Effects of methyl farnesoate injection on fatty acids and amino acids in the hepatopancreas of male narrow-clawed crayfish, Pontastacus leptodactylus

Abstract

To investigate the effects of methyl farnesoate hormone (MF) on the hepatosomatic index, protein, amino acid (AA) and fatty acid (FA) composition of hepatopancreas in male P. leptodactylus two controls (G1 and G2) and three experimental groups (G3, G4, G5) were prepared. Hormone injection was administered 5 times in three different doses (G3: 250 ng/g; G4: 500 ng/g; G5: 1000 ng/g body weight). The results revealed that, the amount of protein determined in the hepatopancreas increased statistically significantly in the G5 (37.8 ± 6.41) group compared to the control (P < 0.05). In crayfish, a total of 18 amino acids and 19 fatty acids were identified in the hepatopancreas and there was no statistically significant difference between the control group and the experimental groups injected with MF. Among the amino acids, arginine and proline were found to be higher in all groups while alanine was found at the lowest levels and there was no difference between the groups (P > 0.05). The findings showed that the hepatosomatic index was significantly increased by MF injection (P < 0.05). In conclusion, it was determined that MF injection had a positive effect on the protein and hepatosomatic index values in the hepatopancreas of P. leptodactylus, but did not cause any difference in the amino acid and fatty acid composition.

Keywords:
Crustacea; Decapoda; reproduction; aquaculture; metabolism

INTRODUCTION

Methyl farnesoate (MF) is a key hormone in crustaceans, structurally similar to insect juvenile hormone III, but distinct due to the absence of an epoxide group. It is secreted by the mandibular organ in various crustaceans (Harlıoğlu and Farhadi, 2017; Harlıoğlu et al., 2020). It plays critical roles in regulating physiological processes such as growth (Qian and Liu, 2019; Reddy and Arifullah, 2021; Zhao et al., 2022), reproduction (Reddy and Reddy, 2015; Hemalatha et al. 2016; Bal and Harlıoğlu, 2022), metamorphosis (Zhao et al., 2021), molting (Reddy et al., 2004; Raghavan and Ayanath, 2019), lipid metabolism (Fu et al., 2022), osmoregulation and stress response (Nagaraju, 2007) in decapod crustaceans. Synthesized by the mandibular organ and present in the hemolymph, MF influences gonadal development and protein synthesis, contributing to reproductive efficiency and metabolic regulation (Pamuru et al., 2022).

Raghavan and Ayanath (2018)stated that MF treatment is effective in promoting ovarian growth and maturation, thus shortening the ovarian cycle time, which is highly advantageous for species important in aquaculture. Similarly, Muhd-Farouk et al. (2019)found that dietary MF was effective in the maturation of the ovary of mud crabs, Scylla olivacea (Herbst, 1796).Laufer et al. (2005)reported that MF is a hormone that regulates both reproduction and morphogenesis.

Methyl farnesoate has been reported to play a significant role in regulating molting processes in the freshwater crab Travancoriana schirnerae (Raghavan and Ayanath, 2019). According to Reddy et al. (2004), this hormone influences both molting and reproductive activities in the crab Oziotelphusa senex [currently Spiralothelphusa senex (Fabricius, 1798)]. Several studies have also investigated the physiological effects of methyl farnesoate on protein and lipid metabolism in decapods. For instance, Hemalatha et al. (2016)demonstrated that MF injection in the freshwater shrimp Macrobrachium rosenbergii (De Man, 1879) led to increased protein and lipid contents in the ovary. Furthermore, MF has been shown to regulate lipid metabolism during ovarian development. Fu et al. (2022) reported that in the mud crab Scylla paramamosain Estampador (1950), MF promotes ovarian maturation by stimulating lipid accumulation in the hepatopancreas.

Pontastacus leptodactylus (Eschscholtz, 1823), commonly known as the narrow-clawed crayfish, holds significant economic value in aquaculture due to its high nutritional quality and market demand (Harlıoğlu and Harlıoğlu, 2004; 2009; Aydın et al., 2012). Thus, the reproduction and cultivation of this species have received more attention in the last two decades (Kulesh and Alekhnovych, 2010; Harlıoğlu et al., 2012; 2013a; 2013b; 2018; Farhadi et al., 2018, Farhadi and Harlıoğlu, 2019, Farhadi et al., 2019; 2020; Roessler et al., 2020).

While the effects of MF on reproductive parameters in female decapods are well documented, limited information exists regarding its influence on male crayfish, particularly concerning protein metabolism and biochemical composition.

