Stability of Nuclear and Mitochondrial Reference Genes in Selected Tissues of the Ambrosia Beetle Xylosandrus germanus

Abstract

The fungus-farming ambrosia beetle Xylosandrus germanus (Blandford) uses a pouch-like structure (i.e., mycangium) to transport spores of its nutritional fungal mutualist. Our current study sought to identify reference genes necessary for future transcriptome analyses aimed at characterizing gene expression within the mycangium. Complementary DNA was synthesized using selected tissue types from laboratory-reared and field-collected X. germanus consisting of the whole body, head + thorax, deflated or inflated mycangium + scutellum, inflated mycangium, and thorax + abdomen. Quantitative reverse-transcription PCR reactions were performed using primers for 28S ribosomal RNA (28S rRNA), arginine kinase (AK), carbamoyl-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1α (EF1α). Reference gene stability was analyzed using GeNorm, NormFinder, BestKeeper, ΔCt, and a comprehensive final ranking by RefFinder. The gene CO1 was identified as the primary reference gene since it was generally ranked in first or second position among the tissue types containing the mycangium. Reference gene AK was identified as a secondary reference gene. In contrast, EF1α was generally ranked in the last or penultimate place. Identification of two stable reference genes will aid in normalizing the expression of target genes for subsequent gene expression studies of X. germanus’ mycangium.

Evaluation of reference genes for quantitative expression analysis in Mylabris sibirica (Coleoptera, Meloidae)

“…Arginine kinase (AK) has a prominent function in invertebrate energy metabolism, which catalyzes the reversible phosphorylation of l-arginine to make up phosphoarginine ( Shi et al, 2012 ). Similar to our results, the arginine kinase gene has been selected as the most suitable gene for expression assessment in Xylosandrus germanus ( Patwa et al, 2021 ), Spodoptera frugiperda ( Han et al, 2021 ) and Spodoptera litura ( Lu et al, 2013 ).…”

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

“…To examine whether candidate genes are expressed in mycangia and the degree of differential expression between body segments, we conducted quantitative RT-PCR. Although mycangia in other species have been dissected from thoracic segments ( Xylosandrus germanus 5 ), in E. validus , the mycangia develop inside the hard head capsule. Further, our previous study suggested their mycangia to be complex therefore we utilized the whole head in this study to avoid any potential bias during the sampling process (Fig.…”

“…Their ability to vector fungal symbionts within specialized organs, called mycangia, has led to many investigations on morphology and function. However, outside phylogenetic research, few studies provide molecular genetic resources 4 , 5 , and the genetic and developmental mechanisms underlying repeated evolution of mycangia is still poorly understood. Mycangia can be found in a variety of body segments with varying degrees of complexity; some mycangia present as shallow pits, while others are more complicated invaginations, pockets, or tubules 3 , 6 .…”