Effects of Light Spectrum on Survival, Growth, Physiological, and Biochemical Indices of Redclaw Cray ﬁ sh ( Cherax quadricarinatus ) Juveniles

. The spectrum is a key environmental factor, and light-emitting diodes (LEDs) can in ﬂ uence the growth and development of crustaceans by altering the composition of the spectrum. This study conducted a 30-day experiment to investigate the effects of ﬁ ve LED spectra (red, yellow, blue, green, and white light) on the growth, antioxidant and immune enzyme activities, stress hormone levels, and the expression of α -amylase ( α -AMY ), ecdysone receptor ( EcR ) and retinoid X receptor ( RXR ) genes in juvenile redclaw cray ﬁ sh ( Cherax quadricarinatus ). The results show that the survival rate of juveniles is markedly higher in the yellow and red-light groups than in the other three groups ( P < 0 : 05). The green light group exhibits the lowest survival rate, yet it demonstrates the highest weight gain rate and speci ﬁ c growth rate. Regarding enzyme activity and hormone levels, the yellow light group shows the lowest malondialdehyde content, with higher superoxide dismutase and acid phosphatase activity than the other groups; no signi ﬁ cant differences are observed in lysozyme activity among the groups ( P > 0 : 05). The melatonin content in the green and blue light groups is signi ﬁ cantly higher than that in the other three groups ( P < 0 : 01). In terms of growth gene expression, the expression of α -AMY , EcR , and RXR in juvenile C. quadricarinatus is regulated by the spectrum. In conclusion, when raised under the yellow light spectrum, juvenile C. quadricarinatus displays elevated survival rates, rapid growth, and robust antioxidant and immune defenses. This study provides important technical parameters for optimizing and enhancing the industrial cultivation of juvenile C. quadricarinatus .


Introduction
The redclaw crayfish (Cherax quadricarinatus), originating from streams in Australia and Papua New Guinea, belongs to the order Decapoda and the family Parastacidae.It is a highvalue freshwater shrimp species that has gradually gained popularity in aquaculture owing to its fast growth, adaptability, and profitable outcomes [1].According to the 2021 Food and Agriculture (FAO) statistics, the total production of redclaw crayfish in countries such as Australia, Cambodia, Malaysia, and Indonesia was approximately 260 tons [2].However, challenges regarding cultivating redclaw crayfish persist, such as low egg-carrying capacity and the immaturity of hatchery technology, which prevent the development of large-scale and industrial production [3,4].Currently, efforts have been made to explore factory-scale cultivation of redclaw crayfish.However, the indoor cultivation process is characterized by various instabilities due to the influence of different environmental factors.Factors such as excessively high or low water temperatures and stocking densities can lead to slow growth and significantly reduce the survival rates of redclaw crayfish juveniles [5,6].Furthermore, maintaining a salinity level between 0.1% and 2% in the water environment enhances the survival and immunity of redclaw crayfish juveniles [7].Currently, research on the influence of various environmental factors affecting factory-scale cultivation of redclaw crayfish remains limited.
In aquaculture, light exposure is closely tied to the growth and survival of aquatic animals, and different spectra play a vital role in their development [8][9][10].For instance, largemouth bass (Micropterus salmoides) experience a significant increase in body weight and exhibit better growth performance in a blue-light environment [10].Furthermore, the impact of spectra on aquatic animals is species-specific [11].In lower-order crustaceans, such as the giant freshwater prawn (Macrobrachium rosenbergii), body length significantly increases when raised under white and green light than blue, yellow, and red light, suggesting that white and green light are beneficial for its growth [12].For the Pacific white shrimp (Litopenaeus vannamei), their juvenile development period shortens under yellow light, indicating the benefit of yellow light on their growth [13].In bivalves such as the discus abalone (Haliotis discus hannai), the survival and specific growth rate (SGR) of juveniles are markedly higher under red and orange light than under green and blue light, enhancing their growth performance [9].The above research indicates that physiological activities such as growth and development in aquatic animals are regulated by changes in the spectrum.However, the mechanisms underlying physiological and biochemical regulation in response to spectrum changes are currently being explored in the scientific community.
Changes in spectra can trigger oxidative stress responses within the bodies of aquatic organisms, resulting in altered antioxidant enzyme activity and stress hormone levels [14,15].For instance, under red light, juvenile yellowtail clownfish (Amphiprion clarkii) had significantly higher serum levels of superoxide dismutase (SOD), catalase (CAT), and melatonin than those in other spectra, indicating that red light-induced oxidative stress responses in these juvenile fish [16].In contrast, juvenile goldfish (Carassius auratus) reared under blue light had significantly increased cortisol levels, suggesting increased stress when cultivated under blue light [17].Besides inducing oxidative stress responses, spectra also affect the expression levels of genes associated with growth or molting in aquatic organisms.For example, juvenile Nile tilapia (Oreochromis niloticus) raised under redlight spectrum conditions exhibited significant upregulation of the α-amylase (α-AMY) gene, promoting their growth [18].Juvenile mud crabs (Scylla paramamosain) exposed to white and blue light exhibited downregulation of the molting inhibitory hormone (MIH) gene and increased molting frequency [19].Currently, research on how light affects newly hatched redclaw crayfish juveniles lasts only 14 days.Given that the factory-scale cultivation period for redclaw crayfish juveniles is approximately 30 days (with the total body length ranges from 3 to 5 cm), this research requires further refinement [20].Hence, the objective of this study was to examine that five distinct spectral conditions impact the survival, growth performance, pertinent enzyme activity, and gene expression levels associated with the growth and development of juvenile redclaw crayfish during a 30-day rearing period.The goal is to better understand the mechanisms underlying the physiological and biochemical responses of C. quadricarinatus juveniles to spectral regulation.

