DOI: 10.1111/are.13897 ORIGINAL ARTICLE Freeze‐dried forms of mosquito larvae for feeding of Siamese fighting fish (Betta splendens Regan, 1910) Karun Thongprajukaew1 | Sittikorn Pettawee1 | Somkiat Muangthong1 | Suktianchai Saekhow1 | Wutiporn Phromkunthong2 1 Department of Applied Science, Faculty of Science, Prince of Songkla University, Songkhla, Thailand Abstract Live diets are preferably used for rearing Siamese fighting fish (Betta splendens 2 Department of Aquatic Science, Faculty of Natural Resources, Prince of Songkla University, Songkhla, Thailand Correspondence Karun Thongprajukaew, Department of Applied Science, Faculty of Science, Prince of Songkla University, Songkhla, Thailand. Email: karun.t@psu.ac.th Regan, 1910) since they provide superior growth over the dry feed. In the current study, three different preparations of mosquito larvae were prepared (frozen at −20°C, F–20; freeze‐dried and kept at 4°C, FD4; freeze‐dried and kept at ambient temperature, FDAT) and were individually fed to 1‐month‐old red male fighting fish (1.18 ± 0.01 g initial body weight) over 6 weeks duration. At the end of experiment, there were no significant differences in growth performance and feed utilization across three dietary treatments (p ˃ 0.05). Specific activity of lipase was significantly lower in fish fed FD4 and FDAT than with the F–20 diet, while no differences in other enzymes were observed. The fish fed with FDAT diet significantly increased in viscerosomatic index relative to F–20 and FD4 treatments. Significant improvements in skin redness and flesh quality (RNA and RNA/protein ratio) were observed in the fish fed with FDAT diet relative to the other treatments. This preferred FDAT treatment also maintained the carcass composition. Analysis of digestive enzymes in FDAT mosquito larvae demonstrated the presence of protein‐, carbohydrate‐, and lipid‐digesting enzymes after 1 month of storage. The findings from our experiments indicate that the freeze‐dried form (FDAT) of mosquito larvae is suitable for rearing Siamese fighting fish. However, effective preparation protocol and appropriate storage times should be further studied. KEYWORDS colour, digestive enzyme, flesh quality, freeze, freeze‐dry, live diet 1 | INTRODUCTION Appoloni, Fernandes, & Millan, 2016; Thongprajukaew, Kovitvadhi, The Siamese fighting fish or betta (Betta splendens Regan, 1910) is a Kovitvadhi, Somsueb, & Rungruangsak‐Torrissen, 2011) and several popular aquarium fish that is commercially produced and exported commercial throughout the world. Selective breeding of this species provides Nuangsaeng, Sriwattanarothai, & Panijipan, 2009). However, live various brilliant colours, tail types, and fin shapes. However, only the diets are commonly used for rearing this species throughout its life male fish are preferred by fish culturists, since the dimorphism of span sex supports its ornamentation differing from the females (Thongpra- Thongprajukaew et al., 2014). Under the natural feeding habits, live jukaew, Kovitvadhi, Kovitvadhi, & Rungruangsak‐Torrissen, 2014). diets of the fighting fish include zooplankton, tubifex worms, Daph- Recently, artificial pellet diets for this species have been formulated nia, mosquito larvae, and small aquatic insects (James & Sampath, (James & Sampath, 2003; Mandal et al., 2010; Sipaúba‐Tavares, 2003; Rainboth, 1996). Aquaculture Research. 2018;1–8. pellet (Goldstein, wileyonlinelibrary.com/journal/are diets 2004; are Lim, currently Dhert, available & (Monvises, Sorgeloos, 2003; © 2018 John Wiley & Sons Ltd | 1 2 | Mosquito larvae (Culex sp.) are the most common live diet for rearing Siamese fighting fish (Thongprajukaew, Kovitvadhi, Engkagul, & Rungruangsak‐Torrissen, 2010a, 2010b ). They contain high THONGPRAJUKAEW ET AL. T A B L E 1 The proximate chemical composition (g/kg of fresh weight) of fresh mosquito larvae. Data are mean of duplicate analyses amount of proteins and lipids that are notably suitable for feeding Chemical component Content carnivorous fish, like Siamese fighting fish (Ghosh, Bhattacharjee, Dry matter 142.9 ± 4.2 Ganguly, Mondal, & Chandra, 2004; Thongprajukaew, Kovitvadhi, Moisture 857.1 ± 6.4 Kovitvadhi, Engkagul, & Rungruangsak‐Torrissen, 2013). Generally, Crude protein 84.8 ± 1.2 live diets contain exogenous enzymes that enhance growth and feed Crude lipid 26.4 ± 0.6 utilization of the reared animals (Cahu & Infante, 2001; Kolkovski, Crude ash 13.8 ± 0.5 2001; Munilla‐Moran, Stark, & Barbour, 1990). However, preparation of the diets requires manpower and space, and is time‐consuming. In Note. Data are expressed as mean ± SEM. addition, it is difficult to preserve the diet for a long time without the use of a deep freezer or during transportation. Live diets com- individually acclimatized in cylindrical plastic beakers (8 cm diame- mercially dried by the private sector are distributed worldwide, but ter × 11.5 cm height) containing 250 ml water, for 10 days. Under published scientific information on them remains unavailable. acclimatization conditions, all the fish were fed ad libitum with the Various drying methods have been used for