Before first feeding, the yolk is utilized for embryo development in most marine fish. As yolk reserves are gradually exhausted, larvae undergo the difficult transition from endogenous to mixed feeding period to obtain energy and required nutrients to support growth and development (Ma et al. 2010; Yúfera et al. 2014). Changes in the behavior of marine fish larvae are closely related to the development of the sensory organs, especially the visual system, which is crucial for feeding and predator defense in larval survival (Lim and Mukai 2014). It is very important for larvae to complete the differentiation of retinal functional cells and mouth-opening before the yolk sac is depleted because food intake requires coordination of food searching, detection, attract, capture and ingestion (Rønnestad et al. 2013, Lim and Mukai 2014, Hu et al. 2018). In this study, the yolk sac of T. rubripes disappeared and the larvae were mouth opening and feeding on rotifers at 2 dah. Histological observation showed that all the ten retinal layers were visible in T. rubripes from 2 dah, indicating that the well-developed visual system provided the necessary conditions for larval feeding. This result is consistent with other reports in teleosts, such as Danio rerio, where the iridophores are scattered over the retina, where the iris will develop and mouth is opening and feeding at 2 dah (Kimmel et al. 1995). In Epinephelus akaara larvae, yolk absorption was complete at 3 dah, and the mouth had opened at 4 dah. Hatched larvae had ONL, INL, and GCL starting from 2 to 3 dah, the retina was differentiated into PRE, PRos/is, ONL, OPL, INL, IPL, GCL and the choroid membrane was pigmented at 4 dah (Kim et al. 2013; Kim et al. 2019). In Engraulis anchoita, the eyes were pigmented and the GCL and the PR were visible in the retina, towards the end of yolk sac stage, when the larvae were 4 mm in length. This stage of acquisition of functionality coincided with the absorption of yolk and the beginning of exogenous feeding (Miranda et al. 2020). In Sparus aurata, the maturation of eye also occurred at 3 to 4 dah and it underwent profound anatomical and physiological alterations, such as the opening of the mouth and anus, the resorption of the yolk sac and functional differentiation of the alimentary canal, liver and pancreas (Parry et al. 2005; Yúfera et al. 2014; Pavón-Muñoz et al. 2016). In contrast, for precocial fish species, such as Hippocampus reidi, Iago omanensis (Triakidae), Chiloscyllium punctatum and Scyliorhinus canicular, the retina was fully developed prior to birth (Fishelson and Baranes 1999; Sánchez-Farías and Candal et al. 2005, 2008; Ferreiro-Galve et al. 2008, 2010a, b, 2012; Harahush et al. 2009; Bejarano-Escobar et al. 2012, 2013; Sánchez-Farías and Candal 2015, 2016; Novelli et al. 2015; Álvarez-Hernán et al. 2019), so the degree of maturation at hatching in the retina of fishes is different in altricial and precocial fish species. And, for the most primitive vertebrates, such as sea lamprey Petromyzon marinus, begin retinal development during embryogenesis but do not complete differentiation until metamorphosis, occurring well after birth at five or more years of age (De Miguel and Anadón 1987; Rodicio et al. 1995; Harahush et al. 2009).