In a previous study, the influences of MF injections on some reproductive parameters (i.e., gonadosomatic index, testicular index, and vasosomatic index), haemolymph MF levels and spermatozoa numbers in vas deference were examined in males of P. leptodactylus by Bal and Harlıoğlu (2022).

This study aims to bridge this knowledge gap by evaluating the effects of MF injection on the hepatosomatic index, protein content, amino acid, and fatty acid profiles in the hepatopancreas of male P. leptodactylus, providing insights into the potential applications of MF in crustacean aquaculture.

MATERIAL AND METHODS

Animals and experimental design

Male crayfish (P. leptodactylus) with a mean body weight, 46.7 ± 3.4 g and mean carapace length 5.7 ± 0.2 cm were captured from Keban Dam Lake (Elazığ, Turkey). Crayfish were placed in 2 x 2 x 1 m concrete ponds and exposed to natural photoperiods (9.75 L : 14.25 D in December and 9.95 L : 14.05 D in January). MF injections were administered between December and January. The oxygen content of the water in the concrete ponds was 7.1 ± 0.2 mg/l, pH was 8.3 ± 0.22 and water temperature was 1.58 ± 0.20 °C and 1.41 ± 0.12 °C in December and January, respectively. Shelters (plastic pipes, 20 cm long and 7 cm in diameter) were placed on the floor of the ponds. Crayfish were daily fed (ad libitum) with a commercial pellet food manufactured by Gürdal, Kahramanmaraş, Turkey, containing 35% crude protein, 8% crude fat, 12% carbohydrates on a dry-weight basis and 3600 kcal/kg gross energy).

Seventy-five crayfish were divided into five different groups. Each experimental group contained 15 samples. The first group served as initial control group (G1) and did not receive any injection. The second group (G2), served as a concurrent control group, and 0.02 ml ethanol was diluted in 0.05 ml saline (physiological solution, 0.85% salt water). The highest ethanol level was 40% of the injection volume (20 μl ethanol + 30 μl physiological solution). MF injections were applied to crayfish at three different doses (Third group, G3: 250 ng/g body weight; fourth group, G4: 500 ng/g body weight and fifth group G5: 1000 ng/g body weight). The lowest dose used in the study is compatible with the levels of MF that can be found naturally in hemolymph for crustaceans and is considered to be close to the physiological range. For the other doses, doses were chosen that would be compatible with research in decapods where physiological effects and biological responses could be determined. All experimental crayfish, except the controls, were injected five times in the second abdominal muscle on days 1, 8, 15, 22, and 29 by means of 1-ml syringes (27 G needle). The doses and times used were determined according to Abdu et al. (2001) , Alfaro et al. (2008) and Rodriquez et al. (2002). A certain amount of time is required for the physiological and biochemical effects of MF injection to manifest. Therefore, sampling one week after the last dose provides sufficient time for the hormone to affect biological processes in the body (e.g. protein synthesis, lipid metabolism, changes in organ weight) that it may influence. Therefore, samples were taken one week after the last MF administration to determine the effect of MF (Raghavan and Ayanath, 2018; Hemalatha et al., 2016). One week after the last dose, the crayfish were ice anesthetized (Fregin and Bickmeyer, 2016) the weight and length were determined, and then dissected with the hepatopancreas stored at -20°C for hepatosomatic index, protein, amino acid and fatty acids analysis. Samples were individually analysed replicates (N = 15).

Determination of hepatosomatic index

Hepatosomatic Index (HSI) values were calculated according to Ravi et al. (1999)using the following formula,

HSI = (Weight of Hepatopancreas / Body Weight) x 100

Protein analysis in the hepatopancreas

Total protein measurement of hepatopancreas samples homogenized in n 10 mL of 50 mM Tris-20 mM Tris-ETDA (Ethylenediaminetetraacetic acid) (pH = 7.4) buffer mixture. 10 µL of tissue homogenate was taken, 4 mL of Lowry's solution was added and combined for 10 min and then 1/1 (v/v) water/folin reagent was added. The samples were kept for 30 min and the absorbance of the crayfish was determined against a blank under 750 nm UV wavelength. Bovine serum albumin was used as the standard. Results were expressed in mg/g (Lowry et al., 1951).