producing a dried F–20 diet twice a day (08.00 and 17.00 hr), under 12‐hr light/12‐hr live diet (Caňavate & Fernández‐Díazde, 2001; de Verga & Böhm, dark. Subsequently, the fish screened for size (1.18 ± 0.01 g initial 1992; Kasiri, Farahi, & Sudagar, 2012). Freeze‐drying is a process for body weight) were distributed individually into new cylindrical plastic removing ice from a material through sublimation and desorption of beakers containing the same water volume as above. The 45 experi- bound water molecules. This technique is used to prepare ingredi- mental units (45 beakers) were set‐up for the three treatments ents or other additives for food and feed productions (de Verga & (F–20, FD4 or FDAT) with 15 fish (n = 15) in each as replicates. All Böhm, 1992; Kubitza & Lovshin, 1997; Moayyedi et al., 2018). It the fish were fed with prepared mosquito larvae for 6 weeks. The causes less damage to substances that are sensitive to heat, such as F–20 was defrosted and presoaked with the water from rearing sys- enzymes, than other processes that incorporate heat treatment with tem to adjust temperature prior to feeding. The FD4 and FDAT drying (de Verga & Böhm, 1992; Fellows, 2016). Therefore, testing forms were gently mixed with the water from rearing system to alternative preparations of mosquito larvae to improve the perfor- increase the moisture content and improve the softness prior to mance quality of reared fish, and perhaps to make the prepared lar- feeding. The water was renewed by dechlorinated stock every day, vae easy‐to‐digest, were the aims of this study. Findings from the and the quality within the studied period was pH 7.06 ± 0.02 (min– current study could be applied in feeding management for rearing max = 6.81–7.32), temperature 27.44 ± 0.04°C (min–max = 27−28° Siamese fighting fish. The processing for preparation of the live diet, C), without the use of a deep freezer, could be suitable for other live 5.26 mg/L). Survival of reared fish was recorded daily. Uneaten diets for the Siamese fighting fish. excess diet was collected 30 min after feeding and dried at 60°C and dissolved oxygen 3.60 ± 0.17 mg/L (min–max = 3.00– until constant weight; the determined weight was used to calculate 2 | MATERIALS AND METHODS 2.1 | Preparation of mosquito larvae the feed conversion ratio (FCR), and protein efficiency ratio (PER). At the end of the experiment, the fish were unfed for 12 hr and then were euthanized by clove oil. Body weight (BW), total length and standard length were recorded prior to measuring the colour Fresh mosquito larvae (Culex sp.) were purchased from a local market coordinates (n = 15 per treatment). Subsequently, visceral organs in Songkhla province of Thailand. The larvae were rinsed with tap and the flesh were collected from the same fish sample (n = 10 per water and then killed by freezing in ice. The proximate composition treatment) while the remaining fish were used for carcass composi- of the fresh mosquito larvae is shown in Table 1. They were soaked tion analysis (n = 5 per treatment). The growth performance and in 5 mg/L of KMnO4 for 30 min (Carpenter, Mashima, & Rupiper, feed utilization parameters were calculated as described below: Survival ð%Þ 2001; Noga, 1996), rinsed with distilled water three times, and fil- ¼ ½Final fish number=initial fish numberŠ  100 tered to remove water. One portion was then kept at −20°C (F–20) until feeding. Two additional equal portions were then freeze‐dried
 Condition factor CF; g cm 3 h i ¼ Live body weight ðgÞ=body length ðcmÞ3  100 (Flexidry; SP Scientific, PA, USA) for 48 hr and then kept at either 4°C (FD4) or ambient temperature, 29–31°C (FDAT). The three preparations were fed to the fish within 1 month after preparation.   Specific growth rate SGR; % BW day 1 2.2 | Feeding trial ¼ ½ðln Wt ln W0 Þ=ðt t0 ފ  100 One‐month‐old red male Siamese fighting fish were purchased from where Wt = mean weight (g) at day t, W0 = mean weight (g) at day a local farm in Nakonpathom province of Thailand. They were t0. THONGPRAJUKAEW | ET AL. 3 continuous values of skin colour recorded were lightness (L*, scale Viscerosomatic index ðVSI; %Þ   Wet weight of visceral organ ðgÞ  100 ¼ Wet body weight ðgÞ from 0 to 100 represent a colour spectrum from black to perfect white), redness (a*, scale describes redness when positive, grey when zero, and greenness when negative), yellowness (b*, scale describes   FCR g feed g gain 1 yellowness when positive, grey when zero, and blueness when nega- ¼ Dry feed consumed ðgÞ=wet weight gain ðgÞ tive), hue (h*, arctan b*/a*), and chroma (C*, (a*2 + b*2)1/2).   