Experimental Operation
2.1.1.Experiment Preparation.After cleaning and disinfecting the aquariums used in the experiment, preaerated recirculating water was added.The dissolved oxygen in the water ranged from 7.25 to 8.66 mg/L, with a pH range of 7.80-8.27,while the temperature was consistently kept at 30 AE 0.5°C.We introduced nontoxic water conditioner tablets (provided by XianDe Biological Technology Co., Ltd., Guangzhou, China) into the filtration tank to improve the water quality [12].The aeration system underwent cleaning using water containing chlorine, and residual chlorine levels were measured in each aquarium after a 24-hr period [21].The experiment commenced when the residual chlorine level did not exceed 0.005 mg/L, at which point the experimental juveniles were introduced.Environmental conditions were kept consistent across all groups.
2.1.2.Experimental Design.The experiment utilized 510 healthy redclaw crayfish juveniles with an initial average weight of 0.03 AE 0.01 g and an initial average length of 1.06 AE 0.14 cm.These juveniles were sourced from Hengzhao Lanlong Aquaculture Co., Ltd.(Jiangmen, China) and acclimated for 1 week at the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences (Guangzhou, China).During this acclimation period, the juveniles were housed in aquariums measuring 130 cm × 52 cm × 60 cm each.The feeding regimen during this temporary rearing period mirrored that of the formal experiment.Black plastic film was applied to cover the outside of the tanks to prevent external light sources from entering.The water depth in the tanks was maintained at 45 cm, and we employed customdesigned lighting fixtures to control the light spectrum for each aquarium.These fixtures utilized 18 W LED lights from Yicai Optoelectronics Industry Co., Ltd.(Jiangmen, China), positioned 30 cm above the water surface to ensure an illumination intensity of 1,000 Lx below the water surface.We designed five experimental groups with different visible light spectra: red (wavelength: 615-650 nm), yellow (wavelength: 580-595 nm), white (wavelength: 450-465 nm), green (wavelength: 495-530 nm), and blue (wavelength: 450-480 nm).Each group consisted of three replicates, with each replicate containing 34 juveniles.The experiment lasted for 30 days, following a natural light cycle with light fixtures set to turn on at 7:00 AM and off at 7:00 PM, providing 12 hr of illumination daily.The juveniles were given commercial crayfish feed (Tongwei Feed Co., Ltd., Chengdu, China) twice a day, at 8:00 AM and 5:00 PM, consisting of crude protein (35%), crude fat (12%), and moisture content (9%).One-third of the  [21].Subsequently, nine redclaw crayfish were chosen from each group for hemolymph, eyestalks, and hepatopancreatic tissue collection.These samples were flashfrozen in liquid nitrogen and stored at −80°C for further analysis, including assessments of antioxidant and immune capabilities, melatonin, and cortisol levels.

