Developmental genetic underpinnings of a symbiosis-associated organ in the fungus-farming ambrosia beetle Euwallacea validus

Abstract

Mutualistic interactions between organisms often mediate the innovation of traits essential to maintain the relationship. Yet our understanding of these interactions has been stymied due to various hurdles in studying the genetics of non-model animals. To understand the genetic mechanisms by which such traits develop, we examined the function of genes breathless (btl), trachealess (trh), and doublesex in the development of a novel fungus-carrying organ (mycangium) that facilitates an obligate relationship between fungus-farming ambrosia beetles and specific fungal partners. Gene knockdown by RNA interference and subsequent micro-computed tomography visualization suggest btl and trh are required for initiation of mycangia and that tubulogenesis may have been co-opted for early mycangial development.

Figure 1

qPCR in female E. validus beetles across development and between head and abdominal tissues. (a) E. validus tissue dissections collected for head (purple) and abdominal (yellow) tissue samples analyzed in qRT-PCR. (b) −ΔCt value for head and abdominal tissues across lifestage. Results between tissues were not significantly different (see Supplemental Table S1 for p-values).

Figure 2

The morphology of mycangia in E. validus. (a) Transverse µCT image showing the major, medial pair of mycangia. One of mycangia is shaded in red (red arrowhead). (b) Transverse µCT image showing the minor (inferior) pair of mycangia-like pouches. One of the structures is shaded in blue (blue arrowhead). (c) An external image of a premature adult female correlates external morphology to plane of digital cross-section. Lines indicate the transverse section plane in a (red) and b (blue).

Figure 3

Representative RNA Interference phenotypes. (a) Planes of virtual dissection for superior (red line corresponding to results b, d, f, h) and inferior (blue line corresponding to results c, e, g, i) cross-sections. Micro-CT scans show intact superior mycangia (red arrow) in the control (b) and dsx-dsRNA treated insects but no equivalent structures within btl-dsRNA (f) or trh-dsRNA (h) treated animals. Inferior mycangia-like spaces (bottom; blue arrow) are visible across negative control (c), dsx-dsRNA (e), btl-dsRNA (g), and trh-dsRNA (i) treatments. Inferior mycangia were not disrupted in any treatment.

Figure 4

Representative phenotype of intact but reduced E. validus mycangia following trh-dsRNA treatment. Mycangia in negative control (a and b) is not affected by treatment while trh-dsRNA treated individual (c and d) exhibits irregular morphology. Red arrows denote mycangia in each digital cross-section.