Effects of different LED light spectra on growth and immunity of the Japanese eel (Anguilla japonica) and giant mottled eel (A. marmorata)

Indoor recirculating aquaculture systems make light control possible and enable the usage of specic coloured lights to promote the growth and immunity of aquaculture species. Five different LED wavelengths (white light, red light [622 nm], green light [517 nm], blue light [467 nm], and dark) were used in this study to evaluate growth and immunity in the glass eel stage of two high-valued anguillid species, Japanese eel ( Anguilla japonica ) and giant mottled eel ( A. marmorata ). There were no signicant differences in growth of the Japanese eel among the groups after 12 weeks of feeding (P > 0.05); the survival rate of each group was over 95%. The giant mottled eel showed better growth in total length and body weight in the red light and dark groups (P < 0.05). Expression levels of immune-related genes were not signicantly different between each group of the Japanese eel and the giant mottled eel (P > 0.05). The growth of the Japanese glass eel was not signicantly sensitive to different LED wavelengths, while the giant mottled glass eel showed better growth under red light and dark environments. Neither eel species showed signicant differences in innate immunity under different LED wavelengths. The regulatory effects of different light spectra on the immune system and endocrine mechanism of sh require further study.


Introduction
Aquaculture has emerged as the most rapidly growing animal food-producing industry in the last two decades (FAO, 2014). Anguillid eels are regarded as an important commercial aquaculture species in East Asia because of their high market demand and nutritional value (Ahn et al., 2015;Shahkar et al., 2015).
The Japanese eel Anguilla japonica is a traditionally reared, highly valued anguillid species. However, the amount of natural resources of A. japonica available is currently only 5% that in the 1970s because of the impact of habitat destruction and over shing (Chen et al., 2014;Dekker, 2004). According to the International Union for Conservation of Nature and Natural Resources (IUCN), A. japonica has been classi ed as "Endangered" in the Red list and needs more attention in the eel aquaculture industry  Currently, there are no studies comparing the effect of lights of different colours on the growth and immune response of A. japonica or A. marmorata. Since RAS is mostly an indoor system, it is easy to control light arti cially. Therefore, we aimed to determine the effect of light spectrum on the growth and immune response in the glass eel stages of the Japanese eel and the giant mottled eel cultured in an indoor RAS system.

Experimental animals and feeding
Glass eels of A. japonica and A. marmorata were caught from eastern Taiwan (A. japonica from the Yilan River, 24.7163 °N, 121.8348 °E, and A. marmorata from Xiugulan River, 23.4612 °N, 121.5008 °E). Eel sampling was approved by the Fishery Agency, Council of Agriculture, Executive Yuan, Taiwan. The specimens were transported at low temperatures through live sh bags lled with oxygen. The health condition of the eels was checked upon arrival at the laboratory located at the Institute of Fisheries Science of National Taiwan University, Taipei. Individuals in good condition were disinfected with 2.5 ppm of potassium permanganate (KMnO 4 ) solution for 10 min to avoid pathogen contamination of the experimental system. After sterilization, the eels were kept in ve sets of indoor RAS systems with ve tanks (30 × 30 × 45 cm) for each set and maintained in freshwater for three days before feeding. Photoperiods were set at 12 h light (7:00-19:00) and 12 h dark.
The initial body weight and total length of A. japonica and A. marmorata (20 A. japonica for each tank; 30 A. marmorata for each tank in triplicates) were measured before experiment started (56.7 ± 2.0 mm, 0.14 ± 0.01 g for A. japonica; 51.04 ± 2.1 mm, 0.15 ± 0.02 g for A. marmorata). An LED (EVERLIGHT Electronics Co., Ltd., Taiwan) was used as the light source to control the background spectra for the experiment. Each set of RAS included ve tanks (30 L water/tank), each exposed to either white light, red light (622 nm), green light (517 nm), or blue light (467 nm) under 100 lx light intensity with photoperiod 12 hours light and 12 hours dakr, or dark (<5 lx). Each RAS tank was covered by a black board to avoid any light in uence from neighbouring tanks or the environment (Fig. 1). The water temperature and pH were controlled between and , respectively, with a water exchange rate of 20 L/day for each RAS; oxygen was dissolved to near saturation by aeration. Fish were fed with blood worms (Chironomus dorsalis larvae), which was often used as glass eel feed, at an amount of 10% of their body weight twice a day for a total of 12 weeks. The remaining feed was removed from the tanks an hour after feeding. The experiment was performed in accordance with the recommendations from the Institutional Animal Care and Use Committee for the care of animals used for experimental or other scienti c purposes (approval number 'NTU-110-EL-00009').