Measurement of Melatonin and Cortisol.
We collected hemolymph and eyestalk samples to measure melatonin and cortisol levels.The measurement procedure was as follows: Hemolymph and eyestalk samples were mixed with phosphatebuffered saline (w : v = 1 : 9), and the mixture was homogenized in a glass mortar containing liquid nitrogen.Afterward, the mixture was centrifuged at 5,000x g for 5 min at 4°C, and the supernatant was collected for further analysis.For melatonin and cortisol measurements, standard curves were prepared using ELISA assay kits (Catalog No. YJ093369; YJ085236; Ji Chun Industrial Co., Ltd., Shanghai, China).The standards were prepared at six different concentrations: 0, 5, 10, 20, 40, and 80 pg/mL for melatonin and 0, 12.5, 25, 50, 100, and 200 ng/mL for cortisol.Subsequently, microplates were incubated in a 37°C incubator for 30 min, and unbound components were washed away with a washing solution.The bound melatonin or cortisol enzyme conjugates were then determined through a reaction with the substrate tetramethylbenzidine in the presence of peroxidase.Finally, the reaction was stopped by adding 100 μL of 0.5 M H 2 SO 4 at 37°C, and the absorbance was read at 450 nm using an absorbance Microplate Reader (SpectraMax 190, Molecular Devices, San Jose, CA, USA).Boston, MA, USA) with a one-way analysis of variance, followed by Tukey's post hoc test for multiple comparisons.Results are presented as mean AE SD.Differences were considered significant at P <0:05 and highly significant at P <0:01.

Impact of Different Light Spectra on Juvenile Crayfish
Growth and Survival Rates.After being reared under different spectral conditions for 30 days, the SGR of redclaw crayfish in the green-light group was markedly higher than that in the blue and red-light groups (P <0:05), while its weight gain rate and total length gain rate increase were markedly higher than those in the white and blue-light groups (P <0:05) and markedly higher than those in the red-light group (P <0:01).The survival rates of juvenile redclaw crayfish in the yellow and red-light groups were significantly higher than those in the blue and white-light groups (P <0:05) and markedly higher than that in the green-light group (P <0:01) (Table 2), with the highest survival rate observed in the yellow-light group at 62%.

Effects of Different Light Spectra on the Antioxidant
Capacity and Immune Parameters of Juvenile Crayfish.In the red-light group, the SOD activity in the hepatopancreas of juvenile crayfish was significantly higher than that in the blue, green, and white-light groups (P <0:05), with no significant differences observed among the other groups (P >0:05) (Figure 1(a)).In the green-light group, the MDA content was significantly higher than that in the red-light group (P <0:05) and markedly higher than that in the yellow-light group (P <0:01), while the blue and white-light groups were significantly higher than the yellow-light group (P <0:05), with no significant differences observed among the other groups (P >0:05) (Figure 1(b)).In the white-light group, the ACP activity of juveniles was significantly higher than that in the yellow-light group (P <0:05) and markedly higher than that in the blue-light group (P <0:01); the red and green-light groups were significantly higher than the blue-light group (P <0:05), with no significant differences observed among the other groups (P >0:05) (Figure 1(c)).Furthermore, no significant differences in LZM enzyme activity were observed between the various groups (P >0:05) (Figure 1(d)).

Effects of Different Light Spectra on Melatonin and
Cortisol Levels in Juvenile Crayfish.The melatonin levels of juveniles in the green and blue-light groups were significantly higher than those in the yellow-light group (P <0:01), and the melatonin level in the yellow-light group was notably higher than that in the red-light group (P <0:01).Notably, no significant differences were observed between the red and the white-light groups (P >0:05) (Figure 2(a)).In the green-light group, the cortisol levels were significantly higher than those in the yellow and red-light groups (P <0:05), with the redlight group significantly higher than the white-light group (P <0:05), while the green, blue, and yellow-light groups were markedly higher than the white-light group (P <0:01) (Figure 2(b)).

