1. INTRODUCTION

The Chinese mitten crab (Eriocheir sinensis) is an important commercially farmed crab species in China. In 2023, the national farmed output reached 888,600 tons.1 E. sinensis has a delicious taste and high nutritional value, and is highly favored by consumers. The breeding methods and feed composition will directly affect the taste, nutrition, and health of crabs.2 Trash fish (TF) is a traditional diet for E. sinensis, playing a crucial role in the growth, nutritional quality, and taste characteristics of the crabs.3 However, using TF as a diet has problems such as unstable sources and uncontrollable quality.4 Over-reliance on TF may restrict the development of the E. sinensis farming industry. Therefore, numerous studies are exploring a wider range of protein sources.5 The 813/2017 EU regulation has approved the use of seven types of insects as animal feed, namely black soldier fly (Hermetia illucens), common housefly (Musca domestica), striped cricket (Gryllodes sigillatus), field cricket (Gryllus assimilis), domestic cricket (Acheta domesticus), small yellow mealworm (Alphitobius diaperinus), and mealworm (Tenebrio molitor, MW).6 The golden apple snail (Pomacea canaliculata, GAS) was introduced to Asia from the Philippines in early 1980 for commercial purposes as a source of protein food.7 Using insects and GAS meat (GASM) as animal feed and sustainable raw materials has many advantages over traditional animal protein, such as lower production costs and environmental friendliness.

Numerous studies have shown that using BSFL, MW, FLM, and GASM protein meal can replace part of dietary fish meal in various aquatic animals, or can be directly used for feeding aquatic animals, without any adverse effects on the growth performance and weight gain rate.7–11 In previous studies, the use of MW meal as a substitute for fish meal in the feed of species such as Nile tilapia (Oreochromis niloticus),12 silver carp (Cyprinus rubrofuscus),13 and mirror carp (Cyprinus carpio var. specularis)14 was found to increase the content of certain amino acids in the muscle, while having no significant effect on the content of free amino acids (FAAs) in the muscle. Yang et al.15 conducted an experiment by replacing part of the dietary fish meal of largemouth bass (Micropterus salmoides) with BSFL meal. They found that a 10%-30% substitution rate did not affect the growth performance and had no adverse effect on the fatty acid composition of the fish bodies. Liu et al.16 conducted an experiment by replacing part of the dietary fish meal with BSFL meal on hybrid croakers (Siniperca chuatsi ♀ × Siniperca scherzeri ♂). They found that a 20% substitution rate could significantly improve the growth performance, without any adverse effects on the muscle quality. In addition, replacing dietary fish meal with 10% GASM meal of catfish (Pangasius sp.) resulted in no negative impact on the growth and feed utilization.5 While fed E. sinensis with GASM, the results showed significant improvements in growth and nutritional quality.17 Substituting part of the dietary fish meal with FLM meal does not affect the growth performance of mirror carp,18 tilapia,19 Chinese soft-shelled turtle (Pelodiscus sinensis),20 and effectively improves the weight gain rate and protein content of the farmed animals.

At present, most experiments have focused on the substitution of dietary fish meal with alternative protein sources. There has been no study on the substitution of TF with insects or GASM on E. sinensis. Therefore, this study aims to investigate the nutritional levels of MW, BSFL, GASM, FLM, and TF, as well as the effects of replacing TF with these four diets on the edible quality and flavor compounds of E. sinensis. It is expected to provide certain theoretical basis and practical reference for the application of MW, BSFL, FLM, and GASM in E. sinensis farming.

2. MATERIALS AND METHODS

2.1. EXPERIMENTAL CRAB AND FEEDING MANAGEMENT

The juvenile E. sinensis were provided by Jiangsu Haoran Bio-Industrial Group Co., Ltd. Crabs were initially reared in canvas pools, with polyvinyl chloride (PVC) pipes serving as shelters. During the rearing period, commercial feed (from Jiangsu Haoran Bio-Industrial Group Co., Ltd., with a crude protein content≥ 40%) was provided. Feeding was stopped 24 hours before the formal experiment, and 400 juvenile crabs with intact appendages, good vitality, in the molting period, and an initial body weight of 4.15 ± 0.10 g were selected for the feeding trial. The 400 crabs were randomly distributed into a circulation water system consisting of 20 PVC tanks (1.0 m × 1.0 m × 0.5 m), with 20 crabs per tank. Crab nests were placed in tanks to serve as shelters. The experiment was divided into 5 groups, with 4 replicates for each group. The five diet types were TF, MW, BSFL, GASM, and FLM. The proximate composition of the diets (Table 1), as well as the composition of amino acids (Table 2) and fatty acids (Table 3), was determined. During the breeding period, all crabs were fed twice a day (at 7:00 and 17:00) to apparent satiation. The feces at the bottom of the tanks were removed by siphoning once a day. The system water was replenished promptly after siphoning, and the water source was the filtered river water. The experiment lasted for 56 days. The water quality parameters were tested by ProQuatro/1020 (YSI, American), the water temperature, dissolved oxygen, pH, ammonia nitrogen, and nitrite were kept at 24−29℃, ≥ 5.8 mgL−1, 7.0–8.5, ≤ 0.2 mgL−1, and ≤ 0.05 mgL−1, respectively. All 20 PVC tanks are within a single circulating water system, and the water quality of each parallel group remains consistent.

Table 1.Proximate composition of the experimental baits (%, fresh tissue).
Item Groups
TF MW BSFL GASM FLM
Moisture 73.51 65.34 71.01 76.35 73.52
Crude protein 17.63 17.51 14.96 16.20 17.33
Crude lipid 4.73 9.46 10.10 3.40 6.70
Ash 3.58 6.87 3.68 3.24 1.80

TF, Trash fish, MW, mealworm, BSFL, black soldier fly larvae, GASM, golden apple snail meat, FLM, fly maggot.