The RPE layer and the PRos/is layer can be observed early from 1 dah in T. rubripes. Although the teleost eye is very similar to the mammalian eye, it is characterized by several unique structures, such as teleost eyes lake eyelids except for the nictitating membranes of certain sharks and most teleost cannot alter the size of their pupil (Kusmic and Gualtieri 2000; Reckel et al. 2002), so the fish retina is more susceptible to potential light-induced damage as they are continuously exposed to intense light. Consequently, alternative protective strategies have developed to cope with high light intensities, including migration of melanin granules and photoreceptor mobility. Acting as an anti-oxidant adjacent to the outer segments of photoreceptor cells, ocular melanin protects the retina against light-induced cell toxicity (Sanyal and Zeilmaker 1988), by migrating in an apical direction in response to light within processes of the RPE and enshroud photoreceptors compared to higher vertebrate (Allen and Hallows 1997). These photoreceptors can move into or out of the deep recesses of the RPE, so the RPE layer develops earlier in T. rubripes, which may be critical to protects the retina against light-induced cell toxicity. The results are consistent with some previous studies in other teleost. For example, in Acanthopagrus latus, Mugil cephalus and Alosa sapidissima, before the retina developed, a thin layer of RPE containing a few melanin particles was clearly observed at the edge of the retina (He et al. 1985; Xu et al. 1988, Gao et al. 2016). In this study, opposing developmental trends were found in the thickness of ONL and OPL, INL and IPL, GCL and OFL in T. takifugu. In teleost, the ONL is composed of the cell bodies of the cones and rods. The OPL contains the processes and synaptic terminals of rods, cones, horizontal cells and bipolar cells. The nuclei of the bipolar cells, amacrine cells, horizontal cells and Müller cells are found in the INL and the IPL consists of the connections between bipolar, amacrine and ganglion cells. The nuclei of ganglion cells form the GCL and the OFL contains the axons of ganglion cells as they collect to form the optic nerve (Fernald 1990; Kolb 2011; Ferreiro-Galve et al. 2008, 2010a, b, 2012; Bejarano-Escobar et al. 2014; Musilova et al. 2019). Cell body and synaptic differentiation becomes more obvious, resulting in a continuous decrease in the thickness of ONL and an increasing thickness of OPL (Ali and Anctil 1977; Kolb 2011). Similarly, with the extension of axons and the continuous branching of dendrites, the thickness of INL decreases and the thickness of IPL increases (Ali and Anctil 1977; Kolb 2011). With the extension of axons and dendrites, the thickness of GCL decreases and the thickness of OFL increases (Ali and Anctil 1977; Kolb 2011). The results obtained in the present study reflect the differentiation process of the retina during the early developmental stage of T. rubripes. The emergence of the plexiform layers occurs almost simultaneously, as have been described in other fast-developing teleost (Parry et al. 2005; Bejarano-Escobar et al. 2012, 2013; Pavón-Muñoz et al. 2016). In contrast, studies conducted in slow-developing species such as elasmobranchs and Sparus trutta have shown that the IPL evolves earlier than the OPL (Ferreiro-Galve et al. 2008, 2010a, b, 2012; Harahush et al. 2009; Bejarano-Escobar et al. 2012; Sánchez-Farías and Candal et al. 2005, 2008).
In a previous study, the ONL/INL ratio was used to estimate the degree of spatial summation of visual information at the first synapse in the retina and compared to crepuscular at 1.4 to 1.7 or nocturnal 2.7 to 3.5 foraging species, diurnal feeding fishes were shown to have a lower summation ratio of 0.5 to 1.4 (Munz and McFarland 1973; Schieber et al. 2012). In nocturnal fishes, the higher ratio increases visual sensitivity by pooling the signals from many photoreceptors, at the expense of spatial resolving power, which reflects the adaptation to dim conditions (Munz and McFarland 1973). In the present study, the ONL/INL ratio of T. rubripes was 1.3 to 2.5 during the early developmental stage, suggesting that the fugu larvae have higher visual sensitivity. As reported previously, the female T. rubripes lays demersal, adhesive eggs in coastal waters at a depth of 10 to 50 m during spring, and juveniles remain in nursery ground areas near the main spawning grounds from spring to summer, and then enter wider areas (Katamachi et al. 2015; Kim et al. 2016; Zhang et al. 2019). The higher ratio of the ONL/INL in T. rubripes reflects an adaptation to their surrounding light conditions. However, Schieber et al. (2012) examined the retinal anatomy of four elasmobranch species with differing ecologies, including Port Jackson shark Heterodontus portusjacksoni, the bull shark Carcharhinus leucas, pink whipray Himantura fai, and the epaulette shark Hemiscyllium ocellatum and found that the ONL:INL ratio may be a less robust indicator of diel activity patterns in elasmobranchs. The ratio of the nucleus number of the ONL layer to the GCL layer reflects the degree of retina network convergence (Xu et al. 1998), and this reflects the degree of visual sensitivity and light sensitivity of fish (Ma et al. 2010). Higher visual sensitivity helps them to distinguish small aquatic organisms such as zooplankton in motion providing the possibility of successful predation (Ma et al. 2010). In this study, no significant difference was observed among the different sampling points, which ranged from 1.9 to 3.7, which suggested that T. rubripes may acquire high visual sensitivity before the larvae open their mouths.
