Opsin Gene Research Articles

Functional diversification process of opsin genes for teleost visual and pineal photoreceptions

Most vertebrates have a rhodopsin gene with a five-exon structure for visual photoreception. By contrast, teleost fishes have an intron-less rhodopsin gene for visual photoreception and an intron-containing rhodopsin (exo-rhodopsin) gene for pineal photoreception. Here, our analysis of non-teleost and teleost fishes in various lineages of the Actinopterygii reveals that retroduplication after branching of the Polypteriformes produced the intron-less rhodopsin gene for visual photoreception, which converted the parental intron-containing rhodopsin gene into a pineal opsin in the common ancestor of the Teleostei. Additional analysis of a pineal opsin, pinopsin, shows that the pinopsin gene functions as a green-sensitive opsin together with the intron-containing rhodopsin gene for pineal photoreception in tarpon as an evolutionary intermediate state but is missing in other teleost fishes, probably because of the redundancy with the intron-containing rhodopsin gene. We propose an evolutionary scenario where unique retroduplication caused a “domino effect” on the functional diversification of teleost visual and pineal opsin genes.

Ips typographus vision system: a comprehensive study.

Aggressive bark beetle species such as the Eurasian spruce bark beetle Ips typographus play a fundamental role in forest ecosystems but can also lead to extensive forest mortality and massive economic damage during outbreaks. Currently I. typographus' eyes, visual perception of the world and recognition of specific targets like host plants are understudied topics. Studying its visual sense can open the way to novel efficient monitoring and management methods, particularly important in avoiding the switch from an endemic to an epidemic condition. In addition, the integration of visual cues in trapping systems may offer new opportunities for surveillance. Vision in I. typographus was investigated by means of morphological analysis, electroretinography (ERG), molecular analysis of opsin genes and behavioural tests. ERG has revealed that the compound eyes contain two classes of photoreceptors, maximally sensitive to UV and green at 370 and 530nm, respectively. The result was further supported by the identification of two relevant opsin genes. Finally, the innate wavelength sensitivity was tested in a Y-maze. Ips typographus consistently preferred UV over non-UV (VIS) light, irrespective of their intensity ratios, but preferred high over low intensity VIS light, consistent with a UV-VIS dichromatic visual system. Overall, the results may open the way to better understand the navigation pattern in tree canopies and the host selection processes of this ecologically and economically important beetle species.

Shining new light on naupliar eyes: A novel molecular phylogeny for Pleuromamma (Family: Metridinidae) and the characterization of luciferase and opsin expression

Pleuromamma (Giesbrecht, 1898) is a cosmopolitan genus of metridinid copepods, with species that perform remarkable diel vertical migrations (DVM) and emit a bioluminescent secretion when disturbed that varies both spectrally and kinetically. Copepod bioluminescence is autogenic and uses luciferase enzymes that catalyze a luciferin, coelenterazine, to produce light. Pleuromamma possess naupliar eyes, relatively simple photosensitive structures used for many visually-guided behaviors. Yet the fundamental molecular unit for vision, the opsin protein, has not been previously described for the family. The light producers and detectors are important to study because DVM is a behavior that mediates significant active elemental fluxes between the upper ocean and midwaters across vast stretches of oceanic habitat, and DVM is guided by visual behaviors, with animals tracking an isolume. Here we provide the first fully resolved molecular phylogeny for Pleuromamma (Family: Metridinidae) and describe the luciferase and opsin gene diversity and expression using de novo assembled transcriptomes. We successfully sequenced and assembled transcriptomes for 10 of 11 described species of Pleuromamma as well as two other metridinid species: Metridia longa and Gaussia princeps. In all species, we obtained coding sequences of one putative rhabdomeric middle wavelength sensitive visual opsin gene, as well as several non-visual opsins – a c-type pteropsin and a tetra-opsin type peropsin. Furthermore, Pleuromamma express luciferases from each of two main evolutionary clades (Luc1 and Luc2), and a single paralog (Luc2a) dominates expression throughout the group. The variation in luciferase number, sequence, and expression among species could lead to different spectral and kinetic properties of bioluminescence and aid in congener recognition.

Day–night expression patterns of opsin genes in the coral Acropora digitifera under natural and LED light conditions

Day–night expression patterns of opsin genes in the coral Acropora digitifera under natural and LED light conditions

Evolution of Opsin Genes in Caddisflies (Insecta: Trichoptera).