Amino acids analysis in the hepatopancreas

For the quantification of free amino acids in the homogenate, the proteins were precipitated with 20% TCA, centrifuged at 5000 rpm and the liquid supernatant was removed under nitrogen flow, mixed with 300 µL N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) and 300 µL acetonitrile, kept at 70 ºC for 40 minutes and 600 µL chlorophosome was added and analysed by GC FID. SLB 5 Ms GC column was used for the analysis. For the quantification of amino acids in proteins, the protein solution was mixed with 6 M HCl solution containing 1% phenol and hydrolysed at 110 °C for 24 hours and then subject to the same experimental treatment and analysed. Analyses were performed using gas chromatography (Buch et al., 2006).

Fatty acid analysis in hepatopancreas

In the fatty acid analysis, lipid extraction was performed according to the technique of Hara and Radin (1978) and preparation of fatty acid methyl esters and gas chromatographic analysis were performed according to Christie (1992) . For the extraction of lipids, 1 g of hepatopancreatic sample was taken and homogenized in 10 ml hexane-isopropanol mixture at a ratio of 3:2 (v/v) for 30 s. The homogenate was put into centrifuge tubes and the supernatant portion of the tissue samples centrifuged at 5000 rpm for 10 min and placed in sealed test tubes. Fatty acids in the lipid extract were converted into methyl esters and analysed by SHIMADZU GC 17 gas chromatography.

Statistical Analysis

The data on the HSI, protein, amino acids, fatty acids provided from P. leptodactylus hepatopancreas was analysed statistically by using one-way analysis of variance (ANOVA), followed by Duncan’s new multiple range test (SPSS 16.0). Significant differences were based on the P < 0.05 level. The results were expressed as means ± standard deviation.

RESULTS

Effect of MF injection on hepatosomatic index, the amount of protein, amino acids and fatty acid composition in the hepatopancreas

The hepatosomatic index value was significantly (F value = 4.51, P < 0.05) higher in the MF exposed experimental groups than in the control groups (Figure 1). While the HSI values were 4.52 ± 0.44 and 4.49±0.46 in the control G1 and G2 groups respectively, these values were 5.04 ± 0.22, 5 ± 0.15 and 5.21 ± 0.36 in the G3, G4 and G5 groups, respectively.

Figure 1.
Effect of different MF doses on hepatosomatic index in male Pontastacus leptodactylus. Letters show groups with significant difference (F value = 4.51, P < 0.05) (mean ± S.D; N = 15).

The amount of protein in the hepatopancreas of crayfish was 27.92 ± 4.54 and 28.24±4.93 mg/g in the control group G1 and G2 groups, respectively. There was no statistically significant difference (P > 0.05) in the protein amount in G3 (34.3 ± 4.35) and G4 (33.54 ± 6.83) groups in comparison to the control groups, but the protein amount determined in the G5 (37.8 ± 6.41) group increased statistically significantly in comparison to the control (F value = 2.37, P < 0.05) (Figure 2).

Figure 2.
Effect of MF injections on protein content in hepatopancreas of male Pontastacus leptodactylus. Letters show significant difference groupings (F value = 2.37, P < 0.05) (mean ± SD; N = 15).

In crayfish, 18 amino acids were identified in the hepatopancreas. There was no statistically significant difference between the control group and the experimental groups injected with MF (P > 0.05) (Tab. 1). Arginine was determined to be the most accumulated amino acid in all groups. Among other amino acids, proline, aspartic acid, cystine, glutamic acid, glycine, valine and leucine were also found in high amounts, but alanine was the least abundant amino acid in the hepatopancreas of P. leptodactylus No statistical difference was found between essential and non-essential amino acids (Tab. 1).

Table 1.
Amino acid amounts determined in the hepatopancreas of male Pontastacus leptodactylus injected with MF (%, total amino acids, mean ± SD, N = 15)

