Fish and Shellfish Immunology 97 (2020) 593–601
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T-2 toxin in the diet suppresses growth and induces immunotoxicity in juvenile Chinese mitten crab (Eriocheir sinensis)
Chunling Wanga, Xiaodan Wanga,∗, Shusheng Xiaoa, Xianyong Bua, Zhideng Lina, Changle Qia, Jian G. Qinb, Liqiao Chena,∗∗ a b
Laboratory of Aquaculture Nutrition and Environmental Health, School of Life Sciences, East China Normal University, 500 Dongchuan Rd, Shanghai, 200241, China School of Biological Sciences, Flinders University, Adelaide, SA, 5001, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: T-2 toxin Eriocheir sinensis juvenile Immunotoxic Growth Hepatopancreas
The T-2 toxin is a trichothecene mycotoxin and is highly toxic to aquatic animals, but little is known on its toxic eﬀect in crustaceans. In the present study, the crab juveniles were fed with diets containing four levels of T-2 toxin: 0 (control), 0.6 (T1), 2.5 (T2) and 5.0 (T3) mg/kg diet for 56 days to evaluate its impact on the juvenile of Chinese mitten crab (Eriocheir sinensis). The crabs fed the T-2 toxin diets had signiﬁcantly lower weight gain and speciﬁc growth rate than those fed the control diet. Moreover, crab survival in T3 group was obviously lower than that in the control. Oxidative stress occurred in all the treatment groups as indicated by higher activities of total superoxide dismutase, glutathione peroxidase, and total antioxidant capacity than those in the control. The total hemocyte count, respiratory burst, phenoloxidase in the hemolymph, and phenoloxidase, acid phosphatase and alkaline phosphatase in the hepatopancreas of crabs fed T-2 toxin were signiﬁcantly lower than those in the control. The transcriptional expressions of lipopolysaccharide-induced TNF-alpha factor, relish, and the apoptosis genes in the hepatopancreas were induced by dietary T-2 toxin. The genes related to detoxication including cytochrome P450 gene superfamily and glutathione S transferase were induced in low concentration, then decreased in high concentration. Dietary T-2 toxin damaged the hepatopancreas structure, especially as seen in the detached basal membrane of hepatopancreatic tubules. This study indicates that dietary T-2 toxin can reduce growth performance, deteriorate health status and cause hepatopancreas dysfunction in crabs.
1. Introduction T-2 toxin is one of the trichothecenes that are a secondary metabolites produced by various fungus species of the genus Fusarium. This toxin is stable in various environmental conditions even by autoclavation and can cause adverse impact such as growth retardation, hepatic damage, immunotoxicity and blood cell reduction [1–4]. The WHO and FAO have listed T-2 toxin a dangerous source of food pollution in nature since 1973 . Because T-2 toxin commonly contaminates grain products including maize, wheat and oats all over the world especially at high temperature and high moisture regions during cultivation and storage . In recent years, the replacement of ﬁsh meal by plant proteins has been accepted as a reality, which increases the risk of mycotoxin contamination such as T-2 toxin in aquatic feed . Therefore, considering the danger and economic loss of T-2 toxin in aquaculture, we face an urgent need to understand the toxic eﬀects of T-2 toxin in aquaculture through feed.
Previous studies have revealed that T-2 toxin can cause growth inhibition, low survival, hepatic injury and immunotoxicity in aquatic animals [8–10]. When the diet is contaminated by T-2, even as low as 0.5 mg/kg, shrimp reduce weight gain . T-2 exposure can also cause weight reduction and kidneys injury in channel catﬁsh . In addition, an exposure to dietary T-2 toxin can exert oxidative stress by increasing the production of free radicals in ﬁsh and chicken [7,11]. Furthermore, T-2 toxin can cause immune system damage, inﬂammation response and apoptosis in Paciﬁc white shrimp, rats and zebraﬁsh [12–14]. Exposure to 0.1 μmol/L or more of T-2 toxin can cause cell apoptosis mainly around the tail area . The dose of 5.3 mg/kg dietary T-2 toxin would decrease non-speciﬁc immunity with a reduction of blood cells and an increase of inﬂammatory cytokines in carp . Apart from the impact of economic loss in aquaculture, dietary T-2 can aﬀect the quality of edible meat of aquatic animals by trace residues. The levels of 17.52 ± 2.87 ng/g and 48.61 ± 3.13 ng/g of
Corresponding author. LANEH, School of Life Sciences, East China Normal University, Dongchuan Road, 200241, Shanghai, PR China. Corresponding author. LANEH, School of Life Sciences, East China Normal University, Dongchuan Road, 200241, Shanghai, PR China. E-mail addresses: [email protected]
(C. Wang), [email protected]
(X. Wang), [email protected]
https://doi.org/10.1016/j.fsi.2019.12.085 Received 13 August 2019; Received in revised form 23 December 2019; Accepted 27 December 2019 Available online 28 December 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved.