PER g gain g protein 1 2.5 | Flesh quality ¼ Wet weight gain ðgÞ=protein intake ðgÞ 2.3 | Determination of digestive enzyme activity 2.5.1 | Protein synthesis capacity 2.3.1 | Extraction of digestive enzymes The scales and skin were carefully removed prior to sampling the The frozen whole visceral organs were extracted in 0.2 M Na2HPO4‐ described in Rungruangsak‐Torrissen (2007). The extinction coeffi- NaH2PO4 buffer (pH 8) at a ratio of 1:10 (w/v), using a microhomoge- cients for calculating RNA and protein were E260 = 40 μg RNA/ml nizer and E280 = 2.1 mg protein/ml respectively. (THP‐220; Omni International, Kennesaw, flesh. The concentrations of RNA and protein were determined as GA, USA). Centrifugation was performed at 15,000 g for 30 min at 4°C prior to collecting the supernatant. The aliquots were used for quantification of protein (Lowry, Rosenbrough, Farr, & Randall, 1951) against the 2.5.2 | Flesh protein linear range of bovine serum albumin, while the remaining portions Ten milligrams of thawed fish flesh (at 0°C for 1 hr) was placed in were kept at –20°C until use for determination of digestive enzymes. an aluminum pan, sealed, allowed to equilibrate at room temperature, and then heated from 20 to 100°C at a rate of 10°C/min 2.3.2 | Digestive enzyme assay against an empty pan, using a differential scanning calorimeter The optimal conditions of assaying pepsin (pH 2 at 40°C), trypsin myosin, actin, and sarcoplasmic proteins, were identified from the (DSC7; Perkin Elmer, Waltham, MA, USA). Flesh proteins, including (pH 8 at 50°C), chymotrypsin (pH 8 at 50°C), amylase (pH 8 at thermal charcateristics (To, onset temperature; Td, denaturation tem- 50°C), and lipase (pH 8 at 40°C) were chosen from a previous study perature; Tc, conclusion temperature) as described in previous in the same species (Thongprajukaew, Kovitvadhi, Engkagul, & Run- reports (Thongprajukaew et al., 2015). Enthalpy (ΔH) of each flesh gruangsak‐Torrissen, 2010a, 2010b ). Activity of pepsin was deter- protein was calculated from the peak areas. mined based on the method of Worthington (1993) using haemoglobin as substrate. One unit (U) of activity was defined as 1.0 increase in absorbance at 280 nm. Activities of trypsin and chy- 2.6 | Proximate chemical composition of carcass motrypsin were determined as described by Rungruangsak‐Torrissen The moisture and ash contents of fish whole body were analysed by Moss Andresen Berg and Waagbo (2006), using N‐α‐benzoyl‐Arg‐p‐ drying at 105°C for 24 hr and incinerating at 600°C for 2 hr respec- nitroanilide (BAPNA) and N‐succinyl‐Ala‐Ala‐Pro‐Phe‐p‐nitroanilide tively (AOAC, 2005). Crude proteins from small samples were (SAPNA) as substrates respectively. Amount of liberated p‐ni- extracted with monophasic solution of phenol and guanidine isothio- troanilide at 410 nm was used to calculate both these enzyme activi- cyanate (TRIzol® reagent; Invitrogen, Carlsbad, CA, USA), and esti- ties. Activity of lipase was assayed according to the method of mated as described in Rungruangsak‐Torrissen (2007). Determination Winkler and Stuckmann (1979), using p‐nitrophenyl palmitate (p‐ of lipid was performed by extraction with ethyl acetate as described NPP) as substrate. Linear range for p‐nitrophenol standard at by Supannapong et al. (2008). 410 nm was used to quantify the enzyme activity. Activity of amylase was assayed as described by Areekijseree et al. (2004), using soluble starch as the substrate. The liberated product was measured spectrophotometrically at 540 nm against the linear range for standard maltose. 2.7 | Determination of digestive enzymes in FDAT diet After storage of the FDAT mosquito larvae for one month, the digestive enzymes were extracted and then analysed for specific 2.4 | Colour measurement of skin activities as described in Section 2.3. All analyses were performed at pH 7 and 25°C. The MiniScan EZ (Hunter Associates Laboratory, Reston VA, USA), a portable reflected‐colour spectrophotometer, was calibrated to white and black standards before measuring the reflected colour of sam- 2.8 | Statistical analysis ples. The euthanized fish was carefully cleaned by soft blotting paper The data were recorded in Microsoft Excel (Microsoft Corporation, and then the colour was measured from the middle part of body. The Redmond, WA, USA) and then subjected to analysis in SPSS Version 4 | THONGPRAJUKAEW ET AL. 14 (SPSS, Chicago, IL, USA). The data are here expressed as mean ± and amylase to trypsin (A/T ratio) (Table 3). The only difference SEM. Arc sine transformation was applied to percentages prior to observed was for lipase specific activity; fish fed FD4 or FDAT had analysis. The normality and homogeneity of variance were checked significantly lower enzyme activities than with the F–20 treatment. before performing one‐way ANOVA. The comparisons of means in the statistical analyses were carried out using Duncan’s multiple range test with significance threshold p ˂ 0.05. 