Plasticity of opsin gene expression in the adult red shiner (Cyprinella lutrensis) in response to turbid habitats.

Vision is very important to fish as it is required for foraging food, fighting competitors, fleeing from predators, and finding potential mates. Vertebrates express opsin genes in photoreceptor cells to receive visual signals, and the variety of light levels in aquatic habits has driven fish to evolve multiple opsin genes with expression profiles that are highly plastic. In this study, red shiners (Cyprinella lutrensis) were exposed to four water turbidity treatments and their opsin genes were cloned to elucidate how opsin gene expression could be modulated by ambient light conditions. Opsin gene cloning revealed that these fish have single RH1, SWS1, SWS2 and LWS genes and two RH2 genes. Phylogenetic analysis also indicated that these two RH2 opsin genes-RH2A and RH2B -are in-paralogous. Using quantitative PCR, we found evidence that opsin expression is plastic in adults. Elevated proportional expression of LWS in the cone under ambient light and turbid treatment indicated that the red shiner's visual spectrum displays a red shift in response to increased turbidity.

The Cone Opsin Repertoire of Osteoglossomorph Fishes: Gene Loss in Mormyrid Electric Fish and a Long Wavelength-Sensitive Cone Opsin That Survived 3R.

Vertebrates have four classes of cone opsin genes derived from two rounds of genome duplication. These are short wavelength sensitive 1(SWS1), short wavelength sensitive 2(SWS2), medium wavelength sensitive (RH2), and long wavelength sensitive (LWS). Teleosts had another genome duplication at their origin and it is believed that only one of each cone opsin survived the ancestral teleost duplication event. We tested this by examining the retinal cones of a basal teleost group, the osteoglossomorphs. Surprisingly, this lineage has lost the typical vertebrate green-sensitive RH2 opsin gene and, instead, has a duplicate of the LWS opsin that is green sensitive. This parallels the situation in mammalian evolution in which the RH2 opsin gene was lost in basal mammals and a green-sensitive opsin re-evolved in Old World, and independently in some New World, primates from an LWS opsin gene. Another group of fish, the characins, possess green-sensitive LWS cones. Phylogenetic analysis shows that the evolution of green-sensitive LWS opsins in these two teleost groups derives from a common ancestral LWS opsin that acquired green sensitivity. Additionally, the nocturnally active African weakly electric fish (Mormyroideae), which are osteoglossomorphs, show a loss of the SWS1 opsin gene. In comparison with the independently evolved nocturnally active South American weakly electric fish (Gymnotiformes) with a functionally monochromatic LWS opsin cone retina, the presence of SWS2, LWS, and LWS2 cone opsins in mormyrids suggests the possibility of color vision.

South American Weakly Electric Fish (Gymnotiformes) Are Long-Wavelength-Sensitive Cone Monochromats.

Losses of cone opsin genes are noted in animals that are nocturnal or rely on senses other than vision. We investigated the cone opsin repertoire of night-active South American weakly electric fish. We obtained opsin gene sequences from genomic DNA of 3 gymnotiforms (Eigenmannia virescens, Sternopygus macrurus, Apteronotus albifrons) and the assembled genome of the electric eel (Electrophorus electricus). We identified genes for long-wavelength-sensitive (LWS) and medium-wavelength-sensitive cone opsins (RH2) and rod opsins (RH1). Neither of the 2 short-wavelength-sensitive cone opsin genes were found and are presumed lost. The fact that Electrophorus has a complete repertoire of extraretinal opsin genes and conservation of synteny with the zebrafish (Danio rerio) for genes flanking the 2 short-wavelength-sensitive opsin genes supports the supposition of gene loss. With microspectrophotometry and electroretinograms we observed absorption spectra consistent with RH1 and LWS but not RH2 opsins in the retinal photoreceptors of E. virescens. This profile of opsin genes and their retinal expression is identical to the gymnotiform's sister group, the catfish, which are also nocturnally active and bear ampullary electroreceptors, suggesting that this pattern likely occurred in the common ancestor of gymnotiforms and catfish. Finally, we noted an unusual N-terminal motif lacking a conserved glycosylation consensus site in the RH2 opsin of gymnotiforms, a catfish and a characin (Astyanax mexicanus). Mutations at this site influence rhodopsin trafficking in mammalian photoreceptors and cause retinitis pigmentosa. We speculate that this unusual N terminus may be related to the absence of the RH2 opsin in the cones of gymnotiforms and catfish.