Table 2.Amino acid composition of the experimental baits (%, fresh tissue).
Item Groups
TF MW BSFL GASM FLM
Histidine 0.29 0.47 0.34 0.22 0.35
Arginine 0.86 0.92 0.66 0.97 0.69
Threonine 0.62 0.65 0.45 0.63 0.50
Valine 0.62 0.87 0.58 0.63 0.47
Methionine 0.41 0.14 0.24 0.23 0.27
Isoleucine 0.60 0.65 0.45 0.57 0.40
Leucine 1.08 1.12 0.76 1.03 0.72
Phenylalanine 0.53 0.53 0.47 0.53 0.66
Lysine 1.67 1.08 0.94 0.71 1.08
EAA 6.68 6.44 4.88 5.52 5.14
Aspartic acid 1.66 1.46 0.91 1.35 1.40
Glutamic acid 2.32 2.04 1.15 2.20 1.63
Serine 0.62 0.82 0.50 0.65 0.55
Glycine 0.58 0.81 0.53 0.92 0.46
Alanine 0.85 1.21 0.68 0.76 0.73
Proline 0.42 1.01 0.53 0.58 0.48
Tyrosine 0.46 1.08 0.74 0.40 0.68
NEAA 6.91 8.44 5.04 6.86 5.93
TAA 13.59 14.88 9.92 12.38 11.06

TF, Trash fish, MW, mealworm, BSFL, black soldier fly larvae, GASM, golden apple snail meat, FLM, fly maggot. EAA, essential amino acids; NEAA, non-essential amino acid; TAA, total amino acids.

Table 3.Fatty acids composition of the experimental baits (% total identified fatty acids).
Item Groups
TF MW BSFL GASM FLM
C6:0 2.33 0.15 0.11 4.88 0.44
C8:0 3.00 0.17 0.27 2.93 0.61
C10:0 0.24 0.03 0.35 0.37 0.06
C12:0 0.69 0.10 14.64 1 0.15
C14:0 0.86 1.50 3.63 1.78 2.33
C15:0 7.24 0.77 0.83 5.73 1.69
C16:0 23.49 19.71 20.79 19.52 25.21
C17:0 0.28 0.21 0.12 1.07 0.22
C18:0 10.37 4.40 3.01 6.65 4.04
C20:0 0.24 0.17 0.10 0.38 0.19
C16:1 1.33 1.19 1.71 0.73 6.60
C18:1 10.76 35.56 30.26 30.51 28.30
C20:1 0.67 0.13 0.06 0.28 0.13
C22:1 0.12 0.01 0.02 0.2 0.04
C20:2 0.85 0.10 0.02 2.19 0.06
C18:2n6 19.39 35.59 23.35 8.51 27.99
C20:3n6 0.49 0.00 0.02 0.96 0.09
C18:3n6 0.10 0.00 0.02 0.07 0.13
C20:4n6 ARA 1.43 0.00 0.33 8.1 0.69
C18:3n3 1.70 0.17 0.12 1.14 0.30
C20:3n3 0.33 0.01 0.01 0.18 0.03
C20:5n3 EPA 1.48 0.01 0.20 0.65 0.62
C22:6n3 DHA 12.62 0.01 0.03 4.36 0.08
SFA 48.74 27.21 43.85 44.31 34.94
MUFA 12.88 36.89 32.05 31.72 35.06
n−6 PUFAs 21.41 35.60 23.71 17.64 28.91
n−3 PUFAs 16.12 0.20 0.36 6.33 1.03
n-6/n-3 PUFAs 1.33 178.00 65.86 2.79 28.07

ARA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.

2.2. SAMPLING

After the feeding trial was completed, crabs in each tank were separately collected. Four crabs from each replicate were randomly selected, placed in a steamer, and steamed for 15 minutes. The steaming treatment of samples was performed to simulate the cooking process, aiming to analyze the flavor compound composition of crabs in an edible state. After steaming, the crab shells were opened, and the steamed hepatopancreas were collected and placed in a 10 ml centrifuge tube for the determination of FAAs, fatty acids, and flavor nucleotides. The crab leg and body muscles were collected and placed in 10 ml centrifuge tubes for the determination of FAAs and flavor nucleotides. Tissue samples from multiple crabs within the same tank were analyzed and pooled as a single sample.

2.3. DIETS COMPOSITION

Crude lipid, crude protein, ash, and moisture of experiment diets were measured following the method of AOAC.21 The moisture and ash content were analyzed by drying the samples at 105°C to constant weight and incinerating the samples at 550°C for 6 h, respectively. Crude protein was determined using an Auto Kjeldahl System (2300-Auto-analyzer, Foss Tecator, Sweden), and the crude lipid content was analyzed by the chloroform–methanol method.

2.4. DIETS HYDROLYZED AMINO ACIDS, MUSCLE AND HEPATOPANCREAS FAAS

2.4.1. PRE-TREATMENT OPERATIONS

Hydrolyzed amino acids: Approximately 1.0 g of the sample was weighed and 50 mL of 0.1% phenol in 6 mol/L hydrochloric acid was added. The mixture was ground into a paste and placed in an oven at 110°C for hydrolysis for approximately 20 h. After hydrolysis, the sample was removed and allowed to cool. A 1 mL aliquot of the hydrolysate was collected and evaporated to near dryness using a nitrogen blower. Subsequently, 1 mL of 0.1 M hydrochloric acid solution was added to dissolve the residue, and the solution was filtered through a membrane prior to derivatization.

Free amino acids (FAAs): Approximately 1.0 g of the sample was weighed and mixed with 10 mL of water. The mixture was ground into a paste, ultrasonicated for 1 h, and centrifuged to obtain the supernatant. The resulting supernatant was collected and prepared for derivatization.