Insects have evolved complex and diverse visual systems in which light-sensing protein molecules called "opsins" couple with a chromophore to form photopigments. Insect photopigments group into three major gene families based on wavelength sensitivity: long wavelength (LW), short wavelength (SW), and ultraviolet wavelength (UV). In this study, we identified 123 opsin sequences from whole-genome assemblies across 25 caddisfly species (Insecta: Trichoptera). We discovered the LW opsins have the most diversity across species and form two separate clades in the opsin gene tree. Conversely, we observed a loss of the SW opsin in half of the trichopteran species in this study, which might be associated with the fact that caddisflies are active during low-light conditions. Lastly, we found a single copy of the UV opsin in all the species in this study, with one exception: Athripsodes cinereus has two copies of the UV opsin and resides within a clade of caddisflies with colorful wing patterns.

Developmental gene expression in the eyes of the pygmy squid Xipholeptos notoides.

The eyes of squids, octopuses, and cuttlefish are a textbook example for evolutionary convergence, due to their striking similarity to those of vertebrates. For this reason, studies on cephalopod photoreception and vision are of importance for a broader audience. Previous studies showed that genes such as pax6, or certain opsin-encoding genes, are evolutionarily highly conserved and play similar roles during ontogenesis in remotely related bilaterians. In this study, genes that encode photosensitive proteins and Reflectins are identified and characterized. The expression patterns of rhodopsin, xenopsin, retinochrome, and two reflectin genes have been visualized in developing embryos of the pygmy squid Xipholeptos notoides by in situ hybridization experiments. Rhodopsin is not only expressed in the retina of X. notoides but also in the olfactory organ and the dorsal parolfactory vesicles, the latter a cephalopod apomorphy. Both reflectin genes are expressed in the eyes and in the olfactory organ. These findings corroborate previous studies that found opsin genes in the transcriptomes of the eyes and several extraocular tissues of various cephalopods. Expression of rhodopsin, xenopsin, retinochrome, and the two reflectin genes in the olfactory organ is a finding that has not been described so far. In other organisms, it has been shown that Retinochrome and Rhodopsin proteins are obligatorily associated with each other as both molecules rely on each other for Retinal isomerisation. In addition, we demonstrate that retinochrome is expressed in the retina of X. notoides and in the olfactory organ. This study shows numerous new expression patterns for Opsin-encoding genes in organs that have not been associated with photoreception before, suggesting that either Opsins may not only be involved in photoreception or organs such as the olfactory organ are involved in photoreception.

Genome-Wide Association Study and transcriptome analysis reveals a complex gene network that regulates opsin gene expression and cell fate determination in Drosophila R7 photoreceptor cells.

An animal's ability to discriminate between differing wavelengths of light (i.e., color vision) is mediated, in part, by a subset of photoreceptor cells that express opsins with distinct absorption spectra. In Drosophila R7 photoreceptors, expression of the rhodopsin molecules, Rh3 or Rh4, is determined by a stochastic process mediated by the transcription factor spineless. The goal of this study was to identify additional factors that regulate R7 cell fate and opsin choice using a Genome Wide Association Study (GWAS) paired with transcriptome analysis via RNA-Seq. We examined Rh3 and Rh4 expression in a subset of fully-sequenced inbred strains from the Drosophila Genetic Reference Panel and performed a GWAS to identify 42 naturally-occurring polymorphisms-in proximity to 28 candidate genes-that significantly influence R7 opsin expression. Network analysis revealed multiple potential interactions between the associated candidate genes, spineless and its partners. GWAS candidates were further validated in a secondary RNAi screen which identified 12 lines that significantly reduce the proportion of Rh3 expressing R7 photoreceptors. Finally, using RNA-Seq, we demonstrated that all but four of the GWAS candidates are expressed in the pupal retina at a critical developmental time point and that five are among the 917 differentially expressed genes in sevenless mutants, which lack R7 cells. Collectively, these results suggest that the relatively simple, binary cell fate decision underlying R7 opsin expression is modulated by a larger, more complex network of regulatory factors. Of particular interest are a subset of candidate genes with previously characterized neuronal functions including neurogenesis, neurodegeneration, photoreceptor development, axon growth and guidance, synaptogenesis, and synaptic function.