Differentiation of visual spectra and nuptial colorations of two Paratanakia himantegus subspecies (Cyprinoidea: Acheilognathidae) in response to the distinct photic conditions of their habitats.

BACKGROUND: Vision, an important sensory modality of many animals, exhibits plasticity in that it adapts to environmental conditions to maintain its sensory efficiency. Nuptial coloration is used to attract mates and hence should be tightly coupled to vision. In Taiwan, two closely related bitterlings (Paratanakia himantegus himantegus and Paratanakia himantegus chii) with different male nuptial colorations reside in different habitats. We compared the visual spectral sensitivities of these subspecies with the ambient light spectra of their habitats to determine whether their visual abilities correspond with photic parameters and correlate with nuptial colorations. RESULTS: Theelectroretinogram (ERG) results revealed that the relative spectral sensitivity of P.h. himantegus was higher at 670 nm, but lower at 370 nm, than the sensitivity of P. h. chii. Both bitterlings could perceive and reflect UV light, but the UV reflection patterns differed between genders. Furthermore, the relative irradiance intensity of the light spectra in the habitat of P. h. himantegus was higher at long wavelengths (480-700 nm), but lower at short wavelengths (350-450 nm), than the light spectra in the habitats of P. h.chii. CONCLUSIONS: Two phylogenetically closely related bitterlings, P. h. himantegus and P. h. chii, dwell in different waters and exhibit different nuptial colorations and spectral sensitivities, which may be the results of speciation by sensory drive. Sensory ability and signal diversity accommodating photic environment may promote diversity of bitterling fishes. UV light was demonstrated to be a possible component of bitterling visual communication. The UV cue may assist bitterlings in genderidentification.

GnRH mRNA levels in male three-spined sticklebacks, Gasterosteus aculeatus, under different reproductive conditions.

In vertebrates, reproduction is regulated by the brain-pituitary-gonad (BPG) axis, where the gonadotropin-releasing hormone (GnRH) is one of the key components. However, very little is known about the possible role of GnRH in the environmental and feedback control of fish reproduction. To investigate this, full-length gnrh2 (chicken GnRH II) and gnrh3 (salmon GnRH) sequences of male three-spined sticklebacks (Gasterosteus aculeatus), which are clustered with the taxa of the same GnRH type as other Euteleostei, were cloned and annotated. gnrh1 is absent in this species. The mRNA levels of gnrh2 and gnrh3 in the sticklebacks' brain were measured under breeding and post-breeding conditions as well as in castrated and sham-operated breeding fish and castrated/sham-operated fish kept under long-day (LD 16:8) and short-day (LD 8:16) conditions. Fully breeding males had considerably higher mRNA levels of gnrh2 and gnrh3 in the thalamus (Th) and in the telencephalon and preoptic area (T+POA), respectively, than post-breeding males. Sham-operated breeding males have higher gnrh3 mRNA levels than the corresponding castrated males. Moreover, higher gnrh2 mRNA levels in the Th and higher gnrh3 mRNA levels in the T+POA and hypothalamus (HypTh) were also found in long-day sham-operated males than in sham-operated fish kept under an inhibitory short day photoperiod. Nevertheless, gnrh2 and gnrh3 mRNA levels were not up-regulated in castrated males kept under long-day photoperiod, which suggests that positive feedbacks on the brain-pituitary-gonad axis are necessary for this response.

The giant mottled eel, Anguilla marmorata, uses blue-shifted rod photoreceptors during upstream migration.

