Colonization dynamics of a defensive insect ectosymbiont

Abstract

Beneficial symbionts are horizontally or vertically transmitted to offspring, relying on host- or microbe-mediated mechanisms for colonization. While multiple studies on symbionts transmitted internally or by feeding highlight host adaptations and dynamics of symbiont colonization, less is known for beneficial microbes colonizing host external surfaces, such as the insect cuticle. Here, we investigate the colonization dynamics of a bacterial symbiont that protects eggs and larvae of Lagria villosa beetles against pathogens. After maternal application to the egg surface, symbionts colonize specialized cuticular invaginations on the dorsal surface of larvae. We assessed the colonization time point and investigated the involvement of the host during this process. Symbionts remain on the egg surface before hatching, providing protection. Immediately after hatching, cells from the egg surface colonize the larvae and horizontal acquisition can occur, yet efficiency decreases with increasing larval age. Additionally, passive or host-aided translocation likely supports colonization of the larval symbiotic organs. This may be especially important for the dominant non-motile symbiont strain, while motility of additional strains in the symbiont community might also play a role. Our findings provide insights into the colonization dynamics of cuticle-associated defensive symbionts and suggest alternate or complementary strategies used by different strains for colonization.

Keywords: animal–microbe interactions; bacterial colonization; motility; symbiosis; transmission.

Figure 1.

Lv-StA symbionts colonize Lagria larvae during or after hatching from the eggs: (a) estimation of internal Lv-StA cells per individual in eggs (days 0, 3, 4), first and second-instar larvae. The lack of cells inside eggs shows that the beetles are colonized only in the larval stages during or after hatching (χ² = 70.822, d.f. = 4, p < 0.001). First instars had significantly fewer cells than second-instar larvae (likelihood ratio = 19.393, p < 0.001). (b) Percentage of larvae showing presence (orange) or absence (blue) of Lv-StA in the dorsal structures based on FISH images (χ² = 7.5208, d.f. = 3, p = 0.057). Larvae were collected for FISH 24-h after Lv-StA infection on originally aposymbiotic late eggs (n = 6), first-instar (n = 6) or second-instar larvae (day 6 (n = 7) and day 7 (n = 6)). (c) FISH of a dissected embryo close before hatching (day 3–4) from a symbiont-infected egg (autofluorescence of the eGFP channel in cyan). The inset shows the larval organs not yet occupied by symbionts. (d) Whole-mount FISH image of a field-collected first-instar larva showing Lv-StB within the organs. (e) Lv-StA cells in the three dorsal organs of an infected first-instar larva. (d,e) Burkholderia-specific staining is shown in green, eubacteria in red, host cell nuclei in blue.

Figure 2.

Simulating transfer of symbionts from eggs to the larval surface using fluorescent beads: (a,b) fluorescent beads in a PBS suspension imaged with bright-field microscopy(a) and in the Cy3-channel (b). (c) A beetle egg after inoculation with fluorescent beads. (d,f) Three whole first-instar larvae that hatched from eggs carrying beads on the outer surface. The individual in (d) shows autofluorescence in the Cy3 channel, most likely due to early cuticle melanization. White arrows aid in distinguishing the beads. (c–f) Dotted lines indicate specimen profiles and the dorsal structures based on the DAPI signal (not shown) or EGFP channel (cyan) autofluorescence of the larval tissues. (g) A section of a 1% agar block with fixed beads. The beads lost fluorescence when fixed in 4% formaldehyde and dehydrated but are visible with bright-field microscopy. (h,i) Sagittal section of a hatched first-instar larva after bead inoculation showing a few (arrowheads in (h)) or no beads (i) within the dorsal structures. Overlay of bright-field microscopy (black and white) and Cy3-channel (red). The red signal corresponds to cuticle autofluorescence.