Transcriptome analysis and RNA interference of cockroach phototransduction indicate three opsins and suggest a major role for TRPL channels

Abstract

Our current understanding of insect phototransduction is based on a small number of species, but insects occupy many different visual environments. We created the retinal transcriptome of a nocturnal insect, the cockroach, Periplaneta americana to identify proteins involved in the earliest stages of compound eye phototransduction, and test the hypothesis that different visual environments are reflected in different molecular contributions to function. We assembled five novel mRNAs: two green opsins, one UV opsin, and one each TRP and TRPL ion channel homologs. One green opsin mRNA (pGO1) was 100–1000 times more abundant than the other opsins (pGO2 and pUVO), while pTRPL mRNA was 10 times more abundant than pTRP, estimated by transcriptome analysis or quantitative PCR (qPCR). Electroretinograms were used to record photoreceptor responses. Gene-specific in vivo RNA interference (RNAi) was achieved by injecting long (596–708 bp) double-stranded RNA into head hemolymph, and verified by qPCR. RNAi of the most abundant green opsin reduced both green opsins by more than 97% without affecting UV opsin, and gave a maximal reduction of 75% in ERG amplitude 7 days after injection that persisted for at least 19 days. RNAi of pTRP and pTRPL genes each specifically reduced the corresponding mRNA by 90%. Electroretinogram (ERG) reduction by pTRPL RNAi was slower than for opsin, reaching 75% attenuation by 21 days, without recovery at 29 days. pTRP RNAi attenuated ERG much less; only 30% after 21 days. Combined pTRP plus pTRPL RNAi gave only weak evidence of any cooperative interactions. We conclude that silencing retinal genes by in vivo RNAi using long dsRNA is effective, that visible light transduction in Periplaneta is dominated by pGO1, and that pTRPL plays a major role in cockroach phototransduction.

Overcoming the loss of blue sensitivity through opsin duplication in the largest animal group, beetles

“…Opsin losses have occurred in other animals, including the American cockroach ( Periplaneta americana ) 28 , deep sea fish 29 , fossorial snakes 30 , caecilians 31 and both nocturnal and aquatic mammals 32–34 . Such losses are typically associated with low-light or spectrally-attenuated environments.…”

Systemic RNAi of V‐ATPase subunit B causes molting defect and developmental abnormalities in Periplaneta fuliginosa

“…However, because RNAi efficacy is variable among insect species (e.g., RNAi experiments were unsuccessful in some studies using Lepidoptera) (Bellés, 2010;Terenius et al, 2011), consideration of the target insect species is required. Previously, RNAi was demonstrated to work well in two cockroach species, P. americana and B. germanica (Cruz et al, 2006;Pueyo et al, 2008;Gomez-Orte & Bellés, 2009;Revuelta et al, 2009;Irles et al, 2013;French et al, 2015;Lozano et al, 2015;Lemonds et al, 2016). For example, the requirement of the melanin pathway for overall body pigmentation in P. americana nymphs and adults was revealed by RNAi of two enzymecoding genes (Lemonds et al, 2016).…”

“…Functional analysis of this unique insect group is expected to greatly contribute to the comparative study of V-ATPases. Additionally, RNAi works well as a powerful reverse genetics tool in two other sanitary pest species, P. americana and B. germanica (Cruz et al, 2006;Pueyo et al, 2008;Gomez-Orte & Bellés, 2009;Revuelta et al, 2009;Irles et al, 2013;French et al, 2015;Lozano et al, 2015;Lemonds et al, 2016). Nevertheless there is no report of RNAi in P. fuliginosa, the most abundant pest cockroach species in Japan (The Japanese Society of Pestology, Personal communication).…”

Evolution of Phototransduction Genes in Lepidoptera

“…A transcriptome study in cockroaches found that trpl was approximately 10 times more abundant than trp (French et al 2015). RNAi of trpl reduced electroretinogram (ERG) response much more than trp after 21 days suggesting that, unlike D. melanogaster, cockroach TRPL rather than TRP has a larger contribution to phototransduction (French et al 2015).…”

“…Instead a lepidopteran paralog appears to carry out a similar function of chromophore binding (Macias-Muñoz et al 2017). Moreover, as observed in the cockroach, while genes such as TRP and TRPL are conserved and expressed, one gene copy (TRPL) might have a greater impact on phototransduction than the other (French et al 2015). Consequently, investigating both gene gain/loss and the expression of phototransduction genes in Lepidoptera might uncover differences in their visual processing that helps them function in different light environments.…”