2.4.2. DERIVATIZATION OF AMINO ACIDS

A total of 200 μL of the clear liquid sample and 200 μL of the amino acid standard solution were transferred separately into 2 mL EP tubes. Subsequently, 20 μL of the positive leucine internal standard solution was accurately added to each tube. Then, 100 μL of triethylamine–acetonitrile solution (pH > 7) and 100 μL of phenyl isothiocyanate–acetonitrile solution were sequentially added to each EP tube. The mixtures were vortexed thoroughly and incubated at 25°C for 1 hour for derivatization. Following incubation, 1 mL of n-hexane was added to each tube, and the mixtures were shaken and allowed to stand for 10 minutes. The upper organic layer was collected and diluted fivefold with water. The diluted solution was then filtered through a 0.22 μm syringe filter prior to analysis.

2.4.3. LIQUID CHROMATOGRAPHY ANALYSIS CONDITIONS

An Agilent 1100 high-performance liquid chromatograph equipped with an ultraviolet detector set at 254 nm was used for the analysis. Separation was performed on a Cussons Amethyst C18-H chromatographic column (250 mm × 4.6 mm, 5 μm). The column temperature was maintained at 40 °C, the flow rate was set at 1 mL/min, and the injection volume was 10 μL. Mobile phase A was prepared by weighing 7.6 g of anhydrous sodium acetate, adding it to 925 mL of water, and dissolving it completely. The pH was then adjusted to 6.5 with acetic acid, followed by the addition of 70 mL of acetonitrile. The solution was mixed thoroughly and filtered through a 0.45 μm membrane filter. Mobile phase B consisted of an 80% aqueous acetonitrile solution.

2.5 MUSCLE AND THE HEPATOPANCREAS NUCLEOTIDES

2.5.1. PRE-TREATMENT OPERATION

Approximately 0.5 g of the sample was weighed and mixed with 1.0 mL of 6% perchloric acid aqueous solution. The mixture was ground into a paste and transferred into an EP tube. Ultrasonic extraction was performed for 20 minutes, followed by centrifugation. Subsequently, 0.5 mL of the supernatant was collected, and the pH was adjusted to 6.5 using sodium hydroxide solution. The volume was then brought up to 1 mL with water, filtered through a needle-type filter, and prepared for analysis.

2.5.2. LIQUID CHROMATOGRAPHY ANALYSIS CONDITIONS

An Agilent 1100 high-performance liquid chromatograph equipped with an ultraviolet detector was used, and the detection wavelength was set at 245 nm. Separation was performed using a Compass C18 (2) reversed-phase chromatographic column (250 mm × 4.6 mm, 5 μm). The column temperature was maintained at 30°C. The flow rate was 0.8 mL/min, and the injection volume was 10 μL. The mobile phase consisted of 0.05 mol/L KH₂PO₄.

2.5.3. STANDARD CURVE DETERMINATION

Precisely weighed each nucleotide standard, dissolved it in water, and prepared 5–6 standard solutions with different concentrations. The peak areas of each standard solution were determined under the chromatographic conditions described above. Standard curves for each nucleotide were constructed by plotting peak area against concentration, and the linear range and correlation coefficient were subsequently calculated.

2.6. DIETS AND HEPATOPANCREAS FATTY ACIDS

2.6.1. PRE-TREATMENT OPERATION

A 0.2 g sample was weighed into a 15 mL centrifuge tube. One milliliter of methanol was added, followed by 2 mL of hydrochloric acid solution (8.3 mol/L). The mixture was thoroughly mixed and placed in an 80°C water bath for hydrolysis for 40 minutes. After hydrolysis, the sample was cooled to room temperature, and 2 mL of methanol was added and mixed well. Subsequently, 2 mL of petroleum ether–ethyl ether (1:1, v/v) was added for extraction. The extraction procedure was repeated three times, and the extracts were combined and dried under a stream of nitrogen to obtain the fat extract.

To the centrifuge tube containing the fat extract, 500 μL of n-hexane was added, followed by 2 mL of 2.5% sulfuric acid–methanol solution. The mixture was reacted in an 80°C water bath for 1 hour. After cooling, 1 mL of n-hexane was added and mixed thoroughly, followed by the addition of 1 mL of saturated sodium chloride solution. The mixture was shaken well, and the upper n-hexane layer was transferred to an EP tube. The extraction was repeated three additional times, and the n-hexane phases were combined. The solvent was evaporated completely under a stream of nitrogen, and the residue was reconstituted to a final volume of 0.5 mL with n-hexane. The solution was vortexed to ensure complete dissolution, and an appropriate aliquot was filtered into a sample vial fitted with an inner liner for analysis. Samples containing higher concentrations of certain analytes were further diluted 10-fold and 100-fold prior to detection.

2.6.3. CHROMATOGRAPHIC ANALYSIS CONDITIONS

Chromatograph: Agilent 6890N gas chromatograph. Chromatographic column: WM-CN100 (100 m * 0.25 mm * 0.2 μm)

Detector (FID) temperature: 260 ℃. Programmed temperature rise: 0-51 min; 140 ℃ - 240 ℃. Split ratio: 10:1.

2.6.4. STANDARD CURVE DETERMINATION

37 fatty acid methyl ester standard substances were accurately weighed and dissolved in n-hexane. Five to six standard solutions with different mass concentrations were prepared. Under the above chromatographic conditions, the peak areas of the 37 fatty acid methyl esters in each standard solution were determined sequentially. The peak area was used as the ordinate and the concentration as the abscissa to construct standard curves for each fatty acid methyl ester. The linear range and correlation coefficient for each fatty acid methyl ester were then determined.

2.7. Statistical Analysis

The experimental data were presented as mean ± standard deviation (n = 4). All data were analyzed using SPSS 22.0 statistical software. A one-way analysis of variance (ANOVA) and Turkey’s multiple range tests were used to determine the statistical significance among groups. Statistical significance was determined at P < 0.05.