3.3 | Skin colouration Significant improvement in redness (a*) was observed in the fish fed with FDAT diet relative to the F–20 treatment; an intermediate 3 | RESULTS value was obtained for the fish fed with FD4 diet (Table 4). The other colour coordinates were similar across the three dietary 3.1 | Survival, growth performance, and feed utilization treatments. No mortality of reared fish was observed within the studied period (Table 2). All growth performances and feed utilization parameters 3.4 | Flesh quality did not differ across the three dietary treatments (p ˃ 0.05, except The highest RNA concentrations and RNA/protein ratio were that VSI significantly increased in the fish fed with FDAT diet rela- achieved by the fish fed with the FDAT diet, followed by FD4 tive to F–20 and FD4 treatments) (Table 2). (Table 5). The lowest RNA concentration was observed in the fish 3.2 | Specific activities of digestive enzymes did not differ from the FD4 group. Regarding muscle protein con- There were no significant differences in specific activities of pepsin, teins, and their total did not significantly differ across the three trypsin, chymotrypsin, and amylase across the three dietary treat- dietary treatments (Table 5). fed with the F–20 diet. However, the RNA/protein ratio in this group tent, the amounts of myosin, actin, actin/myosin, sarcoplasmic pro- ments, nor in the activity ratios trypsin to chymotrypsin (T/C ratio) 3.5 | Carcass composition T A B L E 2 Survival, growth performance, and feed utilization of male Siamese fighting fish fed with various forms of mosquito larvae. The observed parameters were recorded at the end of 6‐week experiment Parameter F–20 FD4 FDAT p‐ value Survival 100 100 100 – IBW (g) 1.17 ± 0.03 1.18 ± 0.03 1.18 ± 0.04 0.355 FBW (g) 2.23 ± 0.13 2.05 ± 0.06 1.97 ± 0.05 0.101 WG (g) 0.92 ± 0.04 0.92 ± 0.04 0.86 ± 0.04 0.355 Total length (cm) 8.08 ± 0.09 7.91 ± 0.10 7.80 ± 0.12 0.341 Standard length (cm) 3.98 ± 0.04 3.97 ± 0.04 3.90 ± 0.05 0.506 CF (g/cm3) 3.42 ± 0.22 3.52 ± 0.12 3.45 ± 0.20 0.923 SGR (% BW/day) 1.28 ± 0.05 1.24 ± 0.31 1.15 ± 0.07 b b a Varying the forms of mosquito larvae did not affect carcass moisture, crude protein, crude lipid, or ash in the Siamese fighting fish (Table 5). 3.6 | Digestive enzyme profiles in FDAT diet Protein‐, carbohydrate‐, and lipid‐digesting enzymes were detected in FDAT form of mosquito larvae after 1 month of storage. The specific activities of pepsin, trypsin, chymotrypsin, amylase, and lipase were 1.47 mU/mg protein, 23.34 mU/mg protein, 26.53 mU/ mg protein, 8.24 U/mg protein, and 22.59 mU/mg protein, on average respectively. 0.252 4 | DISCUSSION VSI (%) 6.86 ± 0.22 6.78 ± 0.27 7.88 ± 0.21 0.003 No differences in growth performance and feed utilization were FCR (g feed/g gain) 1.10 ± 0.07 1.50 ± 0.19 1.54 ± 0.13 0.143 observed across the three dietary treatments, suggesting that all the PER (g gain/g protein) 1.55 ± 0.10 1.23 ± 0.18 1.15 ± 0.08 0.140 fish have high ability to utilize the different preparations of mosquito Note. F–20, frozen and kept at −20°C; FD4, freeze‐dried and kept at 4°C; FDAT, freeze‐dried and kept at ambient temperature; IBW, initial body weight; FBW, final body weight; WG, weight gain; CF, condition factor; SGR, specific growth rate; VSI, viscerosomatic index; FCR, feed conversion ratio; PER, protein efficiency ratio. Data are expressed as mean ± SEM (n = 15). Differences between means were tested with Duncan's multiple range test. Different superscripts in the same row indicate a significant difference (p < 0.05). larvae. The growth and feed utilization from the current study were relatively superior but within the ranges from aquaculture of this species (Mandal et al., 2010; Saekhow, Thongprajukaew, Phromkunthong, & Sae‐khoo, 2018; Sipaúba‐Tavares et al., 2016). Chai, Ji, Han, Dai, and Wang (2013) reported increased VSI in fish fed with diets having optimized protein and lipid contents, relative to other less suitable diet formulations. Other studies have also noted the VSI increasing with dietary lipid levels (Company, Calduch‐Giner, Kaushik, & Perez‐Sanchez, 1999; Yildiz, Sener, & Timur, 2006). On the other hand, the VSI was unaffected by changes in diet, even THONGPRAJUKAEW | ET AL. 5 T A B L E 3 Specific activities of digestive enzymes in male Siamese fighting fish fed with various forms of mosquito larvae. The observed parameters were recorded at the end of 6‐week experiment Digestive enzyme F–20 Pepsin (U/mg protein) FD4 0.20 ± 0.01 Trypsin (mU/mg protein) Chymotrypsin (mU/mg protein) Lipase (U/mg protein) p‐value 0.24 ± 0.01 0.050 0.574 58.50 ± 5.85 50.10 ± 5.46 52.24 ± 5.73 290.99 ± 14.28 282.27 ± 12.48 274.53 ± 11.60 0.677 b b <0.001 1.28 ± 0.06 Amylase (U/mg protein) FDAT 0.26 ± 0.02 a 0.71 ± 0.03 15.50 ± 0.75 12.26 ± 0.95 0.56 ± 0.06 14.07 ± 0.90 0.052 T/C ratio 0.23 ± 0.01 0.18 ± 0.01 0.21 ± 0.05 0.107 A/T ratio 289.92 ± 15.38 321.64 ± 42.32 261.54 ± 18.00 0.301 Note. F–20, frozen and kept at −20°C; FD4, freeze‐dried and kept at 4°C; FDAT, freeze‐dried and kept at ambient temperature; T/C ratio, activity ratio of trypsin to chymotrypsin; A/T ratio, activity ratio of amylase to trypsin. Data are expressed as mean ± SEM (n = 10). Differences between means were tested with Duncan's multiple range test. Different superscripts in the same row indicate a significant difference (p < 0.05). T A B L E 4 Colour parameters of male Siamese fighting fish fed with various forms of mosquito larvae. The observed parameters were recorded at the end of 6‐week experiment Colour parameter F–20 FD4 L* 18.36 ± 0.71 a* These enzymes contribute to digestion and assimilation of the dietary components, especially in the fish larvae (Kolkovski, 2001). Munilla‐Moran et al. (1990) reported 43%–60% and 15%–27% of exogenous proteases and amylase, respectively, in other live diets FDAT p‐ value 