Amino acids (%)	Control group (G1)	Control group (G2)	MF 250 ng/g (G3)	MF 500 ng/g (G4)	MF 1000 ng/g (G5)	F Value
ARGININE	34.0± 2.73	33.60±3.78	34.50±4.7	34.41±5.83	35.57±4.07	0.14
VALINE	4.41±0.89	4.3±0.95	4.54±1.33	6.05±1.99	5.13±0.96	1.57
LEUCINE	4.89±1.54	5.04±1.81	4.27±1.56	5.35±1.02	5.10±1.40	0.36
ISOLEUCINE	2.64±0.95	2.84±1.33	2.30±1.05	3.07±1.37	2.98±1.03	0.34
METHIONINE	3.08±1.22	3.27±1.28	3.65±1.81	3.60±1.32	4.52±0.98	0.83
THREONINE	3.82±0.96	3.68±1.18	3.86±1.15	4.11±1.09	3.78±1.13	0.10
LYSINE	1.07±0.56	1.11±0.70	1.10±0.55	0.77±0.52	0.81±0.50	0,41
HISTIDINE	0.62±0.25	0.54±0.28	1.03±0.14	0.87±0.73	0.91±0.33	1.35
TRYPTOPHAN	0.16±0.09	0.20±0.19	0.34±0.33	0.22±0.40	0.19±0.14	0.34
PHENYLALANINE	0.23±0.1	0.19±0.14	0.27±0.18	0.30±0.26	0.22±0.12	0.27
ƩEAA*	54.94±2.07	54.80±3.78	55.88±4.07	58.79±4.75	59.34±4.93	1.42
ASPARTIC ACID	7.22±0.98	7.34±1.17	7.46±2.29	6.78±1.49	6.99±1.24	0.16
CYSTEINE	6.25±1.18	6.39±1.31	5.98±3.81	6.80±2.92	6.16±2.47	0.073
GLUTAMIC ACID	6.81±1.43	7.01±1.83	4,83±2.37	4.63±3.13	5.34±2.30	1.18
TYROSINE	2.86±1.02	2.96±1.10	2.47±1.21	2.77±2.10	2.34±0.97	0.19
ALANINE	0.08±0.02	0.07±0.03	0.04±0.05	0.06±0.04	0.05±0.04	0.70
SERINE	2.38±0.83ab	2.52±0.81b	1.35±0.46a	2.14±0.66ab	2.26±0.92ab	1.80
PROLINE	12.51±2.65	12.43±2.79	12.20±1.77	11.57±3.75	12.56±2.83	0.10
GLYCINE	5.20±1.09	5.30±1.08	5.10±1.29	4.56±1.72	4.20±0.80	0.73
ƩNEAA**	43.33±2.32	44.05±2.24	39.47±5.50	39.32±5.47	39.92±5.35	1.31
ƩEAA/ ƩNEAA	1.27±0.08 ab	1.24±0.11 a	1.43±0.21 ab	1.51±0.19 b	1.50±0.20 b	2.79

• Note: Within rows, values followed by the different letters are significantly different (P< 0.05). * Essential amino acids, ** Nonessential amino acids,

There was no significant difference in the fatty acid composition in the hepatopancreas of crayfish injected with different doses of MF (P > 0.05). While 19 fatty acids were determined in crayfish, the most abundant fatty acids were palmitic acid (C16:0) in saturated fats, oleic acid (C18:1n-9), linoleic acid (C18:2n-6), eicosapentanoic (C20:5n-3), arachidonic acid (C20:4n-6), linolenic acid (C18:3 n-3) and docosahexanoic acid (C22:6 n-3) in polyunsaturated fats (PUFA) (Table 2). No statistically significant difference was determined between the amounts of total saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in fatty acids (Figure 3). It was determined that there was no significant difference between the groups in terms of total n-3 series, n-6 series fatty acids and n-3 / n-6 ratios (Figure 4).

Table 2.
Fatty acid amounts determined in hepatopancreas of male Pontastacus leptodactylus injected with MF (%, total fatty acids values: mean ± SD, N = 15). Letters indicate significant difference groupings (P < 0.05).