3. RESULT

3.1. MUSCLE FAAS COMPOSITION

Several muscle FAAs with relatively high contents were glutamic acid, glycine, arginine, and proline, while several muscle FAAs with relatively low contents were serine, histidine, tyrosine, and phenylalanine (Table 4). Muscle-free flavor amino acids (FFAAs) in the BSFL and GASM groups reached the same level as that of the TF group (P > 0.05). Among the FFAAs (aspartic acid, glutamic acid, glycine and alanine), muscle free aspartic acid content in the MW group was significantly higher than other groups (P < 0.05). Muscle free glutamic acid content in the TF group was significantly higher than other groups (P < 0.05). Muscle free glycine content of the BSFL and GASM groups reached the same level as that of the TF group (P > 0.05). Muscle free alanine content of the GASM group was significantly higher than the other groups (P < 0.05). Muscle total FAAs (TFAAs) content in the MW group shows no significant diference with that of the TF group (P > 0.05), while the other groups were significantly lower than the TF group (P < 0.05).

Table 4.Muscle FAAs composition of E. sinensis fed with different baits (mg/100 g fresh tissue)
Item Groups
TF MW BSFL GASM FLM
Aspartic acid 23.21±0.94b 29.37±0.74a 24.42±0.92b 22.43±0.50b 24.31±0.55b
Glutamic acid 114.90±3.99a 87.51±2.25b 67.61±2.31c 66.36±5.39c 85.35±5.00b
Histidine 21.44±0.91b 28.17±1.37a 21.22±1.27b 30.61±1.88a 19.45b±0.83b
Glycine 276.30±7.01a 224.43±6.87b 245.45±8.94ab 267.78±12.69a 234.18±18.55b
Histidine 11.16±0.77c 19.62±0.13a 12.74±0.59b 10.14±0.49c 10.94±0.31c
Arginine 320.70±8.51bc 314.12±14.33c 344.25±7.30b 371.65±7.67a 376.32±10.13a
Tyrosine 31.43±1.12a 33.44±1.14a 15.70±0.98b 15.54±1.32b 18.28±0.81b
Aspartic acid 130.93±7.08d 159.89±7.25c 183.11±3.49b 200.27±6.79a 152.60±5.22c
Proline 327.00±6.18a 344.08±11.19a 226.01±5.98b 183.46±15.70c 323.72±15.98a
Tyrosine 11.88±0.49b 17.78±1.31a 8.51±0.76c 7.02±0.55c 8.04±0.63c
Valine 27.52±0.72c 42.48±2.06a 23.93±1.48c 16.01±1.49d 33.8±1.71b
Methionine 98.71±5.19a 9.73±0.37d 19.76±0.99c 34.30±1.52b 24.53±0.94c
Isoleucine 18.39±0.90a 18.01±0.48a 8.89±0.41c 8.18±0.31c 11.07±0.55b
Leucine 28.42±1.31a 29.39±0.73a 13.69±1.07c 14.52±0.74c 17.89±0.93b
Phenylalanine 11.56±0.93a 8.91±0.38b 4.86±0.34c 7.46±0.51b 5.07±0.28c
Lysine 53.35±4.72a 42.65±3.88b 32.62±3.02c 30.81±1.95c 37.07±1.94bc
TFAAs 1506.9±43.8a 1409.6±38.5ab 1252.8±12.7d 1278.2±44.2cd 1382.6±50.9bc
FFAAs 545.33±12.40a 501.20±10.48b 520.59±10.57ab 548.51±15.97a 496.44±25.11b

Note: In the same row, values with different superscripts mean significant difference (P < 0.05).
TFAAs: total free amino acids. FFAAs: free flavor amino acids.

3.2. HEPATOPANCREAS FAAS COMPOSITION

Hepatopancreas FAAs with relatively high contents in each group were leucine, arginine and lysine, while hepatopancreas FAAs with relatively low contents were serine, histidine and aspartic acid (Table 5). The hepatopancreas FAAs and FFAAs in the GASM group show no significant difference with those of the TF group (P > 0.05), while the other three insect diet groups were significantly lower than the TF group (P < 0.05). Among the FFAAs, the GASM group hepatopancreas free aspartic acid and alanine contents reached the same levels as those of the TF group (P > 0.05), and the free glycine content was significantly higher than that of the TF group (P < 0.05). In other insect diet groups, only the FLM showed the free glutamic acid content reached the same level as that of the TF group (P > 0.05).

Table 5.Hepatopancreas FAAs composition of E. sinensis fed with different baits (mg/100 g fresh tissue).
Item Groups
TF MW BSFL GASM FLM
Aspartic acid 20.48±1.18a 8.49±0.86c 15.71±1.32b 19.33±0.93a 14.49±0.89b
Glutamic acid 70.55±6.18a 47.39±3.71c 50.42±4.27c 55.60±5.18bc 66.06±4.22ab
Serine 32.55±2.63a 14.98±1.80b 29.43±1.86a 32.56±3.05a 17.78±1.12b
Glycine 41.42±3.09b 22.95±2.54d 33.39±3.08c 56.58±3.37a 31.66±2.75c
Histidine 26.90±2.29a 18.37±0.82b 21.06±1.44b 30.43±2.09a 18.10±0.80b
Arginine 311.06±15.41b 233.21±12.22c 227.84±10.14c 305.73±7.64a 192.83±10.71d
Threonine 32.54±2.36a 15.50±1.53c 26.98±1.25b 33.40±2.37a 17.74±1.43c
Alanine 105.90±6.47a 78.58±6.63b 59.45±4.16c 99.35±8.41a 63.81±4.75bc
Proline 95.32±7.79a 70.19±3.95b 57.48±4.95b 31.92±3.45c 61.86±4.97b
Tryptophan 77.93±6.89ab 42.36±4.08c 63.57±5.78b 82.19±6.95a 47.26±3.09c
Valine 50.82±3.56ab 25.07±2.57c 41.90±3.84b 51.65±4.94a 27.35±2.22c
Methionine 43.03±3.50a 17.19±2.00d 28.41±1.44c 36.52±2.56b 19.67±0.94d
Isoleucine 40.77±3.15a 22.53±1.91b 37.20±1.93a 42.69±2.63a 21.20±1.57b
Leucine 116.93±9.13a 67.91±4.72c 96.00±5.40b 129.15±8.71a 71.59±3.33c
Phenylalanine 73.20±4.87b 40.83±2.33d 58.85±5.40c 91.36±6.82a 48.15±2.21cd
Lysine 193.43±10.32b 153.72±3.18c 182.16±6.52b 222.73±10.63a 153.87±10.58c
TFAAs 1332.8±80.3a 879.3±42.6c 1029.9±41.8b 1321.2±95.1a 873.4±41.1c
FFAAs 238.35±15.92a 157.41±9.24b 158.98±9.32b 230.86±17.86a 176.01±11.90b

Note: In the same row, values with different superscripts mean significant difference (P < 0.05).
TFAAs: total free amino acids. FFAAs: free flavor amino acids.