17.37 ± 0.50 18.28 ± 0.86 0.519 from fish. No significant change in digestive enzyme activities of Sia- 11.88 ± 0.71b 13.70 ± 0.57ab 14.21 ± 0.63a 0.033 mese fighting fish in the current study suggests negligible effect b* 6.00 ± 0.54 6.93 ± 0.44 6.59 ± 0.61 0.764 from the alternative forms of mosquito larvae. This might be due to h* 24.89 ± 1.58 26.56 ± 0.84 24.34 ± 1.71 0.527 the age of the fish in the current study being one‐month‐old; com- C* 14.08 ± 0.96 14.53 ± 1.19 15.74 ± 0.76 0.474 plete development of digestive system of this species occurs around Note. F–20, frozen and kept at −20°C; FD4, freeze‐dried and kept at 4°C; FDAT, freeze‐dried and kept at ambient temperature; L*, lightness; a*, redness; b*, yellowness; h*, hue; C*, chroma. Data are expressed as mean ± SEM (n = 15). Differences between means were tested with Duncan's multiple range test. Different superscripts in the same row indicate a significant difference (p < 0.05). (rotifers, Artemia, and copepods) relative to the endogenous enzymes 10 days after hatching (Thongprajukaew et al., 2013). Our observations match the findings of Kurokawa, Shiraishi, and Suzuki (1998) and of Pedersen and Hjelmeland (1988). They reported approximately 0.5%–1.0% protease or trypsin activities, respectively, from live diets relative to the activity from fish. However, significantly decreased lipase activity was observed in fish fed with the freeze‐ dried forms of mosquito larvae. This suggests that the fish might physiologically adapt by changing their digestive lipase in response though the experimental diets contained different lipid levels to dietary lipids (Chang, Niu, Jia, Li, & Xu, 2018). Therefore, (Barnes, Brown, & Rosentrater, 2012). Increased VSI in the fish fed increased VSI in FDAT treatment due to insufficient lipase is possi- with FDAT diet form in the current study might be linked with the ble, since some fish store lipid in peritoneal cavity and the used lipid deposition in visceral organs, but not affecting the whole body specimen for digestive enzyme extraction in the current study was lipid. Significant changes of this value might also be associated with whole viscera. Further studies on the lipid catabolism in fish, or fatty body energy storage (Goede & Barton, 1990), which captures the acid profile in the prepared mosquito larvae, should be considered. lipid produced at one time for later release of energy. This character- However, this change had no negative effects on the overall feed istic would provide the energy needed during extended nonfeeding utilization parameters (FCR and PER) or on carcass lipid. periods, such as starvation or mating. One of the most commercially important traits of the male Sia- Live diets contain exogenous enzymes that can enhance growth mese fighting fish is the colouration of body and fins. In addition to and feed utilization of reared animals (Cahu & Infante, 2001; Kolk- the requirements by consumers, these characteristics also affect the ovski, 2001; Munilla‐Moran et al., 1990). However, little is known mating performance (Blakeslee, McRobert, Brown, & Clotfelter, about the enzyme activities in freeze‐dried live diets. In the current 2009; Clotfelter, Ardia, & Mcgraw, 2007). Skin redness in B. splen- study, relatively high level of protein‐, carbohydrate‐, and lipid‐diges- dens is controlled by carotenoids (Clotfelter et al., 2007), and also tive enzymes were observed in the FDAT form. This is in agreement probably by melanines, pterediums, and purines, as in other orna- with some supporting evidences of high proteolytic enzyme activity mental fish (Kop & Durmaz, 2007). Within the chromatophores, in invertebrates constituting fish larval diets, including rotifers, mol- change of colour is in accordance with the habitat, the environment, luscs, Artemia, and others (Dabrowski & Glogowski, 1977a, 1977b). and stimuli (Sugimoto, 2002). In the current study, significant 6 | THONGPRAJUKAEW ET AL. T A B L E 5 The muscle quality and carcass composition of male Siamese fighting fish fed with various forms of mosquito larvae. The observed parameters were recorded at the end of 6‐week experiment Parameter F–20 FD4 FDAT p‐value Muscle (on wet weight) RNA (µg/g) Protein (mg/g) 1,739 ± 65c 1,987 ± 67b 2,511 ± 49a 183.39 ± 14.02 183.28 ± 11.75 192.35 ± 12.77 b b RNA/protein ratio (µg/mg) 8.78 ± 0.94 ΔH Myosin (J/g) 0.54 ± 0.09 8.79 ± 1.23 13.39 ± 1.00 0.54 ± 0.06 <0.001 a 0.51 ± 0.11 0.834 0.015 0.958 ΔH Actin (J/g) 0.30 ± 0.02 0.37 ± 0.05 0.26 ± 0.02 0.115 ΔH Actin/myosin 0.52 ± 0.04 0.60 ± 0.13 0.59 ± 0.11 0.798 ΔH Sarcoplasmic protein (J/g) 0.25 ± 0.03 0.23 ± 0.04 0.28 ± 0.07 0.480 ΣΔH (J/g) 1.10 ± 0.12 0.14 ± 0.06 1.00 ± 0.11 0.928 Carcass (g/kg on wet weight) Moisture 702.2 ± 8.6 707.8 ± 6.3 710.3 ± 12.0 0.824 Crude protein 103.4 ± 9.6 115.3 ± 9.1 104.8 ± 9.1 0.609 Crude lipid 28.2 ± 6.8 23.6 ± 2.7 26.0 ± 4.4 0.805 Crude ash 55.9 ± 2.5 52.7 ± 1.9 53.5 ± 1.5 0.515 Note. F–20, frozen and kept at −20°C; FD4, freeze‐dried and kept at 4°C; FDAT, freeze‐dried and kept at ambient temperature; ΔH, enthalpy. Data are expressed as mean ± SEM (n = 5). Differences between means were tested