Catadromous fishes migrate between ocean and freshwater during particular phases of their life cycle. The dramatic environmental changes shape their physiological features, e.g. visual sensitivity, olfactory ability, and salinity tolerance. Anguilla marmorata, a catadromous eel, migrates upstream on dark nights, following the lunar cycle. Such behavior may be correlated with ontogenetic changes in sensory systems. Therefore, this study was designed to identify changes in spectral sensitivity and opsin gene expression of A. marmorata during upstream migration. Microspectrophotometry analysis revealed that the tropical eel possesses a duplex retina with rod and cone photoreceptors. The λmax of rod cells are 493, 489, and 489 nm in glass, yellow, and wild eels, while those of cone cells are 508, and 517 nm in yellow, and wild eels, respectively. Unlike European and American eels, Asian eels exhibited a blue-shifted pattern of rod photoreceptors during upstream migration. Quantitative gene expression analyses of four cloned opsin genes (Rh1f, Rh1d, Rh2, and SWS2) revealed that Rh1f expression is dominant at all three stages, while Rh1d is expressed only in older yellow eel. Furthermore, sequence comparison and protein modeling studies implied that a blue shift in Rh1d opsin may be induced by two known (N83, S292) and four putative (S124, V189, V286, I290) tuning sites adjacent to the retinal binding sites. Finally, expression of blue-shifted Rh1d opsin resulted in a spectral shift in rod photoreceptors. Our observations indicate that the giant mottled eel is color-blind, and its blue-shifted scotopic vision may influence its upstream migration behavior and habitat choice.

Androgens increase lws opsin expression and red sensitivity in male three-spined sticklebacks.

Optomotor studies have shown that three-spined sticklebacks (Gasterosteus aculeatus) are more sensitive to red during summer than winter, which may be related to the need to detect the red breeding colour of males. This study aimed to determine whether this change of red light sensitivity is specifically related to reproductive physiology. The mRNA levels of opsin genes were examined in the retinae of sexually mature and immature fish, as well as in sham-operated males, castrated control males, or castrated males implanted with androgen 11-ketoandrostenedione (11 KA), maintained under stimulatory (L16:D8) or inhibitory (L8:D16) photoperiods. In both sexes, red-sensitive opsin gene (lws) mRNA levels were higher in sexually mature than in immature fish. Under L16:D8, lws mRNA levels were higher in intact than in castrated males, and were up-regulated by 11 KA treatment in castrated males. Moreover, electroretinogram data confirmed that sexual maturation resulted in higher relative red spectral sensitivity. Mature males under L16:D8 were more sensitive to red light than males under L8:D16. Red light sensitivity under L16:D8 was diminished by castration, but increased by 11 KA treatment. Thus, in sexually mature male sticklebacks, androgen is a key factor in enhancing sensitivity to red light via regulation of opsin gene expression. This is the first study to demonstrate that sex hormones can regulate spectral vision sensitivity.

Grouper interleukin-12, linked by an ancient disulfide-bond architecture, exhibits cytokine and chemokine activities.

Interleukin-12 (IL-12) is a pleiotropic cytokine which bridges innate and adaptive immunity in defense against pathogens. IL-12 proved to be an effective and successful adjuvant to enhance both the innate and adaptive immune responses and could be applicable for a rationale vaccine formulation in fish against pathogen infection. We have cloned the p35 and p40 cDNAs of IL-12 from orange-spotted grouper (Epinephelus coioides). Grouper IL-12 most resembles with sea bass orthologues; moderate to low identity with other teleost and mammalian counterparts. The structural model of grouper IL-12 heterodimer revealed NC(141)F three amino acid patch of grouper p35, which is present in teleost p35 but absent in mammalian and avian p35, and is spatially nearby the conserved cysteine residue located at A-helix of p35 to form a disulfide bond when the 14aa peptide located at loop 1 of grouper p35 was aligned with human corresponding exon 4, instead of exon 5. The results indicated that the loss of this 3aa patch during evolution was compensated by the duplication of exon 4 in mammalian p35 to gain another cysteine residue to form a disulfide bond, evidenced by chicken p35 which does not contain NCF corresponding 3-aa patch nor exon 4 duplication. Accordingly, the inter-chain disulfide bond of IL-12 heterodimer is conserved from teleost to mammalian IL-12. A single chain grouper IL-12 (scgIL-12) construct linked by (G4S)3 was successfully expressed in baculovirus-insect cell system; its identity has been confirmed by LC/MS/MS. In addition, the biological activity of recombinant scgIL-12 (rscgIL-12) are demonstrated for its stimulation of PBL proliferation, chemotactic migration, induction of TNF-α gene expression and a plausible adjuvant effect of prolonged protection against parasite infection in fish. We illustrated the first time in lower vertebrate that grouper IL-12 possesses both cytokine and chemokine activities.

Rutin, a Flavonoid That Is a Main Component of Saussurea involucrata, Attenuates the Senescence Effect in D-Galactose Aging Mouse Model.