3.3. THE MUSCLE AND HEPATOPANCREAS FLAVOR NUCLEOTIDES CONTENT

In muscle, the GASM group 5’-GMP content shows no significant difference with that of the TF group (P > 0.05) (Table 6). The GASM and MW group 5’-AMP content reached the same level as that of the TF group (P > 0.05). In hepatopancreas, the GASM and BSFL group 5’-GMP content shows no significant difference with that of the TF group (P > 0.05); The GASM group 5’-IMP and 5’-AMP content reached the same level as that of the TF group (P > 0.05).

Table 6.Muscle and hepatopancreas flavor nucleotide content of E. sinensis fed with different baits (fresh tissue, mg/100 g).
Item Groups
TF MW BSFL GASM FLM
Muscle
5'-GMP 31.99±1.73a 26.87±1.41bc 21.72±2.32d 31.18±1.52ab 22.24±2.32cd
5'-IMP 108.01±8.39a 80.80±4.93b 63.37±6.04c 83.26±6.07b 67.36±6.27bc
5'-AMP 96.03±5.66a 86.70±5.84a 66.07±4.41b 94.90±4.82a 67.71±4.04b
Hepatopancreas
5'-GMP 29.13±2.35a 15.23±1.30b 25.20±1.62a 28.91±1.71a 15.94±1.18b
5'-IMP 79.42±1.84a 62.00±3.50b 59.20±2.93b 83.86±3.73a 62.51±2.55b
5'-AMP 42.39±2.60a 21.13±2.35c 20.65±1.92c 42.59±2.57a 33.74±3.68b

Note: In the same row, values with different superscripts mean significant difference (P < 0.05).
In the same row, values with different superscripts mean significant difference (P < 0.05).
GMP, guanylic acid sodium salt; IMP, sodium Inosinate; AMP, sodium adenosine triphosphate.

3.4. HEPATOPANCREAS FATTY ACIDS COMPOSITION

The BSFL group had a significantly higher SFA proportion in the hepatopancreas than the other groups (P < 0.05) (Table 7). The MW and FLM group hepatopancreas MUFA proportion were significantly higher than that of the other groups (P < 0.05). The TF group n-3 PUFAs proportion was significantly higher than that of the other groups (P < 0.05). The GASM group n-3 PUFAs proportion was significantly higher than that of the MW, BSFL, and FLM groups (P < 0.05).

Table 7.Hepatopancreas fatty acid composition of E. sinensis fed with different diets (% total identified fatty acids).
item Groups
TF MW BSFL GASM FLM
C6:0 0.03±0.01bc 0.05±0.01b 0.02±0.01c 0.19±0.02a 0.04±0.01bc
C8:0 0.14±0.02b 0.13±0.01b 0.10±0.01c 0.34±0.02a 0.16±0.01b
C10:0 0.01±0.01c 0.01±0.01c 0.18±0.01a 0.05±0.01b 0.01±0.01c
C12:0 0.05±0.01b 0.11±0.01b 16.62±0.53a 0.16±0.02b 0.11±0.01b
C14:0 1.35±0.07c 1.39±0.05c 4.96±0.09a 2.47±0.15b 1.25±0.12c
C15:0 0.47±0.09cd 0.81±0.05b 0.45±0.09d 2.64±0.06a 0.65±0.07bc
C16:0 20.63±1.03b 15.58±0.80c 16.39±1.06c 24.29±0.82a 15.71±0.88c
C17:0 0.13±0.01ab 0.16±0.02ab 0.09±0.01b 0.69±0.50a 0.12±0.01ab
C18:0 2.53±0.14bc 2.83±0.07b 2.25±0.10cd 6.24±0.26a 2.03±0.14d
C20:0 0.01±0.01c 0.13±0.02ab 0.01±0.01c 0.15±0.01a 0.11±0.01b
C14:1 0.10±0.01c 0.07±0.01c 0.51±0.04a 0.30±0.02b 0.07±0.01c
C16:1 5.70±0.29c 4.26±0.33d 6.73±0.14b 9.70±0.16a 6.29±0.24bc
C17:1 0.19±0.01c 0.25±0.02b 0.26±0.01b 0.79±0.03a 0.23±0.03bc
C18:1n9 29.88±1.37b 41.87±1.02a 28.54±1.04b 19.80±1.12c 39.83±1.92a
C20:1 0.92±0.03a 0.31±0.02c 0.11±0.02d 0.45±0.03b 0.38±0.03b
C20:2 1.48±0.04b 1.13±0.06c 0.36±0.03e 3.12±0.20a 0.78±0.03d
C18:2n6 25.63±1.30b 29.82±1.36a 20.78±1.41c 11.44±0.88d 29.84±1.76a
C18:3n6 0.09±0.01a 0.01±0.01c 0.03±0.01b 0.03±0.01bc 0.04±0.01b
C20:3n6 0.30±0.01b 0.05±0.01c 0.05±0.01c 1.18±0.03a 0.07±0.01c
C20:4n6 ARA 0.30±0.01b 0.05±0.01b 0.05±0.01b 1.18±0.03a 0.07±0.01b
C18:3n3 0.18±0.03b 0.15±0.01b 0.17±0.01b 0.26±0.02a 0.25±0.01a
C20:3n3 0.41±0.02a 0.12±0.01d 0.10±0.01d 0.29±0.02b 0.17±0.01c
C20:5n3 EPA 1.22±0.07b 0.57±0.04d 0.64±0.07d 2.45±0.07a 1.03±0.06c
C22:6n3 DHA 8.02±0.10a 0.58±0.06c 0.33±0.03d 2.74±0.06b 0.38±0.07d
SFA 25.36±1.39c 21.21±0.92d 41.07±1.71a 37.23±0.87b 20.19±1.23d
MUFA 38.27±1.17b 47.89±1.43a 36.51±1.25b 34.16±1.51b 47.59±2.26a
n−6 PUFAs 26.52±1.35bc 30.36±1.41ab 21.60±1.47d 23.55±1.27cd 30.85±1.81a
n−3 PUFAs 9.83±0.20a 1.42±0.09d 1.24±0.12d 5.75±0.15b 1.83±0.15c
n-6/n-3PUFAs 2.70±0.09d 21.41±0.35a 17.42+0.45b 4.09±0.11c 16.90±0.37b