Sample Collection and Analyses
The total length (to the nearest 0.1 mm) and body weight (to the nearest 1 mg) were measured every two weeks. The percentage weight gain, condition factor, speci c growth rate, and the survival rate in each group were calculated as follows: Three sh from each tank were randomly selected and sacri ced to obtain head kidney tissues. Three head kidney tissues from the same tank were pooled together and stored in an RNA protecting reagent at −80 before extracting total RNA using an RNA kit (Bioman Scienti c Co. Ltd., Taiwan) for next generation sequencing (NGS). Three other eels from each tank were randomly selected and sacri ced to obtain the head kidney, and pooled together for real-time PCR of immune-related genes.

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The whole genome of A. japonica was successfully assembled in our previous study (http://molas.iis.sinica.edu.tw/jpeel/) (Hsu et al., 2015), and was used as a template to annotate the transcriptome data of A. japonica and A. marmorata. The head kidney samples of the white light, red light, green light, and blue light groups were preserved at −80 and subjected to NGS for transcriptome analysis of the head kidney to identify the immune-related genes in each group. The raw RNA-seq data were ltered using the TrimGalore program (Babraham Bioinformatics, Cambridge, UK) to discard adaptors and low-quality reads (Q < 13). Low-complexity reads (repeat sequences) were then removed using the PRINSEQ program (ver. 0.20.4). Finally, general read properties were generated using the FastQC program (Babraham Bioinformatics, Cambridge, UK). The MAKER2 (https://www.yandell-lab.org/ software/maker.html) pipeline was chosen for gene prediction and the gene transfer format was generated. The input datasets included RNA-Seq data, PacBio Iso-Seq data, the newly established A. japonica genome, available protein sequences of A. japonica, all teleosts from the uniport database, and the pre-existing zebra sh ( The NGS results were further uploaded to the website (http://molas.iis.sinica.edu.tw/jpeel2018/index.php) set up by our laboratory and Academia Sinica for assembly and analysis. This web database is established on the LAMP system architecture (Ubuntu 14.04, Apache 2.04, PHP 5.1 and MySQL 8.0) with the Bootstrap 3 CSS framework (http://getbootstrap.com/), jQuery1.11.1, and jQuery Validation v1.17 to provide an intuitive user experience. The entire system runs in a virtual machine on the cloud infrastructure of the Institute of Information Science, Academia Sinica, Taiwan. The analysis was performed using scripts written in R (3.4.2). According to the assembly with gene transfer format, raw reads generated from RNA-seq were estimated for the expression pro les via an intuitive graphical interface in Docexpress (https://hub.docker.com/r/lsbnb/docexpress _fastqc) with a built-in process of Hisat2 → StringTie → Ballgown. The expression level pro les, in the form of fragments per kilobase of transcript per million mapped reads values, were then submitted to Multi-Omics onLine Analysis System (http://molas.iis.sinica.edu.tw/jpeel2018/index.php) for both eel species.

Real-time PCR
Speci c candidate genes were selected for real-time PCR based on the results of the NGS analysis. TRIzol reagent (Bioman Scienti c Co. Ltd) was used to extract total RNA, and the purity was quanti ed by spectrophotometry (Medclub Scienti c Co. Ltd). Reverse transcription was performed to synthesize complementary DNA (cDNA) for real-time PCR (Bio-Rad). Four immune-related genes, namely, superoxide dismutase (SOD), lysozyme (LZM), peroxidase (POD), and interleukin-6 (IL-6) were selected as the target genes for real-time PCR, and acidic ribosomal protein (ARP) was used as the reference gene. The primers used for real-time PCR are listed in Table 1.

Statistical analysis
All data were analysed by one-way analysis of variance (IBM SPSS Statistics 24.0) to determine the effects of different spectra. Statistical signi cance was set at P < 0.05. A signi cant effect was followed up with the least signi cant difference test to compare the means.