with Duncan's multiple range test. Different superscripts in the same row indicate a significant difference (p < 0.05). improvement in a* was observed in fish fed with FDAT diet relative suggest no negative effects on infrastructure of the flesh, or on to F–20, while there were no differences in L*, b*, h* and C* physiological exercise (Thongprajukaew et al., 2015). However, there between the treatments. This indicates that the preferred form of were no differences in carcass composition across the three dietary preparation (FDAT) can maintain available pigments better than F– treatments. This indicates no negative effects, since all the fish can 20. This might lead to a selection advantage in the body colour, maintain the proximate compositions with any of the alternative since the female fish prefer to associate with the vermilion males preparation forms of mosquito larvae. over those that are pale red (Blakeslee et al., 2009). However, the In conclusion, growth performance, feed utilization, and carcass amounts of carotenoids or total pigments in the diets were not composition were similar across the three dietary forms of mos- examined in the current study. A permanent loss of pigments in F– quito larvae. Adjustments in lipid‐digesting enzymes were observed 20 treatment might occur during repeated freeze–thaw cycles (Rah- in fish fed with different preparation forms of the mosquito larvae; man, Hossain, Rahman, Hashem, & Oh, 2014), since some amount of so further studies on lipid utilization would be warranted. However, the diet remains and is used again in a subsequent meal. On the some improvements in skin colouration and flesh quality were other hand, decreased moisture content in freeze‐dried mosquito lar- observed in the fish fed with freeze‐dried form of mosquito larvae vae reduced the water activity; decreasing hydrolytic rates, and that was stored at ambient temperature. This preferred preparation maintaining the amount of highly sensitive molecules such as pig- form of mosquito larvae as diet contained relatively high level of ments. However, long‐term storage can cause significant loss of pig- digestive enzymes, is easy to prepare, and easy to use on feeding ments in freeze‐dried diet (Syamaladevi, Sablani, Tang, Powers, & the fish. Optimization of the preparation conditions and assessment Swanson, 2011). of the shelf life (appropriate storage time) should be further RNA concentration and RNA/protein ratio have been used as investigated. indirect indices of fish condition and growth (Hart & Reynolds, 2002; Thongprajukaew et al., 2015). Some improvements in flesh quality were observed with the preferred diet: the protein synthesis ACKNOWLEDGEMENTS capacity (RNA concentration), and protein turnover rate (RNA/pro- We acknowledge Assoc. Prof. Dr. Seppo Karrila and the Publication tein ratio) were significantly higher with the FDAT treatment than in Clinic, Research and Development Office, Prince of Songkla Univer- the F–20 and FD4 groups. In fish flesh, myofibrillar protein consti- sity, for advice in manuscript preparation. tuted the major component (39%–56%), followed by sarcoplasmic protein (21%–25%), and connective tissue proteins (6%–21%) (Chaijan, Jongjareonrak, Phatcharat, Benjakul, & Rawdkuen, 2010). No differences in the amount of proteins left in its native state (∆H) ORCID Karun Thongprajukaew http://orcid.org/0000-0002-3534-831X THONGPRAJUKAEW ET AL. REFERENCES AOAC. (2005). Official methods of analysis of AOAC international (18th ed.). Gaithersburg, MD: Association of Official Analytical Chemists. Areekijseree, M., Engkagul, A., Kovitvadhi, U., Thongpan, A., Mingmuang, M., Pakkong, P., & Rungruangsak‐Torrissen, K. (2004). Temperature and pH characteristics of amylase and proteinase of adult freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson 1900. Aquaculture, 234, 575–587. https://doi.org/10.1016/j.aquaculture.2003.12.008 Barnes, M. E., Brown, M. L., & Rosentrater, K. A. (2012). Initial observations on the inclusion of high protein distillers dried grain into rainbow trout diets. Open Fish Science Journal, 5, 21–29. https://doi.org/ 10.2174/1874401X01205010021 Blakeslee, C., McRobert, S. P., Brown, A. C., & Clotfelter, E. D. (2009). The effect of body coloration and group size on social partner preferences in female fighting fish (Betta splendens). Behavioural Processes, 80, 157–161. https://doi.org/10.1016/j.beproc.2008.11.005 Cahu, C., & Infante, J. Z. (2001). Substitution of live food by formulated diets in marine fish larvae. Aquaculture, 200, 161–180. https://doi. org/10.1016/S0044-8486(01)00699-8 Caňavate, J. P., & Fernández‐Díazde, C. (2001). Pilot evaluation of freeze‐dried microalgae in the mass rearing of gilthead seabream (Sparus aurata) larvae. Aquaculture, 193, 257–269. https://doi.org/10. 