Note: In the same row, values with different superscripts mean significant difference (P < 0.05). ARA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.

4. DISCUSSION

4.1. FACTORS AFFECTING THE EDIBLE QUALITY AND FLAVOR

The umami and sweetness of the muscle and hepatopancreas in E. sinensis are highly favored by consumers, mainly due to the presence of a large amount of FAAs, flavor nucleotides, and fatty acids.22 The content of FAAs is a key factor influencing the flavor of meat. For instance, serine, glycine, and alanine have a sweet taste, while aspartic acid and glutamic acid have an inherent sour taste on their own, and this sourness manifests as umami when sodium salt is present.23 Meanwhile, glycine, lysine, phenylalanine, and threonine can undergo Maillard reactions with glucose to produce various aromas.24 Additionally, acidic amino acids and various neutral amino acids can enhance the flavor through the interaction with IMP taste receptors.25 The main components of FAAs in the muscle of E. sinensis are glutamic acid, glycine, arginine, and Proline. Studies on crucian carp (Carassius Auratus)26 and Senegal sole (Solea Senegalens)27 have shown that an increase in the levels of glutamic acid and alanine can significantly enhance the umami flavor of the meat. Moreover, it has been demonstrated that glycine plays a significant role in presenting sweetness in snow crab (Chionoecetes opilio),28 clams,29 and scallops.30 Among the flavor nucleotides, Wang et al.31 reported that the contents of AMP, IMP, and GMP are important flavor substances that affect the edible quality of E. sinensis, with AMP mainly increasing sweetness, and IMP and GMP mainly enhancing umami flavor.

Turchini et al.32 mentioned in their study that the change in the fatty acid composition of fish fillets would affect the total volatile compounds, thereby affecting the flavor. n−3PUFAs are healthy lipids that improve the absorption, digestion, and transport of nutrients and are very important for growth performance. It can enhance the nutritional value of crabs and may also generate volatile flavor substances through lipid oxidation, making the crab meat aroma more intense. Experts from the Food and Agriculture Organization of the United Nations (FAO) proposed that the ratio of n-6/n-3 PUFAs in food should be between 5:1 and 10:1. Fillmore et al.33 and El-Ansary et al.34 suggested that it is beneficial to human health when the ratio of n-6/n-3 PUFAs in the human body is 1:1. Currently, as human fat intake continues to increase, the intake of n-3 PUFAs has significantly decreased, while the intake of n-6 PUFAs has continuously increased. The ratio of n-6/n-3 PUFAs has reached as high as 30:1.35 This severe imbalance ratio will eventually cause lipid metabolism disorders, thereby triggering a series of metabolic diseases such as obesity and diabetes.36 Therefore, the appropriate ratio of n-6/n-3 PUFAs in the fatty acid composition is of great significance for flavor and nutritional quality. An appropriate n-6/n-3 ratio helps maintain lipid metabolism balance, prevents excessive oxidation that can generate unpleasant odors, and simultaneously enhances the freshness, aroma, and harmony of the crab meat’s taste.

4.2. THE INFLUENCE OF MW ON THE EDIBLE QUALITY AND FLAVOR

In recent years, many studies have conducted experiments on replacing dietary fish meal with MW. Xu et al.37 pointed out that the nutritional components of defatted mealworm meal are similar to those of fish meal and have a strong fishy smell, which has a certain feeding-enticing effect and a relatively small impact on feeding volume. In this experiment, it was also found that the feeding volume of E. sinensis in the MW group was comparable to that in the TF group. Regarding the composition of the feed, the crude protein content of MW (17.51%) was consistent with that of TF (17.63%), while the crude fat (9.46%) and ash (6.87%) contents were higher than those of TF (4.73%, 3.58%), which was consistent with the research results of Christos et al.38 Therefore, MW can provide sufficient protein, fat and mineral sources for E. sinensis.

In this experiment, the muscle TFAAs content in the MW group reached the same level of the TF group, while the FFAAs content was significantly lower than that of the TF group (Table 4), which is consistent with the experimental results of Zhang et al.39 The hepatopancreas TFAAs and FFAAs contents in the MW group were significantly lower than those in the TF group. This result is consistent with the experimental results of Iaconisi et al.,40 who replaced 50% of the dietary fish meal with MW meal for Sparus aurata L., resulting in a decrease in muscle FFAA content. In terms of the diet amino acid composition, these flavor amino acids in MW have no significant difference from those in TF, and the content of alanine (1.21) and glycine (0.81) in MW is higher than that in TF (0.85, 0.58). The reason for the decrease in the muscle and hepatopancreas FFAAs content may lie in the differences in the absorption and metabolism of diet components by the E. sinensis, further research is needed. In the MW group, except for the muscle 5’-AMP content, which reached the same level as that of the TF group, the contents of other umami nucleotides in the hepatopancreas and muscle were significantly lower than those in the TF group, which may also affect the flavor performance of the crabs.

