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.
Animal phototransduction proceeds through light absorption by rhodopsins (opsins linked to retinal molecules), which activate ion channels via G-protein coupled second messenger pathways (Fain et al., 2010). Insect and other arthropod compound eyes contain the opsin molecules in microvilli of photoreceptor cells. Rich genetic and molecular tools have made Drosophila compound eyes the best-understood model of insect phototransduction (Hardie and Postma, 2008).
In Drosophila, photon absorption by rhodopsin causes photoisomerization to metarhodopsin, which activates a heterotrimeric Gq-protein, initiating a cascade leading to activation of IP3 and diacylglycerol. Linkages from this cascade to opening of transient receptor potential (dTRP) and TRP-like (dTRPL) ion channels that carry the receptor current are still debated, and both chemical (Chyb et al., 1999; Huang et al., 2010) and mechanical (Hardie and Franze, 2012) intermediate steps have been proposed
In Drosophila, dTRP and dTRPL channels are thought to carry approximately equal parts of light-activated current under physiological conditions (Reuss et al., 1997).
Although major features of Drosophila phototransduction may apply to other insect species, there are probably many variations to accommodate the different visual requirements of this large and diverse group of animals. However, the study of such mechanisms has been hindered by the lack of powerful methods that can be used in Drosophila, including deletion/inactivation mutants and targeted mutations.
Here we used the American cockroach, Periplaneta americana, which has a very different lifestyle to Drosophila, including mainly terrestrial, secluded habitats, reliance on chemical and mechanical information via prominent antennae and cerci, and preference for dark or crepuscular visual environments (Cameron, 1961).
Evidence already exists that the anatomy and physiology of Periplaneta compound eyes are adapted to dim light (Heimonen et al., 2006, 2012), and recent electrophysiological data suggested that these differences include a larger role for TRPL than TRP channels (Immonen et al., 2014).
To explore phototransduction mechanisms we created a transcriptome of Periplaneta retina and assembled mRNA sequences for opsins, TRP and TRPL genes (French, 2012). To investigate the roles of each protein, we used in vivo RNA interference (RNAi) based gene silencing to suppress translation of these genes by injecting double stranded RNA (dsRNA) into the head hemolymph.