Growth rate
The mean initial total length and body weight of the eels from each tank were not signi cantly different before the start of the experiment ( Table 3, Table 4). The growth of A. japonica showed no signi cant difference in the total length ( Fig. 2) and body weight (Fig. 3) among the different groups (P > 0.05) after 12 weeks of feeding ( Table 3). The percentage weight gain, condition factor (K), and survival rate also did not show signi cant differences among the groups (P > 0.05) ( Table 3).
Although A. marmorata grew much slower than the Japanese eel, its growth rate was signi cantly different among each of the treatment groups (P < 0.05) ( Fig. 4; Fig. 5). The mean total length and body weight were signi cantly higher in the dark and red light groups than in the other groups (P < 0.05) ( Table   4). The speci c growth rate and percentage weight gain in these groups were also signi cantly higher than those in the green light group. The survival rate was signi cantly higher in the red light group than in the green light and dark groups. However, there was no signi cant difference in the condition factor (K) among the groups ( Table 4). The fastest growing period of the giant mottled eel occurred from the sixth to the eighth week ( Fig. 4; Fig. 5). On the other hand, the white, green, and blue light groups showed some growth retardation during the eighth to the tenth week. The red light group showed no decrease in growth during the entire experimental period.

Real-time PCR
Real-time PCR was conducted for precise quanti cation to compare whether the innate immunity of both eel species was affected by different light spectra. The target genes of real-time PCR were SOD, LZM, POD, and IL-6, and acidic ribosomal phosphoprotein (ARP) was used as a reference gene. The results for A. japonica indicated that although the dark group showed higher SOD expression, there was no signi cant difference among the groups (P > 0.05) (Fig. 6). The expression levels of LZM in the red light and dark treatment groups were higher than in others, but there were no signi cant differences among groups (P > 0.05) (Fig. 7). The white light group showed the highest expression of IL-6; however, there was no signi cant difference among the groups (P > 0.05) (Fig. 8). The expression of POD was highest in the green light group but was not signi cantly different from the other groups (P > 0.05) (Fig. 9).
The real-time PCR results of the giant mottled eel showed that SOD expression was higher in the dark group than in the other groups, but there was no signi cant difference among the groups (P > 0.05) (Fig.  6). The expression level of LZM in white light and red light groups was the highest; however, there was no signi cant difference among the groups (P > 0.05) (Fig. 7). The red light and white light groups also showed higher expression levels of LZM than the other groups, but without a signi cant difference (P > 0.05) (Fig. 8). The POD expression levels in the blue light group were lower than those in others; however, there was no signi cant difference among the groups (P < 0.05) (Fig. 9). Moreover, comparison of the qPCR results between both eel species showed no signi cant differences (P > 0.05) in the expression levels of the four immune-related genes under all light spectra (Figs. 6-9).

Discussion
An earlier study has shown that some sh showed different growth rates under a speci c background light spectrum, but the most suitable spectrum differed among species. There were no signi cant differences among the groups in the growth experiment of the glass eel stage of the Japanese eel. Interestingly, Japanese eels in the blue light group showed better feeding motivation than the other groups. Red light could stimulate the feeding motivation in Nile tilapia Oreochromis On the other hand, the giant mottled eel clearly showed signi cantly better growth in the dark and red light groups (P < 0.05). Aquatic creatures use photoreceptor cells with the highest photosensitivity at a speci c wavelength (λmax) to detect underwater objects. λmax can maximize visual acuity, such as in deep-sea sh (Bowmaker, 1990), or maximize visual contrast, such as in sh inhabiting shallow water or coastal areas (Lythgoe, 1979). Therefore, sh tend to live in environments with the best spectral conditions (Downing & Litvak, 2001). Light of speci c colours may enhance their growth potential by facilitating food capture or detection (Pérez et al., 2019). In addition, there may be some potential nonvisual effects of light on endocrine secretion in non-mammalian vertebrate brains, such as that of growth hormone or thyroid hormone (Jonathan et al., 2019). The giant mottled eel shows a blue-shifted rod photoreceptor during its upstream migration stage (Wang et al., 2014), which is insensitive to red light. Moreover, eels are nocturnal animals that prefer to stay away from light. Therefore, it is likely that red light or dark surroundings may reduce stress for the giant mottled eel, resulting in better overall growth. The Japanese eel seems to be more insensitive to the environmental spectrum, and this may have resulted in the lack of signi cant difference in growth among the treatment groups.
Most of the mortality of the two anguillid species in this study, especially that of the giant mottled eel, resulted due to their escape from the tank ( Table 3; Table 4). This might be because the eels are less adapted to a speci c wavelength, increasing their stress levels and eliciting an escape response. The Japanese eel may be more tolerant to lights of different colour, and is thus well-adapted to the environment, resulting in a high survival rate. The escape rate was generally high for the giant mottled eel, especially in the dark group. However, it also had the largest growth rate, and stress did not seem to be an important factor. Alternatively, an earlier study suggested that the escape behaviour may be a natural instinct for the giant mottled eel (Matsuda et al., 2016), considering that it prefers to migrate in its early life stage.
Interestingly, the body colour of the giant mottled eel in the red light and dark groups was slightly lighter than those of the others, which is similar to the results of an earlier study (Shin & Choi, 2014). It has been shown that the pigments in sh can respond to the wavelength of the colour of their environmental background (Bayarri et al., 2002). Bio lm attachments were found on the tank wall in some groups in our study. There were attachments with a dark brown muddy bio lm on the bottom of the blue, green, and white light tanks, while the red and dark tanks had no attachments. This may have caused the darker body colour in red and dark groups to adapt the environment without dark attachment.