The hepatopancreas SFA and n-3 PUFA content in the MW group was significantly lower than in the TF group, whereas MUFA and n-6 PUFA content were significantly higher in the MW group. Studies on large yellow croaker (Larimichthys crocea),41 European sea bass (Dicentrarchus labrax L.),42 common catfish (Ameiurus melas Raf.),43 rainbow trout,44 tilapia,45 and blackspot sea bream (Pagellus bogaraveo),46 it was found that as the proportion of dietary fish meal replaced by the MW meal increased, the n-3 PUFAs and the ratio of n-3/n-6 PUFAs in the experimental animals showed a downward trend, while the content of linoleic acid and n-6 PUFAs significantly increased. Mastoraki et al.42 reported that the MW linoleic acid content was much higher, consistent with the results of this experiment. Sánchez-Muros et al.47 documented that this is mainly because most terrestrial insects typically rely on plant-based substances as their main food source and lack direct sources of n-3 PUFAs. As a result, they are significantly deficient in EPA and DHA. This phenomenon is consistent with the results in this experiment (Table 3), and is in agreement with the difference in the proportion of fatty acids in the crab hepatopancreas in the MW group. However, EPA and DHA have significant biological functions in aquatic organisms. After replacing TF with MW for feeding, the crabs’ fatty acid nutritional requirements could not be met. The specific impacts resulting from this and the corresponding improvement plans still need to be further explored.

4.3. THE INFLUENCE OF BSFL ON THE EDIBLE QUALITY AND FLAVOR

Studies have shown that the protein content of BSFL is comparable to that of other insect and plant protein sources.48 Although BSFL is in the middle to lower range among insects, the crude fat content of BSFL meal is higher than that of most insect meals, even higher than fish meal and soybean meal. After de-fatting, the crude protein content of defatted BSFL meal can be significantly increased.47,48 In the present study, the BSFL fat content was higher than that of the other four diets, which is consistent with previous studies.

In this experiment, the muscle TFAAs content of the BSFL group was significantly lower than that of the TF group, while the muscle FFAAs content reached the same level as that of the TF group (Table 4). The total hepatopancreas TFAAs and FFAAs contents of the BSFL group were significantly lower than those of the TF group (Table 5). This might be due to the BSFL total amino acid content (9.92%) and the essential amino acid content (4.88%) being significantly lower than those of the TF (13.59% and 6.68%), resulting in insufficient supply, or it might be related to the protein synthesis mechanism of the crabs and their utilization efficiency of the protein in the diet. However, Qiu et al.49 found in their study that feeding male E. sinensis with BSFL resulted in a significant increase in the content of FFAAs. Contrary to the results of this experiment, this might be due to the tissue-specific absorption differences between male and female crabs, and further research on the correlation is needed. In terms of nucleotide content, the contents of 5’-GMP, 5’-IMP, and 5’-AMP in the muscle of the BSFL group, as well as the contents of 5’-IMP and 5’-AMP in the hepatopancreas, were significantly lower than those in the TF group. This may affect the overall synergistic effect of flavor.

In this experiment, the hepatopancreas SFA content in the BSFL group was significantly higher than in the TF group, whereas the n-6 and n-3 PUFAs content was significantly lower than in the TF group (Table 7). Study by Belforti et al.50 also indicated that as the increasing proportion of dietary fish meal was replaced by BSFL meal, the proportion of EPA and DHA in trout fillets significantly decreased, which was consistent with the results of this experiment. This was also demonstrated in feeding experiments conducted on rainbow trout51,52 and largemouth bass.53 The fundamental reason is that the content of n-3 PUFAs in BSFL (0.36%) is significantly lower than that in TF (16.12%), leading to insufficient accumulation of n-3 PUFAs. Therefore, although BSFL has certain characteristics in terms of certain amino acids and fatty acids components, feeding them to crabs has a significant negative impact on the content of crab FFAAs and n-3 PUFAs, which may affect the flavor and nutritional value of the crabs.

4.4. THE INFLUENCE OF FLM ON THE EDIBLE QUALITY AND FLAVOR

FLM is classified as animal feed, and numerous studies have reported that it has great potential for development.54,55 The detection results show that the ash content of FLM (1.80%) is lower than that of TF (3.58%), while the fat content (6.70) is higher than that of TF (4.73). The difference in ash content between the two is consistent with the results of Ogunji et al.56 and Jabir et al.,57 where the ash content of fish decreased as the increase in dietary fish meal was replaced by FLM meal. The report by Weatherup et al.58 also noted that replacing dietary fish meal with FLM meal increased fish lipid levels and decreased ash content. In Chinese mud crabs (Scylla paramamosain),59 Pacific white shrimp (Litopenaeus vannamei),60,61 Chinese soft shelled turtles,62 and tilapia,63 dietary fish meal replaced by FLM meal resulted in a significant increase in body fat content as the amount of FLM meal increased, while protein content remained basically unchanged, which is consistent with the MW nutritional composition results in this study.

In this experiment, the muscle and hepatopancreas TFAAs and FFAAs in the FLM group were significantly lower than those in the TF group (Table 4, Table 5). Shi et al.63 pointed out in their report that the content of Met and lysine in FLM meal was much lower than that in fish meal, which is consistent with the results of this experimental study (Table 2). It indicated that the deficiency of essential amino acids in FLM will affect the growth and metabolic performance of E. sinensis, thereby influencing the edible quality of E. sinensis. Regarding the content of flavor nucleotides, the 5’-GMP, 5’-IMP, and 5’-AMP contents in the muscle and hepatopancreas of the FLM group were significantly lower than those in the TF group. This may have led to a reduction in the flavor of the E. sinensis.