Saussurea involucrata (Kar. et Kir.), known as the snow lotus, grows in the Tian Shan and A'er Tai areas of China. It has recently been reported that the ethyl acetate extract of S. involucrata (SI-2) can inhibit proliferation and induce apoptosis in PC-3 human prostate cancer cells. This study investigated the protective effect of ethyl acetate extract of S. involucrata (SI-2) or rutin, a flavonoid extracted from ethyl acetate extract of S. involucrata (SI-2), on D-galactose- (D-gal-) induced brain injury in mice. Administering SI-2 or rutin (30 mg/kg/d and 30 mg/kg/d) for 6 weeks, concomitant with D-gal injection, significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, the result showed that the percentages of cleaved caspase-3 and PARP in the D-gal-treated mice were much higher than those in the control. Pretreatment using SI-2 or rutin decreased the expression of cyclooxygenase-2 via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. Furthermore, our results also showed that oral administration of rutin to these mice significantly improved behavioral performance in a step-through passive avoidance task and these results suggest that SI-2 or rutin exerts potent antiaging effects on D-gal in mice via antioxidative mechanisms.

Effects of early visual experience on the background preference in juvenile cuttlefish Sepia pharaonis.

Although cuttlefish are capable of showing diverse camouflage body patterns against a variety of background substrates, whether they show background preference when given a choice of substrates is not well known. In this study, we characterized the background choice of post-embryonic cuttlefish (Sepia pharaonis) and examined the effects of rearing visual environments on their background preferences. Different rearing backgrounds (enriched, uniformly grey and checkerboard) were used to raise cuttlefish from eggs or hatchlings, and four sets of two-background-choice experiments (differences in contrast, shape, size and side) were conducted at day 1 and weeks 4, 8 and 12 post-hatch. Cuttlefish reared in the enriched environment preferred high-contrast backgrounds at all post-embryonic stages. In comparison, those reared in the impoverished environments (uniformly grey and checkerboard) had either reversed or delayed high-contrast background preference. In addition, cuttlefish raised on the uniformly grey background, exposed to a checkerboard briefly (0.5 or 3 h) at week 4 and tested at week 8 showed increased high-contrast background preference. Interestingly, cuttlefish in the enriched group preferred an object size similar to their body size at day 1 and week 4, but changed this preference to smaller objects at week 12. These results suggest that high-contrast backgrounds may be more adaptive for juvenile cuttlefish, and visually enriched environments are important for the development of these background preference behaviours.

Arl6ip1 plays a role in proliferation during zebrafish retinogenesis.

OBJECTIVE: Arl6ip1 has been reported to play a role in ocular development, but its regulatory function as it relates to proliferation is unclear. Therefore, this study aimed to evaluate how Arl6ip1 may regulate the proliferative behavior of retinal progenitor cells during zebrafish embryogenesis. METHOD: Arl6ip1 was specifically knocked down by introducing morpholino nucleic acid oligomers. The DNA content of cells dissociated from morphant eyes was analyzed by fluorescence-activated cell sorting (FACS). Retinal cells in the S- and late G2/M-phase were detected by labeling with BrdU and then immunostaining with anti-BrdU antibody and by immunostaining with phospho-histone H3 antibody, respectively. We also examined the expressions of shh,p57kip2, and cyclin D1 in retinas of experimental animals. The bidirectional plasmid pGFP:HSE:p57kip2 was used to rescue the defect in cell cycle exit. RESULTS: FACS analysis showed that the >2C DNA content per cell in the eyes of arl6ip1 morphants was 2-fold greater than that of the wild type. Following functional Arl6ip1 knockdown, anti-BrdU- and anti-phospho-histone H3-positive signals were higher and the retinal progenitors kept expressing cyclin D1 but not shh or p57kip2, suggesting that eye progenitor cells remained in an early progenitor state and could not exit the cell cycle to progress to differentiation. Interestingly, overexpression of p57kip2, which enables exit from the cell cycle, led to a reduction of anti-BrdU-positive signals in the retinas of arl6ip1 morphants. CONCLUSION: Arl6ip1 not only affects signals controlling eye development but also plays an important role in the proliferation of retinal progenitor cells.

A comparative study on the visual adaptations of four species of moray eel.