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modiﬁed-masked toxins (mT-2s) were found in the hepatopancreas of shrimp following exposure to diﬀerent levels of T-2 toxin for 20 d, posing a potential health risk to consumers . It is therefore necessary to understand the detoxiﬁcation mechanism to alleviate the toxic effects of T-2 toxin and ensure seafood safety to consumers. The liver is the main detoxifying organ for T-2 toxin [16,17] and the cytochrome p450 enzymes (phase Ι) and glutathione-S-transferase (phase Π) in the liver play an important role in biotransformation and detoxiﬁcation of xenobiotics such as T-2 toxin [18,19]. The Chinese mitten crab, Eriocheir sinensis, is a native and important freshwater economic species and its nutrition requirement for growth performance [20–22], metabolism [23,24], immunity [25–27] and disease resistance [28–30] have been well documented. However, little is known on the impact of toxic materials from feed of crabs. A recent study shows that environmental toxin such as benzo[a]pyrene (BaP) can disturb immune system more than metabolic system in crabs . In the diet ingredients of crabs such as maize and wheat are easily contaminated by mycotoxins like T-2 toxin . Up to date, the impacts of T-2 toxin on other aquatic animals such as Paciﬁc white shrimp , black tiger shrimp  and rainbow trout  have been reported, but no data have been reported on its toxicity in crabs. Therefore, in the present study, the eﬀects of the T-2 toxin on growth performance, oxidative stress, immune function and biotransformation enzyme activities were investigated in the juvenile of Chinese mitten crab to gain insight into the T-2 toxic eﬀect and its inﬂuence on meat quality in crabs.
Table 1 Formulation and proximate composition of experimental diets (g kg−1 dry basis). Ingredients
Fish meal Casein Gelatine Corn starch Fish oil Soybean oil Vitamin premixa Mineral premixb Cholesterol Soylecithin CMC Choline chloride Betaine Cellulose BHT T-2 toxin(mg) Total Moisture Crude protein Crude lipid Ash T-2 toxin (mg/kg)
230 213 68 250 20 20 30 30 5 10 20 5 10 88.5 0.5 0 1000 82.4 402.0 60.7 78.8 0
230 213 68 250 20 20 30 30 5 10 20 5 10 88.5 0.5 0.625 1000 86.0 405.2 61.6 75.2 0.5
230 213 68 250 20 20 30 30 5 10 20 5 10 88.5 0.5 2.5 1000 83.7 401.8 63.3 78.8 2.0
230 213 68 250 20 20 30 30 5 10 20 5 10 88.5 0.5 5 1000 83.9 403.3 61.6 80.3 4.6
a Vitamin premix (per 100 g premix): retinol acetate, 0.043 g; thiamin hydrochloride, 0.15 g; riboﬂavin, 0.0625 g; Ca pantothenate, 0.3 g; niacin, 0.3 g; pyridoxine hydrochloride, 0.225 g; para-aminobenzoic acid, 0.1 g; ascorbic acid, 0.5 g; biotin, 0.005 g; folic acid, 0.025 g; cholecalciferol, 0.0075 g; αtocopherol acetate, 0.5 g; menadione, 0.05 g; inositol, 1 g. All ingredients are ﬁlled with α-cellulose to 100 g. b Mineral premix (per 100 g premix): KH2PO4, 21.5 g; NaH2PO4, 10.0 g; Ca (H2PO4)2, 26.5 g; CaCO3, 10.5 g; KCl, 2.8 g; MgSO4·7H2O, 10.0 g; AlCl3·6H2O, 0.024 g; ZnSO4·7H2O, 0.476 g; MnSO4·6H2O, 0.143 g; KI, 0.023 g; CuCl2·2H2O, 0.015 g; CoCl2·6H2O, 0.14 g Calcium lactate, 16.50 g; Fe-citrate, 1 g. All ingredients are diluted with α-cellulose to 100 g.
