For the road: calibrated maternal investment in light of extracellular symbiont transmission

Abstract

Faithful transmission of beneficial symbionts is critical for the persistence of mutualisms. Many insect groups rely on extracellular routes that require microbial symbionts to survive outside the host during transfer. However, given a prolonged aposymbiotic phase in offspring, how do mothers mitigate the risk of symbiont loss due to unsuccessful transmission? Here, we investigated symbiont regulation and reacquisition during extracellular transfer in the tortoise beetle, Chelymorpha alternans (Coleoptera: Cassidinae). Like many cassidines, C. alternans relies on egg caplets to vertically propagate its obligate symbiont Candidatus Stammera capleta. On average, each caplet is supplied with 12 symbiont-bearing spheres where Stammera is embedded. We observe limited deviation (±2.3) in the number of spheres allocated to each caplet, indicating strict maternal control over symbiont supply. Larvae acquire Stammera 1 day prior to eclosion but are unable to do so after hatching, suggesting that a specific developmental window governs symbiont uptake. Experimentally manipulating the number of spheres available to each egg revealed that a single sphere is sufficient to ensure successful colonization by Stammera relative to the 12 typically packaged within a caplet. Collectively, our findings shed light on a tightly regulated symbiont transmission cycle optimized to ensure extracellular transfer.

Keywords: host–microbe coevolution; maternal investment; symbiont transmission; symbiosis.

Figure 1.

Structure and content of symbiont-bearing egg caplets. (a) The tortoise beetle, Chelymorpha alternans. (b) Eggs deposited on the underside of an Ipomoea batatas leaf, each topped with a caplet at the anterior pole. (c) Scanning electron microscopy (SEM) image outlining the exterior features of the egg caplet. (d) Fluorescence in situ hybridization (FISH) cross-section of a caplet (green: autofluorescence) and its enclosed spheres where Stammera (magenta: 16 rRNA probe) is embedded. (e,f) SEM images of an inverted caplet detached from the egg. Viewing direction is tilted by approximately 90°. (g) SEM cross-section of the egg caplet. Abbreviations: c, caplet; m, membrane; s, sphere. Scale bars are included for reference. (Online version in colour.)

Figure 2.

(a) Symbiont population dynamics in the caplet during embryo development in the egg following the quantification of 16S rRNA gene copy numbers. Lines represent medians, boxes indicate 25–75 percentiles, and whiskers denote range. Different letters above boxes indicate significant differences (LM, p = 0.016). (b) FISH cross-sections of egg caplets (green: autofluorescence) and its enclosed spheres where Stammera (magenta: 16 rRNA probe) is embedded 7, 9 and 10 days following oviposition. Membrane is intact up until day 10. Scale bar is included for reference. (Online version in colour.)

Figure 3.

(a) Stammera infection frequencies in C. alternans larvae following experimental manipulation of the egg caplet (Pearson's χ²-squared test, χ² = 68.62, d.f. = 3, p < 0.001). Bar coloration signifies the experimental treatment. Number of samples = 87 larvae; Caplet intact (24), Caplet removed (19), Caplet reapplied (23) and Spheres reapplied (21). Whiskers denote the 95% binomial confidence intervals. (b) Larval survivorship (to adult eclosion) following experimental removal the egg caplet. Line coloration signifies the experimental treatment. Asterisks indicate significant differences between treatments (Cox's model, p < 0.001). (Online version in colour.)

Figure 4.

(a) Stammera relative abundance following the quantification of 16S rRNA gene copy numbers in 6- and 12-day-old larvae. Box coloration signifies the experimental treatment. Lines represent medians, boxes indicate 25–75 percentiles and whiskers denote range. Different letters above boxes indicate significant differences in gene copy numbers (6-day-old larvae: LM, p = 0.014; 12-day-old larvae: LM, p = 0.27). (b) Larval survivorship (to adult eclosion) across treatments. Line coloration signifies the experimental treatment. The asterisks indicate significant differences between treatments (Cox's model, p = 0.3). n.s.: not significant. (Online version in colour.)