Regulation of opsin and circadian clock genes on mate-finding behavior of the day-flying red moth, Phauda flammans (Walker)

ABSTRACT First, significantly higher mate-finding success was found under light condition than under constant darkness condition in Phauda flammans, a typical diurnal moth. We speculate that mate-finding behavior in P. flammans may be influenced by the light-sensitive opsin genes Long wavelength opsin (PfLW), Ultraviolet opsin (PfUV) and Blue opsin (PfBL), which are potentially regulated by both light-cues and endogenous circadian rhythms. Second, the circadian clock genes Period (PfPer), Timeless (PfTim), Cryptochrome1 (PfCry1), Cryptochrome2 (PfCRY2), Cryptochrome3 (PfCry-like), Clock (PfClk), Cycle (PfCyc), Vrille (PfVri), and Slimb (PfSli) were identified in P. flammans. Third, circadian rhythms in the relative expression levels of opsin and circadian clock genes were demonstrated via quantitative real-time PCR analysis, with peak expression coinciding with the mate-finding peak. Notably, the relative expression of PfLW in males P. flammans was significantly higher than that in females P. flammans at the mate-finding peaks Zeitgeber time (ZT) 8 and ZT 10 under light, while the expression of the opsin gene PfBL showed a similar pattern at ZT 10 under light. Additionally, the expression of the clock gene PfCry-like was significantly higher in males than in females at ZT 8 and ZT 10 under light, while PfPer, PfTim, PfClk and PfCyc exhibited similar male-biased expression patterns at ZT 10 under light. Conversely, PfCry1 and PfVri expression was significantly higher in females than in male at ZT 8 under light. In conclusion, sex differences were detected in the expression of opsin and circadian clock genes, which indicated that light-mediated regulation of these genes may contribute to the daytime mate-finding behavior of P. flammans.

Dynamic Expansions and Retinal Expression of Spectrally Distinct Short-Wavelength Opsin Genes in Sea Snakes.

The photopigment-encoding visual opsin genes that mediate color perception show great variation in copy number and adaptive function across vertebrates. An open question is how this variation has been shaped by the interaction of lineage-specific structural genomic architecture and ecological selection pressures. We contribute to this issue by investigating the expansion dynamics and expression of the duplicated Short-Wavelength-Sensitive-1 opsin (SWS1) in sea snakes (Elapidae). We generated one new genome, 45 resequencing datasets, 10 retinal transcriptomes, and 81 SWS1 exon sequences for sea snakes, and analyzed these alongside 16 existing genomes for sea snakes and their terrestrial relatives. Our analyses revealed multiple independent transitions in SWS1 copy number in the marine Hydrophis clade, with at least three lineages having multiple intact SWS1 genes: the previously studied Hydrophis cyanocinctus and at least two close relatives of this species; Hydrophis atriceps and Hydrophis fasciatus; and an individual Hydrophis curtus. In each lineage, gene copy divergence at a key spectral tuning site resulted in distinct UV and Violet/Blue-sensitive SWS1 subtypes. Both spectral variants were simultaneously expressed in the retinae of H. cyanocinctus and H. atriceps, providing the first evidence that these SWS1 expansions confer novel phenotypes. Finally, chromosome annotation for nine species revealed shared structural features in proximity to SWS1 regardless of copy number. If these features are associated with SWS1 duplication, expanded opsin complements could be more common in snakes than is currently recognized. Alternatively, selection pressures specific to aquatic environments could favor improved chromatic distinction in just some lineages.

Sws2 Gene Positively Regulates Melanin Production in Plectropomus leopardus Skin via Direct Regulation of the Synthesis of Retinoic Acid.

Opsins are a class of transmembrane proteins encoded by opsin genes, and they play a variety of functional roles. Short wavelength-sensitive opsin 2 (sws2), one of the five classes of visual opsin genes, mainly senses blue light. Previous research has indicated that sws2 is essential for melanocyte formation in fish; however, its specific role in skin color differentiation remains to be elucidated. Here, we identified the sws2 gene in a prized reef-dwelling fish, Plectropomus leopardus. The full-length P. leopardus sws2 gene encodes a protein consisting of 351 amino acids, and exhibits substantial homology with other fish species. The expression of the sws2 gene was widespread across P. leopardus tissues, with high expression in eye and skin tissues. Through immunohistochemistry and in situ hybridization analyses, we discovered that the sws2 gene was primarily localized in the rod and cone cells of the retina, and epidermal cells of the skin. Furthermore, dsRNA interference was used for sws2 gene knockdown in living P. leopardus to elucidate its function in skin color differentiation. Black-color-related genes, melanin contents, and tyrosinase activity in the skin significantly decreased after sws2 knockdown (p < 0.05), but red-color-related genes and carotenoid and lutein contents significantly increased (p < 0.05). Retinoic acid injection produced the opposite results. Our results suggested that the sws2 gene influences P. leopardus skin color regulation by affecting vitamin synthesis and melanin-related gene expression levels. This study establishes a foundation for elucidating the molecular mechanisms by which sws2 regulates melanocyte formation in fish skin.

