RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

Abstract

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases. 11.

Probing the conserved roles of Cut in the development and function of optically different insect compound eyes

“…To ensure a comparable stage and preparation for T. marmoratus , we injected cut dsRNA in late third instar larvae, and stained for Cut in early developing whole pupal retina. Consistent with previous findings (Rathore et al ., 2020), we found knockdowns in T. marmoratus to be highly successful, with 10 of 12 injected individuals showing reduced Cut expression when compared to controls (n = 8). However, complete cut knockdown was rare, likely because of cut ’s vital role in the development of multiple organ systems in insects (Bauke et al ., 2015; Corty et al ., 2016; Klann et al ., 2021).…”

“…These similarities led us to hypothesize a conserved functional role for this transcription factor between these two eye types. To test for such conserved roles, we used a loss-of-function approach, applying a Semper cell-restricted knockdown in D. melanogaster (Charlton-Perkins et al, 2017) and a generalized dsRNAi injection in late stage T. marmoratus larvae (Rathore et al ., 2020).…”

“…2 for primers) from whole tissue cDNA. This amplicon was used for dsRNA synthesis as described in (Rathore et al ., 2020). Then, 100 ng of cut dsRNA was injected in late-stage third instar larvae to ensure cut knockdown early during compound eye development (which is initiated during the pre-pupation stages in T. marmoratus larvae; personal observation).…”

Probing the conserved roles of cut in the development and function of optically different insect compound eyes

“…These similarities led us to hypothesize a conserved functional role for this transcription factor between these two eye types. To test for such conserved roles, we used a loss-of-function approach, applying a Semper cell-restricted knockdown in D. melanogaster (Charlton-Perkins et al, 2017) and a generalized dsRNA injection in late stage T. marmoratus larvae (Rathore et al, 2020).…”

“…Figure S2 for primers) from whole tissue cDNA. This amplicon was used for dsRNA synthesis as described in (Rathore et al, 2020). Then, 100 ng of cut dsRNA was injected in late-stage third instar larvae to ensure cut knockdown early during compound eye development (which is initiated during the pre-pupation stages in T. marmoratus larvae; personal observation).…”