Lysozyme (LZM)
is an important enzyme that shows antiviral, antibacterial, and anti-in ammatory activities (Saurabh & Sahoo, 2010). LZM also combines and metabolizes advanced glycosylation end products produced from reactive oxygen species that would otherwise accumulate and cause harm to . The real-time PCR results of both eel species revealed that although the dark group showed the highest SOD value, there were no signi cant differences among the groups (P > 0.05) ( Fig. 6; Fig. 9). SOD and POD have been used as biomarkers of stress in previous studies ( IL-6 is a chemical secreted by the immune system (Tanaka et al., 2014). It can stimulate the body tissues to activate immune mechanisms, help the growth of cells, promote the activation of immune cells of the acquired immune system, and direct blood cells to help macrophages to destroy the source of infection (Stefan et al., 2017). An increase in its concentration can lead to a cytokine storm (Ana et al., 2020). The results of real-time PCR for both eel species showed no signi cant differences among the groups (Fig. 8).
This suggested that the expressions of the innate immune genes were not affected by different light spectra in either eel species. The results also showed no signi cant difference in expression levels between both eel species, which indicates that different spectra only affect the growth, but do not change the immune expression in the Japanese eel and the giant mottled eel.

Conclusion
Growth is not affected by the wavelength of light in Japanese eels. However, it is recommended to avoid rearing giant mottled eels in a short-wavelength environment, and a red light or dark background is more suitable for their growth. Neither of the eel species showed any signi cant difference in the expression of four innate immune-related genes (LZM, SOD, POD, and IL-6). The regulatory effects of different light spectra on the immune system and endocrine mechanism of sh require further study.

Declarations
The authors declare that they have no con icts of interests.

Ethics approval
The experiment was performed in accordance with the recommendations from the Institutional Animal Care and Use Committee for the care of animals used for experimental or other scienti c purposes (approval number 'NTU-110-EL-00009').

Consent to participate
Not applicable.

Consent for publication
Not applicable.

Data Availability
The data that support the ndings of this study are available from the corresponding author, Yu-San Han, upon reasonable request.    Different letters indicate signi cant differences between groups (p < 0.05). Different letters indicate signi cant differences between groups (P < 0.05). Figure 1 Graph of a set of recirculating aquaculture systems (RAS) used in this study. The ve tanks were each 40 L in volume and covered by a black board. W: white light; R: red light (622 nm); G: green light (517 nm); B: blue light (467 nm).

Figure 2
Page 20/23 The total length of Japanese eel reared in different light spectra for 12 weeks. W: white light; Black: dark; B: blue light; G: green light; R: red light The total length of giant mottled eel reared in different light spectra for 12 weeks. W: white light; Black: dark; B: blue light; G: green light; R: red light. Different letters indicate signi cant differences between The SOD expression levels of Japanese eel and giant mottled eel reared in different light spectra. W: white light; B: blue light; G: green light; R: red light; black: dark. Different letters indicate signi cant differences between different spectra groups of the same eel species (P < 0.05).

Figure 7
The LZM expression levels of Japanese eel and giant mottled eel reared in different spectra. W: white light; B: blue light; G: green light; R: red light; black: dark. Different letters indicate signi cant differences between different spectra groups of the same eel species (P < 0.05).

Figure 8
The IL-6 expression levels of Japanese eel and giant mottled eel reared in different spectra. W: white light; B: blue light; G: green light; R: red light; black: dark. Different letters indicate signi cant differences between different spectra groups of the same eel species (P < 0.05).