1016/S0044-8486(00)00492-0 Carpenter, J. W., Mashima, T. Y., & Rupiper, D. J. (2001). Exotic animal formulary (2nd ed.). Philadelphia, PA: W.B. Saunders Company. Chai, X. J., Ji, W. X., Han, H., Dai, Y. X., & Wang, Y. (2013). Growth, feed utilization, body composition and swimming performance of giant croaker, Nibea japonica Temminck and Schlegel, fed at different dietary protein and lipid levels. Aquaculture Nutrition, 19, 928–1935. Chaijan, M., Jongjareonrak, A., Phatcharat, S., Benjakul, S., & Rawdkuen, S. (2010). Chemical compositions and characteristics of farm raised giant catfish (Pangasianodon gigas) muscle. LWT‐Food Science and Technology, 43, 452–457. https://doi.org/10.1016/j.lwt.2009.09.012 Chang, J., Niu, H. X., Jia, Y. D., Li, S. G., & Xu, G. F. (2018). Effects of dietary lipid levels on growth, feed utilization, digestive tract enzyme activity and lipid deposition of juvenile Manchurian trout, Brachymystax lenok (Pallas). Aquaculture Nutrition, 24, 694–701. Clotfelter, E. D., Ardia, D. R., & Mcgraw, K. J. (2007). Red fish, Blue fish: Trade–offs between pigmentation and immunity in Betta splendens. Behavioral Ecology, 18, 1139–1145. https://doi.org/10.1093/beheco/ arm090 Company, R., Calduch‐Giner, J. A., Kaushik, S., & Perez‐Sanchez, J. (1999). Growth performance and adiposity in gilthead sea bream (Sparus aurata): Risks and benefits of high energy diets. Aquaculture, 171, 279–292. https://doi.org/10.1016/S0044-8486(98)00495-5 Dabrowski, K., & Glogowski, J. (1977a). Studies on the proteolytic enzymes of invertebrates constituting fish food. Hydrobiologia, 52, 171–174. Dabrowski, K., & Glogowski, J. (1977b). Studies on the role of exogenous proteolytic enzymes in digestion processes in fish. Hydrobiologia, 54, 129–134. de Verga, V., & Böhm, J. (1992). The effect of freeze‐dried zooplankton as a dry feed additive for Danube salmon (Hucho hucho L.) fry. Aquaculture, 108, 155–168. https://doi.org/10.1016/0044-8486(92) 90325-F Fellows, P. J. (2016). Food processing technology: Principles and practice (4th ed.). Kent: Woodhead Publishing/Elsevier Science. Ghosh, A., Bhattacharjee, I., Ganguly, M., Mondal, S., & Chandra, G. (2004). Efficacy of some common aquarium fishes as biocontrol agent of preadult mosquitoes. Buletin Penelitian Kesehatan, 32, 144–149. Goede, R. W., & Barton, B. A. (1990). Organismic indices and an autopsy‐ based assessment as indicators of health and condition of fish. American Fisheries Society Symposium, 8, 93–108. | 7 Goldstein, R. J. (2004). The betta handbook. New York, NY: Barron’s Educational Series Inc. Hart, P. J. B., & Reynolds, J. D. (2002). Handbook of fish biology and fisheries volume I: Fish biology. Oxford: Blackwell Science. James, R., & Sampath, K. (2003). Effect of animal and plant protein diets on growth and fecundity in ornamental fish, Betta splendens (Regan). Israeli Journal of Aquaculture – Bamidgeh, 55, 39–52. Kasiri, M., Farahi, A., & Sudagar, M. (2012). Growth and reproductive performance by different feed types in fresh water angelfish (Pterophyllum scalare Schultze, 1823). Veterinary Research Forum, 3, 175–179. Kolkovski, S. (2001). Digestive enzymes in fish larvae and juveniles—implications and applications to formulated diets. Aquaculture, 200, 181–201. https://doi.org/10.1016/S0044-8486(01)00700-1 Kop, A., & Durmaz, Y. (2007). The effect of synthetic and natural pigments on the color of the cichlids (Cichlasoma severum sp., Heckel 1840). Aquaculture International, 16, 117–122. Kubitza, F., & Lovshin, L. L. (1997). The use of freeze‐dried krill to feed train largemouth bass (Micropterus salmoides): Feeds and training strategies. Aquaculture, 148, 299–312. https://doi.org/10.1016/ S0044-8486(96)01426-3 Kurokawa, T., Shiraishi, M., & Suzuki, T. (1998). Qualification of exogenous protease derived from zooplankton in the intestine of Japanese sardine Sardinops melanoticus larvae. Aquaculture, 161, 491–499. Lim, L. C., Dhert, P., & Sorgeloos, P. (2003). Recent developments in the application of live feeds in the freshwater ornamental fish culture. Aquaculture, 227, 319–331. https://doi.org/10.1016/S0044-8486(03) 00512-X Lowry, O. H., Rosenbrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275. Mandal, S. C., Sahu, N. P., Kohli, M. P. S., Das, P., Gupta, S. K., & Munilkumar, S. (2010). Replacement of live feed by formulated feed: Effect on the growth and spawning performance of Siamese fighting fish (Betta splendens, Regan, 1910). Aquaculture Research, 41, 1707– 1716. Moayyedi, M., Eskandari, M. H., Rad, A. H. E., Ziaee, E., Khodaparast, M. H. H., & Golmakani, M.‐T. (2018). Effect of drying methods (electrospraying, freeze drying and spray drying) on survival and viability of microencapsulated Lactobacillus rhamnosus ATCC 7469. Journal of Functional Foods, 40, 391–399. https://doi.org/10.1016/j.jff.2017.11. 016 Monvises, A., Nuangsaeng, B., Sriwattanarothai, N., & Panijipan, B. (2009). The Siamese fighting fish: Well‐known generally but little‐ known scientifically. ScienceAsia, 35, 8–16. https://doi.org/10. 2306/scienceasia1513-1874.2009.35.008 Munilla‐Moran, R., Stark, J. R., & Barbour, A. (1990). The role of exogenous enzymes in digestion in cultured turbot larvae (Scophthalmus maximus L.). Aquaculture, 3–4, 337–350. https://doi.org/10.1016/ 0044-8486(90)90159-K Noga, E. J. (1996). Fish disease: Diagnosis and treatment. St. Louis, MO: Mosby‐Year Book. Pedersen, B. H., & Hjelmeland, K. (1988). Fate of trypsin and assimilation efficiency in larval herring Clupea harengus following digestion of copepods. Marine Biology, 97, 467–476. https://doi.org/10.1007/ BF00391042 Rahman, M. H., Hossain, M. M., Rahman, S. M. E., Hashem, M. A., & Oh, D.