In this experiment, the proportions of SFA and n-3 PUFA in the crab fed with FLM were significantly lower than those in the TF group, while the proportions of MUFA and n-6 PUFAs were significantly higher than those in the TF group (Table 7). This is consistent with the fatty acid composition of TF compared to FLM. Turchini et al.64 and Borgogno et al.65 have reported that differences in fatty acid composition may affect the sensory characteristics of aquatic products at the aroma and flavor levels. Direct feeding of FLM to crabs is bound to have a negative impact on the crabs’ edible quality. FLM, like BSFL and MW, are all terrestrial insects, which usually rely on plant-based substances as their main food source and lack direct sources of n-3 PUFAs. Their nutritional composition is significantly deficient in EPA and DHA. The fatty acids in aquatic animals are usually accumulated through digestion and absorption from their food, so directly feeding them terrestrial insects is likely to cause a deficiency of highly unsaturated fatty acids in their bodies, thereby affecting the edible quality.

4.5. THE INFLUENCE OF GASM ON THE EDIBLE QUALITY AND FLAVOR

In recent years, the rice-calamusin symbiotic system, which involves raising E. sinensis in rice fields to prey on pests such as GAS, has become increasingly common. This system not only protects rice and increases yields, but also breeding E. Sinensis as a by-product to generate economic benefits, creating a virtuous cycle,66 demonstrating the potential of GAS as a food source for E. sinensis. Currently, studies on aquatic animals such as pasupati catfish (Pangasius sp.),5 striped catfish (Pangasianodon hypophthalmus),67,68 tiger shrimp (Penaeus monodon),69 and tilapia70 have shown that dietary fish meal can be part replaced by the GASM meal has no negative impact on the growth performance of the farmed animals. As aquatic organisms, the growth environment and physiological metabolism of the GAS have its own uniqueness, and there is also a certain similarity with TF.7 The crude protein content of GASM is 16.20%, which is similar to that of TF (17.63%), indicating that GASM can provide relatively abundant protein to meet the basic requirements of the growth and development of E. sinensis. Its crude fat content is 3.40%, lower than that of other diet. This may help reduce excessive fat accumulation, which could affect the crab’s taste and flavor. In addition, the ash content of GASM is 3.24%, similar to that of TF (3.58%), indicating that it can meet the physiological needs such as the development of the exoskeleton of E. sinensis.

It is rare to find reports on the influence of GASM as a diet or feed ingredient on the flavor and quality of aquatic animals. The content of FFAAs in the hepatopancreas and muscle of crabs in the GASM group reached the same level as that of the TF group (Tables 4 and 5). The muscle-free alanine and the hepatopancreas-free glycine were even significantly higher than those in the TF group (Tables 4 and 5). This suggests that the crabs in the GASM group exhibited flavor profiles comparable to those in the TF group. Meanwhile, compared with the TF group, the crabs fed with GASM had a relatively minor impact on the composition of flavor nucleotides. Apart from the 5’-IMP content in the muscle being lower than that of the TF group, there were no significant differences in the contents of other taste nucleotides in the muscle and hepatopancreas between the two groups. Nucleotides are synergistic enhancers of umami,25 indicating that the crabs fed with GASM can effectively retain the umami substances, supporting their flavor quality.

In the present study, the proportion of SFA in the hepatopancreas of crabs fed with GASM was significantly higher than that in the TF group, while the proportion of n-3 PUFAs was significantly lower than that in the TF group. Wang et al.17 also documented that feeding E. sinensis with GASM could significantly increase the proportion of SFA and reduce the proportion of n-3 PUFAs in the hepatopancreas. Although the proportion of n-3 PUFAs in the hepatopancreas (5.75) was significantly lower than that in the TF group (9.83), it was still significantly higher than that in the MW (1.42), BSFL (1.24), and FLM (1.83) groups. At the same time, Bombeo-Tuburan et al.69 showed that GASM is a good source of EPA. Therefore, from the perspective of fatty acid composition, the GASM group has a significant substitution advantage over the three types of insect diets and has greater potential as an alternative to the E. sinensis diet. Regarding the flavor substance content in the muscle and hepatopancreas of E. sinensis, GASM also has the potential to replace TF.

5. CONCLUSION

In this experiment, three insect diets have a significant negative impact on the edible quality and flavor of the crabs. The GASM diet showed almost no effect on the muscle and hepatopancreas FFAAs and nucleotides. The proportion of hepatopancreas n-3 PUFAs in the GASM group was significantly lower to the TF group, but it was still significantly higher than those of the three insect groups. In conclusion, based on the measured biochemical indicators, among the four types of diet on the edible quality and flavor, GASM has the greatest potential to replace TF as a diet for E. sinensis.

ACKNOWLEDGMENTS

This work was financially supported by Jiangsu Agri-animal Husbandry Vocational College school level research project (NSF2025ZR08) and Jiangsu Haorun Biological Industry Group Co., Ltd. (S20240619).

AUTHORS’ CONTRIBUTIONS

Conceptualization: Wenxiang Yao (Equal), Chunyan Zhang (Equal). Methodology: Wenxiang Yao (Equal), Chunyan Zhang (Equal). Investigation: Wenxiang Yao (Equal), Chunyan Zhang (Equal). Writing – review & editing: Wenxiang Yao (Equal), Chunyan Zhang (Equal). Project administration: Wenxiang Yao (Equal), Xinze Li (Equal), Xiaoya Wu (Equal), Guoan Hua (Equal), Sizhe Zhao (Equal), Chunyan Zhang (Equal). Funding acquisition: Wenxiang Yao (Equal), Xinze Li (Equal), Xiaoya Wu (Equal), Chunyan Zhang (Equal). Formal Analysis: Ningyang Sun (Equal), Guoan Hua (Equal), Sizhe Zhao (Equal). Writing – original draft: Ningyang Sun (Lead).

ETHICS STATEMENT

All animal care and use procedures were approved by the Institutional Animal Care and Use Committee of Jiangsu Agri-animal Husbandry Vocational College (permit number: jsahvc-2024-25), and all authors clearly indicated that such guidelines have been followed.

CONFLICTS OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

DATA AVAILABILITY

All data generated or analyzed during this study are included in this article.