The vertebrate visual opsin genes are expressed in the retina and are responsible for facilitating visual perception. The expression of visual genes like rhodopsin, LWS, SWS2 and green opsin, and non-visual genes like opsin3 and opsin5 in T. rubripes were detected early from 1 dah, suggesting the fugu larvae may be able to detect different wavelength spectra, especially detecting the short wavelength light could increase the contrast of prey against the water background (Hargrave et al. 1983; Loew et al. 1993; Browman et al. 1994). A similar pattern was also observed in most fish species, including Clupea pallasi, (Sandy and Blaxter 1980), Pseudopleuronectes americanus (Mader and Cameron 2004), and Oncorhynchus nerka (Flamarique and Hawryshyn 1996). Previous studies have shown that the SWS, including ultraviolet/violet or short-wavelength sensitive type 1 cone opsins (SWS1; approx. 360–430 nm) and blue or short-wavelength sensitive type 2 cone opsins (SWS2; approx. 430– 460 nm) (Jacobs et al. 1996; Van et al. 2006). The light-sensitive pigments of SWS1 can absorb the maximum spectral range of incident light within the spectrum of ultraviolet, which plays a significant role in foraging, communication and mate selection. The SWS1 genes have been isolated from a surprisingly wide range of vertebrates, including lampreys, teleosts, amphibians, reptiles, birds, and mammals (Van et al.2006). However, previous studies have shown that no sequence related to the SWS1 gene was found in the transcriptome of T. rubripes eyeball tissue, indicating that this gene was lost in a common Tetraodontiform ancestor after the Percomorph radiation (Neafsey et al. 2005). Fish possess SWS2 opsin, which is absent in most mammals, including humans, except for a few marsupials and monotreme species. The expression levels of the fish non-vision genes, rod opsin, opsin3 and opsin5 were measured. Rod opsin can restore visual functions in retinal degeneration and mutations in rod opsin cause neurodegenerative blindness retinitis pigmentosa (Athanasiou et al. 2012; Rennison et al. 2012). Rod opsin-treated mice can detect visual stimuli in a dimly lit room, including a flicker at a range of frequencies (up to 10Hz), differences in luminance commonly encountered in visual scenes (Jasmina et al. 2015). In this study, rod opsin was expressed from 1 dah in T. rubripes and gradually increased, which suggested that the protection function of the retina in T. rubripes was gradually increasing. The opsin3, also known as cerebral opsin or panopsin, is a protein encoded by the opsin3 gene in humans (Jiao et al. 2012) and may confer photosensitivity in extraocular tissues that are considered light-insensitive in vertebrates (Sugihara et al. 2018). The opsin5 is a new type of short-wavelength sensitive photopigment in the brain of fish (Nakane et al. 2010) and as a photoreceptor, it can regulate seasonal changes in physiological behavior (Nakane et al. 2014). In T. rubripes larvae, opsin3 and opsin5 were expressed from 1 dah, implying that although vision seems to be the primary sense playing a key role in foraging activity and feeding, non-retinal photoreceptors such as the pineal organ and deep brain photoreceptors and non-visual opsins may be involved in fugu larval growth and development. Previous studies also have shown that the non-visual opsins opsin3 and opsin5 are expressed in inner retinal cells of chick retina at early phases of development and remain expressed in the mature retina at post-natal day 1 and 10 (Rios et al. 2019). Similarly, non-visual opsins including opsin4a, tmtopsa, tmtopsb, and opsin3 are broadly expressed in the zebrafish larva central nervous system (Fernandes et al. 2013; Hang et al. 2016). TMT -opsin, opsin4 and other non-visual opsins expression were detected in the telencephalon (rostral forebrain) of Oryzias latipes (Fischer et al. 2013). How these light-sensitive structures mediate photoperiodic signals and entrain key physiological events needs to be further clarified in fish.
In conclusion, the results obtained here suggest that the maturation of the eye of T. rubripes occurred during the transition period from endogenous to mixed feeding. The findings obtained here will provide implications for vision-based survival skills during the early life stages after hatching, and for the overall ecology and fitness of T. rubripes.