Effects of Different Light Spectra on Gene Expression
Levels in Juvenile Crayfish.The results of the ecdysone receptor (EcR) expression levels in juvenile redclaw crayfish under different spectral groups showed that the white-light group had significantly higher expression levels than the other four groups (P <0:01).Specifically, the green-light group had significantly higher expression levels than the yellow-light group (P <0:05), while the blue-light group had markedly higher expression levels than the yellow-light group (P <0:01), with no significant differences observed among the other groups (P >0:05) (Figure 3(a)).Comparisons of the retinoid X receptor (RXR) expression levels among juvenile redclaw crayfish in different spectral groups revealed that the red-light group had markedly higher expression levels than the other four groups (P <0:01), with no significant differences observed among the other four groups (P >0:05) (Figure 3(b)).The results of α-AMY expression levels in juvenile redclaw crayfish under different spectral groups showed that the expression levels in the yellow and green-light groups were significantly higher than those in the white and blue-light groups (P <0:05), and markedly higher than those in the red-light group (P <0:01), with no significant differences observed among the other groups (P >0:05) (Figure 3(c)).

Discussion
Spectra directly impact the growth, development, and survival of crustaceans [12,22].Different crustacean species exhibit varying degrees of photosensitivity [23,24], which may be closely related to their environmental niches during different developmental stages [25][26][27].Previously, Cheng et al. [20] found that within 14 days post-hatching, juvenile redclaw crayfish exhibited higher survival rates under red light and experienced accelerated growth rates under blue light but demonstrated decreased survival rates and slower growth under green light [20].However, findings from this investigation reveal that under yellow light spectrum cultivation conditions, the survival rate of juvenile redclaw crayfish  Aquaculture Research was highest after 30 days posthatching, with a higher SGR.Conversely, under green light spectrum cultivation regimes, juvenile redclaw crayfish had the lowest survival rate but achieved the highest final body weight and SGR.Despite no significant difference in survival rates between the red-light and yellow-light groups, the average weight gain rate and total length gain rate within the red-light group were notably lower compared to those observed in the yellow and green-light groups.This may be due to the different durations of the two experiments; this study lasted 16 days longer than the experiment designed by Cheng et al. [20].Consequently, the continuous development of the compound eyes of crustaceans and their corresponding changes in sensitivity to different spectra and internal response mechanisms may occur, and the optimal light conditions for growth may also change accordingly [27].This may also be due to differences in water transparency caused by variations in the plankton and organic matter content in the aquaculture water, resulting in different survival and development effects of juvenile C. quadricarinatus under the same light conditions, as demonstrated in the two studies, which warrants further investigations in the future.Nevertheless, it is important to mention that in additional studies concerning crustaceans, yellow light has been demonstrated to enhance the survival rate of Pacific white shrimp (L.vannamei) and expedite the growth of swimming crab (Portunus trituberculatus) juveniles [13,28].Conversely, red light has been found to be detrimental to the growth, development, and survival of giant freshwater prawn (M.rosenbergii) and Amazon river prawn (Macrobrachium amazonicum) [12,29].To a certain extent, these studies illustrate that the yellow light spectrum is advantageous for the growth and survival of larvae in most crustacean species, while red light negatively impacts the normal life of many species.
The antioxidant capacity plays a crucial role in determining the growth performance of aquatic organisms.In aquaculture, alterations in the spectrum can trigger oxidativereductive reactions in crustaceans, leading to fluctuations in the activity of antioxidant enzymes and the levels of oxidative products [12,19].Among these, SOD and MDA serve as vital markers for the antioxidant capacity of aquatic organisms [30,31].The findings from our experiment reveal that the SOD activity of juvenile redclaw crayfish under green, blue, and white light spectra is markedly lower  Aquaculture Research compared to that under the red light spectrum.SOD is a critical antioxidant enzyme that maintains the oxidative-reductive balance of the body by scavenging harmful superoxide anion radicals [32].The markedly higher SOD in the red-light group than that in the other groups indicates that individuals living under red light may be constantly facing oxidative-reductive imbalances, leading to excessive energy consumption, which affects their growth [19].MDA is the end product of lipid peroxidation in organisms, which can cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids and exhibits cytotoxicity.Its content reflects the oxidative level and stress status within the organism [33].In this experiment, the MDA levels in the yellow-light group are lower compared to those in the green, blue, and white-light groups.This suggests that juvenile redclaw crayfish living under yellow light conditions maintain a more favorable oxidative-reductive balance and experience lower stress levels.A series of reports have also indicated that the reduction in SOD activity and the elevation in