The genomic evolution of visual opsin genes in amphibians

Among tetrapod (terrestrial) vertebrates, amphibians remain more closely tied to an amphibious lifestyle than amniotes, and their visual opsin genes may be adapted to this lifestyle. Previous studies have discussed physiological, morphological, and molecular changes in the evolution of amphibian vision. We predicted the locations of the visual opsin genes, their neighboring genes, and the tuning sites of the visual opsins, in 39 amphibian genomes. We found that all of the examined genomes lacked the Rh2 gene. The caecilian genomes have further lost the SWS1 and SWS2 genes; only the Rh1 and LWS genes were retained. The loss of the SWS1 and SWS2 genes in caecilians may be correlated with their cryptic lifestyles. The opsin gene syntenies were predicted to be highly similar to those of other bony vertebrates. Moreover, dual syntenies were identified in allotetraploid Xenopus laevis and X. borealis. Tuning site analysis showed that only some Caudata species might have UV vision. In addition, the S164A that occurred several times in LWS evolution might either functionally compensate for the Rh2 gene loss or fine-tuning visual adaptation. Our study provides the first genomic evidence for a caecilian LWS gene and a genomic viewpoint of visual opsin genes by reviewing the gains and losses of visual opsin genes, the rearrangement of syntenies, and the alteration of spectral tuning in the course of amphibians’ evolution.

Characteristics of pectens in diurnal and nocturnal birds and a new functional proposal relating to non-visual opsins.

The pecten is a fold-structured projection at the ocular fundus in bird eyes, showing morphological diversity between the diurnal and nocturnal species. However, its biological functions remain unclear. This study investigated the morphological and histological characteristics of pectens in wild birds. Additionally, the expression of non-visual opsin genes was studied in chicken pectens. These genes, identified in the chicken retina and brain, perceive light periodicity regardless of visual communication. Similar pleat numbers have been detected among bird taxa; however, pecten size ratios in the ocular fundus showed noticeable differences between diurnal and nocturnal birds. The pectens in nocturnal brown hawk owl show extremely poor vessel distribution and diameters compared with that of diurnal species. RT-PCR analysis confirmed the expression of Opn5L3, Opn4x, Rrh and Rgr genes. Insitu hybridization analysis revealed the distribution of Rgr-positive reactions in non-melanotic cells around the pecten vessels. This study suggests a novel hypothesis that pectens develop dominantly in diurnal birds as light acceptors and contribute to continuous visual function or the onset of periodic behaviour.

Expansion and Functional Diversification of Long-Wavelength-Sensitive Opsin in Anabantoid Fishes

Gene duplication is one of the most important sources of novel genotypic diversity and the subsequent evolution of phenotypic diversity. Determining the evolutionary history and functional changes of duplicated genes is crucial for a comprehensive understanding of adaptive evolution. The evolutionary history of visual opsin genes is very dynamic, with repeated duplication events followed by sub- or neofunctionalization. While duplication of the green-sensitive opsins rh2 is common in teleost fish, fewer cases of multiple duplication events of the red-sensitive opsin lws are known. In this study, we investigate the visual opsin gene repertoire of the anabantoid fishes, focusing on the five lws opsin genes found in the genus Betta. We determine the evolutionary history of the lws opsin gene by taking advantage of whole-genome sequences of nine anabantoid species, including the newly assembled genome of Betta imbellis. Our results show that at least two independent duplications of lws occurred in the Betta lineage. The analysis of amino acid sequences of the lws paralogs of Betta revealed high levels of diversification in four of the seven transmembrane regions of the lws protein. Amino acid substitutions at two key-tuning sites are predicted to lead to differentiation of absorption maxima (λmax) between the paralogs within Betta. Finally, eye transcriptomics of B. splendens at different developmental stages revealed expression shifts between paralogs for all cone opsin classes. The lws genes are expressed according to their relative position in the lws opsin cluster throughout ontogeny. We conclude that temporal collinearity of lws expression might have facilitated subfunctionalization of lws in Betta and teleost opsins in general.