‐H. (2014). Effect of repeated freeze‐thaw cycles on beef quality and safety. Korean Journal for Food Science of Animal Resources, 34, 482–495. https://doi.org/10.5851/kosfa.2014.34.4.482 Rainboth, W. J. (1996). Fishes of the Cambodian Mekong. FAO species identification field guide for fishery purposes. Rome: FAO. Rungruangsak‐Torrissen, K. (2007). Digestive efficiency, growth and qualities of muscle and oocyte in Atlantic salmon (Salmo salar L.) fed on diets with krill meal as an alternative protein source. Journal of Food 8 | Biochemistry, 31, 509–540. https://doi.org/10.1111/j.1745-4514. 2007.00127.x Rungruangsak‐Torrissen, K., Moss, R., Andresen, L. H., Berg, A., & Waagbo, R. (2006). Different expressions of trypsin and chymotrypsin in relation to growth in Atlantic salmon (Salmo salar L.). Fish Physiology and Biochemistry, 32, 7–23. https://doi.org/10.1007/ s10695-005-0630-5 Saekhow, S., Thongprajukaew, K., Phromkunthong, W., & Sae‐khoo, H. (2018). Minimal water volume for intensively producing male Siamese fighting fish (Betta splendens Regan, 1910). Fish Physiology and Biochemistry, 44, 1075–1085. https://doi.org/10.1007/s10695-0180495-z Sipaúba‐Tavares, L. H., Appoloni, A. M., Fernandes, J. B. K., & Millan, R. N. (2016). Feed of Siamese fighting fish, Betta splendens (Regan, 1910) in open pond: Live and formulated diets. Brazilian Journal of Biology, 76, 292–299. https://doi.org/10.1590/15196984.11514 Sugimoto, M. (2002). Morphological color changes in fish: Regulation of pigment cell density and morphology. Microscopy Research and Technique, 58, 496–503. https://doi.org/10.1002/jemt.10168 Supannapong, P., Pimsalee, T., A‐komol, T., Engkagul, A., Kovitvadhi, U., Kovitvadhi, S., & Rungruangsak‐Torrissen, K. (2008). Digestive enzymes and in vitro digestibility of different species of phytoplankton for culture of the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus. Aquaculture International, 16, 437–453. https://doi.org/10. 1007/s10499-007-9156-4 Syamaladevi, R. M., Sablani, S. S., Tang, J., Powers, J., & Swanson, B. G. (2011). Stability of anthocyanins in frozen and freeze‐dried raspberries during long‐term storage: In relation to glass transition. Journal of Food Science, 76, E414–E421. https://doi.org/10.1111/j.1750-3841. 2011.02249.x Thongprajukaew, K., Kovitvadhi, U., Engkagul, A., & Rungruangsak‐Torrissen, K. (2010a). Characterization and expression levels of protease enzymes at different developmental stages of Siamese fighting fish (Betta splendens Regan, 1910). Agriculture and Natural Resources, 44, 411–423. Thongprajukaew, K., Kovitvadhi, U., Engkagul, A., & Rungruangsak‐Torrissen, K. (2010b). Temperature and pH characteristics of amylase and lipase at different developmental stages of Siamese fighting fish (Betta splendens Regan, 1910). Agriculture and Natural Resources, 44, 210–219. THONGPRAJUKAEW ET AL. Thongprajukaew, K., Kovitvadhi, U., Kovitvadhi, S., Engkagul, A., & Rungruangsak‐Torrissen, K. (2013). Evaluation of growth performance and nutritional quality of diets using digestive enzyme markers and in vitro digestibility in Siamese fighting fish (Betta splendens Regan, 1910). African Journal of Biotechnology, 12, 1689–1702. Thongprajukaew, K., Kovitvadhi, S., Kovitvadhi, U., & Rungruangsak‐Torrissen, K. (2014). Pigment deposition and in vitro screening of natural pigment sources for enhancing pigmentation in male Siamese fighting fish (Betta splendens Regan, 1910). Aquaculture Research, 45, 709– 719. Thongprajukaew, K., Kovitvadhi, U., Kovitvadhi, S., Somsueb, P., & Rungruangsak‐Torrissen, K. (2011). Effects of different modified diets on growth, digestive enzyme activities and muscle compositions in juvenile Siamese fighting fish (Betta splendens Regan, 1910). Aquaculture, 322–323, 1–9. https://doi.org/10.1016/j.aquaculture.2011.10.006 Thongprajukaew, K., Rodjaroen, S., Yoonram, K., Sornthong, P., Hutcha, N., Tantikitti, C., & Kovitvadhi, U. (2015). Effects of dietary modified palm kernel meal on growth, feed utilization, radical scavenging activity, carcass composition and muscle quality in sex reversed Nile tilapia (Oreochromis niloticus). Aquaculture, 439, 45–52. https://doi.org/ 10.1016/j.aquaculture.2015.01.021 Winkler, U. K., & Stuckmann, M. (1979). Glycogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. Journal of Bacteriology, 138, 663–670. Worthington, V. (1993). Worthington enzyme manual. Enzymes and related biochemicals. Lakewood, NJ: Worthington Chemical. Yildiz, M., Sener, E., & Timur, M. (2006). Effect of seasonal change and different commercial feeds on proximate composition of sea bream (Sparus aurata). Turkish Journal of Fisheries and Aquatic Sciences, 6, 99–104. How to cite this article: Thongprajukaew K, Pettawee S, Muangthong S, Saekhow S, Phromkunthong W. Freeze‐dried forms of mosquito larvae for feeding of Siamese fighting fish (Betta splendens Regan, 1910). Aquac Res. 2018;00:1–8. https://doi.org/10.1111/are.13897