MDA content is detrimental to the cellular oxidative-reductive balance in crustaceans and have negative effects on normal physiological metabolism [19,34], which is also reflected in the enzyme activity and growth results of this study.ACP is an important nonspecific immune enzyme in crustaceans.In the immune response, it forms a hydrolytic enzyme system to degrade and eliminate foreign substances invading the body, thereby achieving immune defense [35].It was found that the ACP activity levels in the white light spectrum cultivation environment were the highest among the groups, indicating that under different light spectrum cultivation conditions, juvenile redclaw crayfish produced a series of stress responses.Furthermore, overall, yellow light may be beneficial to the oxidativereductive balance of cells in juvenile redclaw crayfish, positively impacting physiological metabolism by avoiding damage to the immune system and energy consumption.
Melatonin is a common and conserved biogenic amine that is synthesized and secreted in organisms ranging from unicellular organisms to mammals.In invertebrates, light exposure can transmit photic information through the visual ganglia, regulating melatonin synthesis and secretion [36,37].Melatonin holds considerable importance in upholding the internal environment of crustaceans, regulating diverse biological functions such as reproduction, molting, limb regeneration, antioxidative stress, and plasma glucose homeostasis [38].For instance, the cleaner shrimp (Lysmata amboinensis), reared under red light, perceives the red-light environment as darkness, leading to a significant increase in melatonin levels compared to blue light conditions, thus affecting its biological rhythms [39].On the contrary, our study findings reveal that juvenile crayfish reared under blue and green light spectra have the highest melatonin levels, while those reared under red light have the lowest melatonin levels.Similar results have been found in studies on fish melatonin, where Nile tilapia (O.niloticus) exhibited decreased melatonin levels under red light spectrum cultivation conditions, indicating a weaker perception of red light and, consequently, poorer growth performance [40].Hence, different light spectra may have species-specific effects on melatonin secretion and synthesis.In juvenile crayfish reared under red light, melatonin secretion and synthesis may be inhibited, indicating a weaker sensitivity to red light, which in turn results in poor growth performance under red light.
Cortisol is a vital and conserved stress hormone, and it is considered one of the indicators reflecting the stress status of animals, including crustaceans.Elevated plasma cortisol levels, mediated by stress, can reduce the energy required for muscle growth in animals, including crustaceans, leading to growth inhibition and decreased survival rates [41][42][43].Changes in light conditions represent a stressor for aquatic animals, triggering stress responses and inducing cortisol synthesis and release [19,[44][45][46].Previous studies have shown that juvenile mud crabs (S. paramamosain) exhibit elevated cortisol levels under purple light spectrum cultivation conditions, indicating intensified stress, resulting in decreased survival rates and slower growth rates [19].However, our study revealed that juvenile crayfish reared under green-light conditions had elevated cortisol levels, decreased survival rates, the fastest growth rate, and the largest final body weight.Thus, we hypothesized that elevated cortisol levels do not necessarily inhibit the growth of aquatic animals and may even promote growth.Villamizar et al. [45] reported that zebrafish (Danio rerio) raised under blue or violet light conditions exhibited increased cortisol levels and higher stress; however, they had high survival rates and significantly accelerated growth rates [45].Zou et al. [46] reported that olive flounder larvae (Palichthys olivaceus) raised under green-light conditions had the highest cortisol levels, markedly surpassing those in the white-light group.Although the larvae experienced increased stress, they showed accelerated growth.However, the survival of larvae reared under white and green light did not significantly differ [46].In summary, our results suggest that changes in light spectra can regulate cortisol levels in juvenile crayfish.Elevated cortisol levels in crayfish raised under green-light conditions accelerated growth and reduced survival rates.An appropriate increase in cortisol levels in the organism can promote the growth of certain aquatic animals; however, excessively high cortisol levels can inhibit both survival and growth.
EcR, RXR, and α-AMY are associated with the molting and digestive absorption capacity of crustaceans and serve as indicators of their growth status [47][48][49].EcR and RXR form heterodimers that promote development, reproduction, and molting in organisms [50].α-AMY is involved in energy production by hydrolyzing α-1,4-glycosidic bonds in polysaccharides such as starch, branched starch, and glycogen, subsequently affecting nutrient absorption and metabolism in the organism [51].Reports indicate that light conditions can influence the molting frequency, digestive absorption, and gene expression related to these processes in crustaceans, thus affecting their growth performance [19,52].In this study, the group reared under red light exhibited the highest RXR expression and the lowest α-AMY expression but had the slowest growth rate.In contrast, the group exposed to yellow light exhibited the highest expression of the α-AMY and the lowest expression of the EcR, yet it displayed the fastest growth rate.Moreover, the expression levels of the