The goal of this study was to investigate how the eyes of different species of moray eel evolved to cope with limitations to vision imposed on them by the photic environments in which they reside. The comparative retinal histological structures and visual pigment characteristics including opsin gene sequences, of four species of moray eel inhabiting diverse habitats (i.e., shallow-water species, Rhinomuraena quaesita and Gymnothorax favagineus, and deep-sea species, Gymnothorax reticularis and Strophidon sathete) were examined. The histological sections showed that retinal layer structures of R. quaestia are significantly different from those of the other three species which likely reflects the effects of distribution depth on the structures. The maximal absorbance wavelength (λ(max)) of photoreceptor cells, as measured by microspectrophotometry (MSP), showed a close correlation between the λ(max) and the intensity/spectral quality of the light environment where each species lives. The spectra-shift, between shallow and deep-sea species, observed in the rods cells results from amino acid substitution in Rh1 gene, while that in cones most likely results from differential expression of multiple Rh2 genes.

Activity and function of rabbit muscle-specific creatine kinase at low temperature by mutation at gly268 to asn268.

Carp muscle-specific creatine kinase M1 isoenzyme (M1-CK) seems to have evolved to adapt to synchronized changes in body temperature and intracellular pH. When gly(268) in rabbit muscle-specific creatine kinase was substituted with asn(268) as found in carp M1-CK, the rabbit muscle-specific CK G286N mutant specific activity at pH 8.0 and 10°C was more than 2-fold higher than that in the wild-type rabbit enzyme. Kinetic studies showed that K(m) values of the rabbit CK G268N mutant were similar to those of the wild-type rabbit enzyme, yet circular dichroism spectra showed that the overall secondary structures of the mutant enzyme, at pH 8.0 and 5°C, were almost identical to the carp M1-CK enzyme. The X-ray diffraction pattern of the mutant enzyme crystal revealed that amino acid residues involved in substrate binding are closer to one another than in the rabbit enzyme, and the cysteine283 active site of the mutant enzyme points away from the ADP binding site. At pH 7.4-8.0 and 35-10°C, with a smaller substrate, dADP, specific activities of the mutant enzyme were consistently higher than the wild-type rabbit enzyme and more similar to the carp M1-CK enzyme. Thus, the smaller active site of the RM-CK G268N mutant may be one of the reasons for its improved activity at low temperature.

Visual contrast modulates maturation of camouflage body patterning in cuttlefish (Sepia pharaonis).

Camouflage is the primary defense behavior in cephalopods. It is known that cuttlefish immediately after hatching are capable of showing various body patterns for concealing themselves, however recent studies suggest that maturation of camouflage body patterns is faster for cuttlefish (Sepia officinalis) reared in enriched environments than those reared in impoverished environments. Since camouflage patterning in cephalopods is predominately visually driven, this study specifically investigates effects of the rearing background contrast on the maturation of body patterns in cuttlefish (Sepia pharaonis). Newly hatched animals were separated into two cohorts, one reared in a uniform-gray background (low-contrast, or L group) and the other raised in a black/white checkerboard background (high-contrast, or H group). At Weeks 2, 4, 6, 8, 10, and 12, cuttlefish were placed individually either on uniform or checkerboard substrates to examine their body patterns. Animals from both L and H groups appear to show moderate disruptive patterns on the checkerboard and less disruptive on the uniform background at Week 2. Throughout development, however, cuttlefish from the H group showed stronger disruptive patterns than that of the L group on the checkerboard background at Weeks 10 and 12. In interesting findings, cuttlefish from both L and H groups showed similar strength but different disruptive components on the uniform background in later postembryonic stages. These results suggest that the maturation of camouflage body patterns in S. pharaonis is at least in part affected by visual contrast of their rearing backgrounds, although environmental complexity or social interaction is also likely to be involved in this process. This also implies that early visual experience could exert its effect on the seemingly preprogrammed behaviors such as camouflage body patterning in cephalopods.

Tiger shrimp (Penaeus monodon) penaeidin possesses cytokine features to promote integrin-mediated granulocyte and semi-granulocyte adhesion.