Differential Regulation of Opsin Gene Expression in Response to Internal and External Stimuli.

Determining how internal and external stimuli interact to determine developmental trajectories of traits is a challenge that requires the integration of different subfields of biology. Internal stimuli, such as hormones, control developmental patterns of phenotypic changes, which might be modified by external environmental cues (e.g. plasticity). Thyroid hormone (TH) modulates the timing of opsin gene expression in developing Midas cichlid fish (Amphilophus citrinellus). Moreover, fish reared in red light accelerate this developmental timing compared to fish reared in white light. Hence, we hypothesized that plasticity caused by variation in light conditions has coopted the TH signaling pathway to induce changes in opsin gene expression. We treated Midas cichlids with TH and crossed this treatment with two light conditions, white and red. We observed that not only opsin expression responded similarly to TH and red light but also that, at high TH levels, there is limited capacity for light-induced plasticity. Transcriptomic analysis of the eye showed that genes in the TH pathway were affected by TH, but not by light treatments. Coexpression network analyses further suggested that response to light was independent of the response to TH manipulations. Taken together, our results suggest independent mechanisms mediating development and plasticity during development of opsin gene expression, and that responses to environmental stimuli may vary depending on internal stimuli. This conditional developmental response to external factors depending on internal ones (e.g. hormones) might play a fundamental role in the patterns of phenotypic divergence observed in Midas cichlids and potentially other organisms.

Mutant kri1l causes abnormal retinal development via cell cycle arrest and apoptosis induction

Damage to the ribosome or an imbalance in protein biosynthesis can lead to some human diseases, such as diabetic retinopathy (DR) and other eye diseases. Here, we reported that the kri1l gene was responsible for retinal development. The kri1l gene encodes an essential component of the rRNA small subunit processome. The retinal structure was disrupted in kri1l mutants, which resulted in small eyes. The boundaries of each layer of cells in the retina were blurred, and each layer of cells was narrowed and decreased. The photoreceptor cells and Müller glia cells almost disappeared in kri1l mutants. The lack of photoreceptor cells caused a fear of light response. The development of the retina started without abnormalities, and the abnormalities began two days after fertilization. In the kri1l mutant, retinal cell differentiation was defective, resulting in the disappearance of cone cells and Müller cells. The proliferation of retinal cells was increased, while apoptosis was also enhanced in kri1l mutants. γ-H2AX upregulation indicated the accumulation of DNA damage, which resulted in cell cycle arrest and apoptosis. The kri1l mutation reduced the expression of some opsin genes and key retinal genes, which are also essential for retinal development.

Investigating the Influence of Varied Light-Emitting Diode (LED) Wavelengths on Phototactic Behavior and Opsin Genes in Vespinae.

The phototactic behavior of insects is commonly used to manage pest populations in practical production. However, this elusive behavior is not yet fully understood. Investigating whether the opsin genes play a crucial role in phototaxis is an intriguing topic. Vespinae (Hymenoptera: Vespidae) are a common group of social wasps that are closely associated with human activities. Efficiently controlling wasp populations while maintaining ecological balance is a pressing global challenge that still has to be resolved. This research aims to explore the phototactic behavior and key opsin genes associated with Vespinae. We found significant differences in the photophilic rates of Vespula germanica and Vespa analis under 14 different light conditions, indicating that their phototactic behavior is rhythmic. The results also showed that the two species exhibited varying photophilic rates under different wavelengths of light, suggesting that light wavelength significantly affects their phototactic behavior. Additionally, the opsin genes of the most aggressive hornet, Vespa basalis, have been sequenced. There are only two opsin genes, one for UV light and the other for blue light, and Vespa basalis lacks long-wavelength visual proteins. However, they exhibit peak phototaxis for long-wavelength light and instead have the lowest phototaxis for UV light. This suggests that the visual protein genes have a complex regulatory mechanism for phototactic behavior in Vespinae. Additionally, visual protein sequences have a high degree of homology among Hymenoptera. Despite the hypotheses put forward by some scholars regarding phototaxis, a clear and complete explanation of insect phototaxis is still lacking to date. Our findings provide a strong theoretical basis for further investigation of visual expression patterns and phototactic mechanisms in Vespinae.

The expression and function of opsin genes related to the phototactic behavior of Tuta absoluta

The expression and function of opsin genes related to the phototactic behavior of Tuta absoluta

Diversity and Molecular Evolution of Nonvisual Opsin Genes across Environmental, Developmental, and Morphological Adaptations in Frogs.

