Fig. 7: Competition reinforces specificity between tortoise beetles and Stammera capleta.

A Illustration of dual-infected Chelymorpha alternans embryos. The native symbiont corresponds to S. capleta from C. alternans, while the non-native symbiont stems from Chelymorpha gressoria. B Fluorescence in situ hybridization (FISH) cross-section of foregut symbiotic organs in 5-day-old dual-infected C. alternans larvae. Bii–iv correspond to a close-up view of the foregut symbiotic organ. Bi, ii correspond to the merged channels images, while Biii corresponds to the native symbiont channel image, and Biv to the non-native channel image. The image is from a single individual and is shown for illustrative purposes (see Fig. S8). Probes: S. capleta from C. alternans (magenta: 16S rRNA), S. capleta from C. gressoria (green: 16S rRNA), C. alternans host (dark blue: 18S rRNA), and DAPI-stained DNA (orange). Symbiont-targeting probes are highly specific and used simultaneously during hybridization. Scale bars are included for reference. C Population dynamics of native and non-native symbionts in the symbiotic organs of dual-infected C. alternans beetles during development, quantified via groEL gene copy numbers. Box colors indicate either the native symbiont (magenta) or non-native symbiont (green). Lines represent medians, boxes indicate the interquartile range (25–75%), whiskers denote the range, and dots show replicates (1-day-old larvae, n = 3; 5-day-old larvae, n = 3; 12-day-old larvae, n = 5; 50-day-old adults in the foregut symbiotic organs, n = 6; 50-day-old adults in the ovary-associated glands, n = 4). Stars or “n.s.” indicate significant or non-significant differences between symbionts (LM, p < 0.001, F _(4,24) = 83.61; post hoc comparisons using least-squares means with Tukey correction). D Relative growth rate of non-native symbionts compared to native symbionts in the foregut symbiotic organs of dual-infected C. alternans larvae. The slope is used to calculate the selection coefficient (ω), shown overlaid on the plot. E Infection frequencies of native and non-native S. capleta in larvae derived from dual-infected C. alternans adults. Box colors indicate the experimental treatment: larvae from eggs laid by dual-infected C. alternans beetles. Three replicates were collected per treatment (n = 48). Bars represent the mean proportion infected, whiskers represent 95% binomial confidence intervals, and dots represent the average of each replicate. Significant differences between symbionts are indicated with stars (GLM (binomial), df = 1, p<0.001, χ2 = 133.08). g gut, fso foregut symbiotic organs. Source data are provided as a Source Data file.