Recent studies indicated that antimicrobial peptides (AMPs) play multiple roles in both innate and adaptive immune functions. The penaeidin of tiger shrimp Penaeus monodon shows an antimicrobial activity against Gram-positive bacteria and filamentous fungi. To study immunomodulation functions of the penaeidin, we transfected shrimp hemocytes in primary culture with penaeidin-specific small interfering RNA (siRNA-3) and observed a concomitant 20% reduction in adhesive hemocytes compared with mock-transfected cells. The addition of biosynthesized or chemically synthesized penaeidin or penaeidin proline-rich domain (PRD) to the culture medium of penaeidin knock-down hemocytes led to a full recovery in the number of adhesive hemocytes. The effect of penaeidin knock-down on the expression of tiger shrimp cell adhesion-associated molecules was examined using real-time Q-PCR. Results demonstrated 91% and 64% decreases in the expression of integrin-beta and collagen, respectively, and a 396% increase in the expression of collagenase. The addition of chemically synthesized penaeidin after penaeidin knock-down hemocytes normalized the expression of these genes. The addition of the integrin-beta ligand competitor RGDS to mock-transfected hemocytes decreased the number of adhesive hemocytes similar to penaeidin knock-down. In conclusion, penaeidin possesses an integrin-beta-mediated cytokine feature that promotes shrimp granulocyte and semi-granulocyte adhesion. This is the first report about functional shrimp cytokine.

Ontogenetic changes in color vision in the milkfish (Chanos chanos Forsskål, 1775).

The milkfish (Chanos chanos Forsskål, 1775) is a euryhaline fish widely distributed in tropical and subtropical Indo-Pacific waters. It is unique in having in the cephalic region adipose eyelid tissue that begins to develop in the larval stage and is completely formed by the Juvenile stage. The formation of the adipose eyelids coincides with the onset of active swimming ability. Larval and juvenile milkfish have different dietary modes and habitats. This study was aimed to investigate ontogenetic changes in color perception ability with the use of microspectrophotometry (MSP). Larval milkfish had rod cells and red, green, blue, and violet cone cells, while juvenile milkfish lost the violet cone cells, and the blue cones shifted to shorter wavelengths. Histological sections showed the presence of cone cells of the single type (but no double or twin types) in the retina, which implies that the milkfish may not have polarized vision.

Adaptation of visual spectra and opsin genes in seabreams.

Three species of seabreams, Acanthopagrus berda, Acanthopagrus schlegelii and Pagrus major, living at different depths, were chosen to investigate how visual spectra and opsin genes evolve in response to various photic environments. The lambda max of photoreceptors and opsin genes were measured and cloned from these species. Eight to twelve nm spectral shifts in the rod and blue cone cells were observed between the deep-sea, P. major, and shallow-sea species, A. berda and A. schlegelii. Furthermore, the deep-sea P. major has lost its red light vision. Six opsin genes, Rh1, Rh2A, Rh2B, SWS1, SWS2 and LWS, were identified from all three seabream species, with the LWS genes of P. major having undergone pseudogenization. These data indicate that the photic environment of habitats select for the physiology of visual spectra and coding of opsin genes.

Acoustically evoked potentials in two cephalopods inferred using the auditory brainstem response (ABR) approach.

It is still a matter of debate whether cephalopods can detect sound frequencies above 400 Hz. So far there is no proof for the detection of underwater sound above 400 Hz via a physiological approach. The controversy of whether cephalopods have a sound detection ability above 400 Hz was tested using the auditory brainstem response (ABR) approach, which has been successfully applied in fish, crustaceans, amphibians, reptiles and birds. Using ABR we found that auditory evoked potentials can be obtained in the frequency range 400 to 1500 Hz (Sepiotheutis lessoniana) and 400 to 1000 Hz (Octopus vulgaris), respectively. The thresholds of S. lessoniana were generally lower than those of O. vulgaris.

Adaptive evolution of cone opsin genes in two colorful cyprinids, Opsariichthys pachycephalus and Candidia barbatus.

Opsariichthys pachycephalus and Candidia barbatus are two phylogenetically related freshwater cyprinids that both exhibit colorful, yet quite different nuptial coloration. This study was designed to test the hypothesis that differences in nuptial coloration between two species could reflect differences in color perception ability and the opsin genes that coded for it. Genes encoding the visual pigments of these two species were cloned and sequenced, lambda(max) of cone photoreceptors and the reflectance spectra of their body coloration were measured to test the hypothesis. The 14-nm spectral shift between green-light-sensitive photoreceptors of these two cyprinids is found to correlate well with differences in their reflective spectra. The spectral shift could result from differential expression of opsin genes and the interactive effects of the amino acid replacements in various minor sites. These results support our hypothesis that nuptial coloration is tied to color perception ability and opsin genes.