note the additional presence of symbionts in the queen ovaries (ov). (d) Section of the abdomen of an adult queen, with symbionts visible in the ovaries (ov). (e and f) Ovaries of an adult queen. Symbionts are mainly localized in the nurse cells (nc), but enter the developing oocyte (oc), probably during nurse cell depletion (arrowhead). Scale bars: 100 μm (a, b and d), 50 μm (c) and 20 μm (e and f).