Fatty acids	Control group (G1)	Control group (G2)	MF 250 ng/g (G3)	MF 500 ng/g (G4)	MF 1000 ng/g (G5)	F Value
C14:0	2.46±0.53	2±0.61	1.64±0.58	3.22±2.25	2.43±1.66	0.94
C14:1	0.5±0.25	0.67±0.24	0.77±0.01	0.61±0.04	0.64±0.10	0.27
C15:0	0.39±0.06	0.42±0.04	0.35±0.01	0.36±0.17	0.53±0.19	1.15
C15:1	0.37±0.08ab	0.35±0.08ab	0.27±0.05a	0.33±0.05ab	0.42±0.06b	1.43
C16:0	13.76±2.91	14.88±2.48	15.8±1.27	15.2±3.56	15.38±0.93	0.74
C16:1n-7	9.44±2.21	7.45±2.50	9.88±1.10	9.28±1.56	8.89±2.39	0.89
C17:0	5.4±2.41b	4.41±1.40ab	1.91±1.40a	3.8±2.97ab	2.56±2.67ab	1.91
C17:1	1.25±0.35	1.52±0.02	1.37±0.14	1.48±0.65	1.35±0.32	0.25
C18:0	7.69±0.56	8.56±1.89	6.6±1.46	9.11±5.01	9.49±5.53	1.08
C18:1n-7	4.93±0.81a	5.05±0.64a	6.93±0,94b	4.67±1.04a	5.84±0.32ab	1.67
C18:2n-6	13.84±3.86	12.32±5.43	11.18±2.37	10.54±3.86	10.11±2.18	0.93
C18:3n-3	4.04±0.62	4.03±0.57	4.35±0.50	3.98±0.81	5.03±1.49	0.36
C20:1	1.3±0.23	1.46±0.64	1.75±0.48	1.11±0.30	1.23±0.43	1.58
C20:2	0.91±0.22	1.05±0.11	1.1±0.10	1.11±0.03	1.16±0.15	1.33
C20:4n-6	3.58±0.70ab	4.73±1.11b	3.46±0.26a	3.62±1.35ab	3.36±0.45a	1.91
C20:5n-3	3.62±0.58ab	4.3±0.61b	3.14±0.14a	3.02±0.68a	3.24±0.59a	4.2
C22:5	0.91±0.30a	1.57±0.66b	1.26±0.39ab	0.84±0.33a	1.17±0.46ab	2.1
C22:6	2.92±0.25a	3.63±0.44ab	4.02±0.61b	3.47±0.67ab	3.44±0.49ab	2.97
C18:1n-9	22.2±3.02	21.29±2.75	23.22±1.78	23.29±1.27	22.88±1.58	1.82

Figure 3.
The amount of saturated fatty acids, polyunsaturated fatty acids and monounsaturated fatty acids (%, mean±standard deviation) determined in hepatopancreas of control groups (G1 and G2) and MF injected crayfish.

Figure 4.
Total N-3 series, N-6 series fatty acids and N-3/N-6 ratios (%, mean±standard deviation) determined in hepatopancreas of control groups (G1 and G2) and MF injected crayfish.

DISCUSSION

The effects of MF applications on female decapods have been investigated by Chan et al. (2005); Medesani et al. (2012); Xie et al. (2015); Hemalatha et al. (2016); Liu et al. (2016); Raghavan et al. 2018; Zhang et al. 2024), but information on the effects of MF in male decapods is more limited (Alfaro et al., 2008; Kalavathy et al., 1999; Nagaraju and Borst, 2008; Bal and Harlıoğlu, 2022). On the other hand, little information is available about its effects on amino acids and fatty acids in decapod metabolism (Homola and Chang, 1997; Paulson and Skinner, 1988).

The hepatopancreas is the main organ responsible for the deposition and metabolism of nutrients in decapod crustaceans and has an important role in lipid accumulation in the ovary during gonadal development. This organ is important for the absorption, storage, digestion and synthesis of digestive enzymes. During growth and reproduction, stored nutrients are transferred to muscle, gonads and other tissues. It is also known that the hepatopancreas has a substantial act in the synthesis of vitellogenin and sex steroid hormones and in the maturation of growth and gonads (Wang et al., 2014). Homola and Chang (1997)stated that MF promotes protein synthesis in decapods. Pamuru et al. (2022)reported that MF increases hemolymph protein contents and hepatopancreatic index in male crab O. senex reared in Ca(OH)₂ EDTA medium.

Han et al. (2006) investigated the effects of cyclic AMP (cAMP, cAMP, cyclic adenosine monophosphate)) signalling on protein synthesis in the blue crab (Callinectes sapidus Rathbun, 1896) Y-organs, which secrete ecdysteroid hormones that control growth and moulting. In vitro studies showed that cAMP analogues and agents that increase cAMP levels suppress protein synthesis in Y-organs. Overall protein synthesis was found to be affected. The results suggest that in C. sapidus, cAMP plays an important role in the regulation of protein synthesis associated with ecdysteroid production in Y-organs. In this study, MF was found to be effective at the protein level in hepatopancreas.

In vitro applications of farnesoic acid and methyl farnesoate sesquiterpenoid hormones were carried out by Luan et al. (2025)on the hepatopancreas of Neocaridina davidi (Bouvier, 1904) shrimps. Sesquiterpenoid hormones such as methyl farnesoate are known to control development and reproduction in insects and crustaceans. Transcriptomic analyses revealed a total of 65 and 112 differentially expressed genes in the hepatopancreas treated with MF and FA, respectively, 3 hours after treatment. Although this research in shrimp did not determine exactly how the two sesquiterpenoid hormones regulate immune responses, it was determined that MF and FA regulate different sets of genes in decapod crustaceans. MF was reported to regulate immune-related proteins such as hemocyanin and potentially enhance immune capacity during reproductive maturation. In decapods, sesquiterpenoid hormones appear to regulate the hepatopancreas.