Nonvisual opsins are transmembrane proteins expressed in the eyes and other tissues of many animals. When paired with a light-sensitive chromophore, nonvisual opsins form photopigments involved in various nonvisual, light-detection functions including circadian rhythm regulation, light-seeking behaviors, and seasonal responses. Here, we investigate the molecular evolution of nonvisual opsin genes in anuran amphibians (frogs and toads). We test several evolutionary hypotheses including the predicted loss of nonvisual opsins due to nocturnal ancestry and potential functional differences in nonvisual opsins resulting from environmental light variation across diverse anuran ecologies. Using whole-eye transcriptomes of 81 species, combined with genomes, multitissue transcriptomes, and independently annotated genes from an additional 21 species, we identify which nonvisual opsins are present in anuran genomes and those that are also expressed in the eyes, compare selective constraint among genes, and test for potential adaptive evolution by comparing selection between discrete ecological classes. At the genomic level, we recovered all 18 ancestral vertebrate nonvisual opsins, indicating that anurans demonstrate the lowest documented amount of opsin gene loss among ancestrally nocturnal tetrapods. We consistently found expression of 14 nonvisual opsins in anuran eyes and detected positive selection in a subset of these genes. We also found shifts in selective constraint acting on nonvisual opsins in frogs with differing activity periods, habitats, distributions, life histories, and pupil shapes, which may reflect functional adaptation. Although many nonvisual opsins remain poorly understood, these findings provide insight into the diversity and evolution of these genes across anurans, filling an important gap in our understanding of vertebrate opsins and setting the stage for future research on their functional evolution across taxa.

A ubiquitous tire rubber additive induced serious eye injury in zebrafish (Danio rerio)

Previous studies have indicated that tire wear particles (TWPs) leachate exposure induced serious eye injury in fish through inhibiting the thyroid peroxidase (TPO) enzyme activity. However, the main TPO inhibitors in the leachate were still unknown. In this study, we identified 2-Mercaptobenzothiazole (MBT) as the potential TPO inhibitor in the TWPs leachate through references search, model prediction based on Danish QSAR and ToxCast database, molecular docking, and in vivo assay. We further explored the toxic mechanism of MBT under environmentally relevant concentrations. The decreased eye size of zebrafish larvae was mainly caused by the decreased lens diameter and cell density in the inner nuclear layer (INL) and outer nuclear layer (ONL) of the retina. Transcriptomics analysis demonstrated that the eye phototransduction function was significantly suppressed by inhibiting the photoreceptor cell proliferation process after MBT exposure. The altered opsin gene expression and decreased opsin protein levels were induced by weakening thyroid hormone signaling after MBT treatment. These results were comparable to those obtained from a known TPO inhibitor, methimazole. This study has identified MBT as the primary TPO inhibitor responsible for inducing eye impairment in zebrafish larvae exposed to TWPs leachate. It is crucial for reducing the toxicity of TWPs leachate in fish.

OpsinLW2 serves as a circadian photoreceptor in the entrainment of circadian locomotor rhythm of a firebrat

Photic entrainment is an essential function of the circadian clock, which enables organisms to set the appropriate timing of daily behavioral and physiological events. Recent studies have shown that the mechanisms of the circadian clock and photic entrainment vary among insect species. This study aimed to elucidate the circadian photoreceptors necessary for photic entrainment in firebrats Thermobia domestica, one of the most primitive apterygote insects. A homology search of publicly available RNA sequence (RNA-seq) data from T. domestica exhibited a cryptochrome 2 (cry2) gene and three opsin genes, opsin long wavelength 1 (opLW1), opLW2, and opUV, as candidate circadian photoreceptors. We examined the possible involvement of these genes in photic entrainment of firebrat locomotor rhythms. Firebrats had the highest entrainability to the light–dark cycle of green light. Treatment with dsRNA of the candidate genes strongly downregulated the respective targeted genes, and in the case of opsin genes, other untargeted genes were occasionally downregulated to various degrees. Under constant light, most control firebrats became arrhythmic, whereas a fraction of those treated with double RNAi of the two opLWs remained rhythmic. Behavioral experiments revealed that the transient cycles necessary for re-entrainment to shifted light cycles were lengthened when opLW2 expression was reduced. These results suggest that opLW2 is involved in the photic entrainment of circadian rhythm in firebrats.