Aquaculture Research
EcR and α-AMY were notably elevated in the white and green-light groups, surpassing those in the other four groups (P <0:01).These findings imply that the spectrum not only induces alterations in the gene expression levels associated with molting and digestion in redclaw crayfish juveniles but also directly affects their growth performance.We speculate that changes in the spectrum may lead to variations in the expression levels of associated genes, thereby affecting the growth rate of juveniles.Specifically, the yellow light spectrum increases the expression level of the α-AMY, thereby promoting juvenile growth.This result suggests that the yellow light spectrum may have a positive impact on the intensive farming of redclaw crayfish juveniles.

Conclusions
This study examined the impacts of five distinct light spectra on the survival, growth, and pertinent physiological indicators of juvenile redclaw crayfish.The findings demonstrate that the survival rate of juvenile redclaw crayfish is highest in the yellow and red-light groups and lowest in the green-light group.Regarding antioxidant enzyme activity, juveniles in the yellow-light group exhibited elevated levels of SOD and ACP activity, along with the lowest MDA content.Furthermore, in terms of hormones, melatonin levels in juvenile redclaw crayfish were markedly higher in the green and blue-light groups compared to the other three groups (red, white, and yellow light) (P <0:01), while cortisol levels in the green-light group were markedly higher than that in the other three groups (red, white, and yellow-light groups) (P <0:05).Lastly, regarding gene expression levels, the expression of RXR, α-AMY, and EcR was highest in the red, yellow, green, and white-light groups, respectively.These results provide valuable technical support for optimizing and enhancing the factory farming of juvenile redclaw crayfish.

Table 1 .
Real-time qPCR was performed using the Step One RNA Extraction, cDNA Synthesis, and Quantitative PCR (qPCR) Analysis.Total RNA in hepatopancreatic tissues was extracted using Trizol Reagent (Invitrogen, Waltham, MA, USA).RNA integrity was evaluated via 1% agarose gel electrophoresis, and the concentration and purity were determined using a Nanodrop-2000 microspectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).To remove genomic DNA, 1 µg of total RNA underwent DNase I digestion with an enzyme from New England Biolabs (Ipswich, MA, USA) for 15 min.Subsequently, first-strand cDNA synthesis was conducted using the M-MLV reverse transcriptase reagent kit (Invitrogen, Waltham, MA, USA) following the manufacturer's instructions.Primer sequences for target and reference genes utilized in this study are provided in s, extension at 72°C for 30 s, and a final extension step at 72°C for 10 min.Melt curve analysis involved subjecting the samples to 95°C for 5 s, 60°C for 30 s, and 95°C for 15 s to obtain dissociation curves.2.4.Statistical Analysis.The experimental data were analyzed using GraphPad Prism version 8.0.2 (GraphPad Software,

TABLE 1 :
Primers used for qPCR in this study.

TABLE 2 :
Corresponding growth index under different spectra.