In the present study, MF injections promoted the protein content in the hepatopancreas and hepatopancreatic index of male P. leptodactylus in all groups that received MF (G3, G4 and G5). The highest values were obtained in the G5 group.

In the literature, there is no study investigating the effect of MF on the amount of amino acids and fatty acids in the hepatopancreas of decapods, but studies on the effects of different hormones have been conducted. For example, Coccia et al. (2010)investigated the effects of estradiol and progesterone on amino acid and fatty acid profiles in the hepatopancreas of the crayfish Cherax albidus Clark (1936). Their findings indicated that estradiol administration led to an increase in saturated fatty acid content, accompanied by a decrease in polyunsaturated (PUFA) and monounsaturated fatty acids (MUFA). In contrast, treatment with progesterone resulted in elevated levels of both PUFA and MUFA. In this study, it was determined that MF did not cause a significant difference in saturated fats, PUFA and MUFA amounts. In addition, Fu et al. (2022)investigated the exogenous administration of JH III, MF, farnesoic acid and methoprene to the hepatopancreas of mud crabs (S. paramamosain) on lipid metabolism during gonad development. The results of Fu et al. (2022) showed that MF, tested in vitro, effectively induced lipid accumulation, especially triglycerides and lysophospholipids, in the hepatopancreas of mud crabs in preparation for ovarian development.

In a different study, Farhadi et al. (2020) studied the role of serotonin exposure on the breeding characteristics and haemolymph MF amount in P. leptodactylus. They found that there was no difference between the amount of amino acids and fatty acids in the hepatopancreas of male P. leptodactylus in which the level of MF in the hemolymph increased with serotonin injection (Farhadi et al., 2020). Similarly, in the present study, MF injections did not improve the amino acid and fatty acid parameters in the hepatopancreas in male P. leptodactylus.

CONCLUSION

This investigation revealed that MF injection substantially affected hepatopancreatic protein and hepatosomatic index values in male P. leptodactylus. Studies have reported that MF has a regulatory role on lipid and protein metabolism in relation to reproduction and development in decapods. Future research could further investigate the effects of MF injection on abdominal meat, ovary, testis and vas deferens (e.g., protein, amino acids and fatty acids) in male and female crayfish depending on their growth stage.

ACKNOWLEDGEMENTS

This study was conducted as a part of Mehmet BAL’s masters thesis “Effect of Methyl Farnesoate on some Reproductive Efficiency Parameters in Male Astacus leptodactylus” supported by the Scientific and Technological Research Council of TURKEY, TÜBİTAK (Project No: TOVAG-117O915). The authors of this article are very grateful to TÜBİTAK-TOVAG as this article would not have been possible without this support. The authors are also very grateful to Prof Dr. Ökkeş Yılmaz for his invaluable assistance in the laboratory. The crayfish in this experiment were treated in agreement with the experimental protocol approved by the Fırat University Animal Experimentation Ethics Committee operating under the 2006 Turkish code of practice for the care and use of animals for scientific purposes (July 28, 2017, Protocol No:2017/90).

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• 1
  Zoobank: http://zoobank.org:pub:E7B440CF-A2F0-40D1-9A72-53D8ED686DA2
• Consent for publication
  All authors declare that they have reviewed the content of the manuscript and gave their consent to submit the document.
• Data availability
  All study data are included in the article.
• Funding and grant disclosures
  This study was conducted as a part of Mehmet BAL’s masters thesis, “Effect of Methyl Farnesoate on some Reproductive Efficiency Parameters in Male Astacus leptodactylus”, in the Department of Fish Cultivation, Faculty of Fisheries, Graduate School of Natural and Applied Sciences, Elazığ/TURKEY, supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK, Project No: TOVAG-117O915).
• Study association
  Not applicable.
• Study permits
  The crayfish in this experiment were treated in agreement with the experimental protocol approved by the Fırat University Animal Experimentation Ethics Committee operating under the 2006 Turkish code of practice for the care and use of animals for scientific purposes (July 28, 2017, Protocol No:2017/90).

Edited by

• Associate Editor:
  Sandro Santos

• Editor-in-chief:
  Christopher Tudge

Data availability

All study data are included in the article.

Publication Dates

• Publication in this collection
  11 May 2026
• Date of issue
  2026

History

• Received
  04 Apr 2025
• Accepted
  09 Sept 2025