2. Materials and methods 2.1. Experimental diets Four practical diets were formulated with diﬀerent concentrations of T-2 toxin: 0 (control), 0.6 (T1), 2.5 (T2), 5.0 (T3) mg/kg diet, which were designed according to the LC50 of juvenile Paciﬁc white shrimp at the same speciﬁcation as the crabs we use . The ingredients and proximate composition are presented in Table 1. The T-2 toxin crystalline (Pribolab Pte, Ltd. Singapore; purity > 98%) was dissolved in ethanol and added to the diet. Same amount of ethanol was also added to the control diet. The mixtures were subsequently blended and extruded into 2.5-mm-diameter pellets by a double helix plodder (F-26, SCUT industrial factory, Guangdong, China). Through the extruding process, the extrusion temperature and gelatin and sodium carboxymethyl cellulose pellets in the diets improved the starch digestibility and water stability of diets. These pellets were air-dried to < 10% moisture and stored at −20 °C until use. The proximate composition analysis of diets was analyzed according to the Association of Oﬃcial Analytical Chemists (AOAC) procedures . Moisture content was analyzed by drying the samples at 105 °C until a constant weight. Crude protein was measured by the Kjeldahl method using Kjeltec™ 8200 (Kjeltec, Foss, Sweden). Lipid was determined by the method of soxhlet apparatus with allihn condenser. Ash content were analyzed by burning the dry samples in a muﬄe furnace (PCD-E3000 Serials, Peaks, Japan) at 550 °C for 5 h. And T-2 toxin content is detected by liquid chromatography-tandem mass spectrometry methods (LC-MS/MS) .
daily at 08:00 and 18:00 for 8 weeks. Two hours after feeding, the uneaten feed and feces were siphoned out. Dead crabs were weighed and recorded during the experiment. The water condition was maintained at 26.0 ± 1.0 °C, 8.5 ± 1.0 mg/L dissolved oxygen and 7.5 ± 0.4 pH throughout the experiment. During the trial, the water was exchanged at 30% of the tank volume daily and natural light-dark cycle was used.
2.3. Sample collection At the end of the 8-week feeding trial, all the crabs were fasted for 24 h, and then the crabs of each bucket were counted and weighed. Crabs were subsequently anaesthetized with ice slurry and then hemolymph was taken with a 1-mL syringe from the joints of walking legs. One part of hemolymph was placed at 4 °C for 24 h and centrifuged at 4500 rpm and 4 °C (5415 R, Eppendorf, Germany) for 10 min to get the serum which was stored at −80 °C for enzyme analysis. The other part was diluted with an equal volume of anticoagulant solution (0.1 M glucose, 30 mM citrate, 26 mM citric acid, 0.14 M NaCl, 10 mM EDTA) to count the numbers of total hemocytes (THC) and determinate the respiratory burst. The hepatopancreas of crabs from each group were quickly separated. Six of hepatopancreas were weighed in a group for calculating the hepatosomatic index. Three of them were ﬁxed in 4% paraformaldehyde for histological analysis and the rest were stored at −80 °C for further analysis of gene expressions and enzymatic activities. All research protocols on live crabs in this study were approved by the Committee on the Ethics of Animal Experiments of East China Normal University.
2.2. Experimental crab and feeding management The juvenile crabs were obtained from a local farm in the Chongming County, Shanghai, China. Crabs were acclimated for 7 days before the experiment. A total of 700 crabs (mean weight 2.00 ± 0.05 g) were randomly divided into four groups with ﬁve replicates and thirty-ﬁve crabs each in plastic buckets (200 L, 82 × 62 × 58 cm). In each bucket, four arched tiles and six groups of plastic pipes (12 cm long and 3 cm diameter, six pipes per group) were used to reduce attacking. All crabs were fed to apparent satiation twice 594
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Table 2 Primer pair sequences for real-time qPCR in Chinese mitten crab. Target gene
Primer sequence forward(5’ - 3′)
Primer sequence reverse(5’ - 3′)
LITAF Relish CYP2 CYP3 CYP4 GST Bax Bcl-2 caspase-8 caspase-3 CncC β-actin
TAAAGGCAAGGGAGGCTTCG TCAGGATTCGGTGGCAACTC ACCGCCGCTTCACCTTAC CTCCACGACTACAAGATGTTACGC AAGACTTCGTGGAGGTGTTC CTGTCATCACCATAAAGAAGGCCA GTCAGTGAACCTCAGCTGCAT ATAAGGTGGTTCGCTCCGTC CATGGTGATGAGAATGAC CATTCGCCAGCCTTGCCCACA GCATCCTTCTGGTACCTCGTT CAGGAAATGACCACTGCCGC
GAATGGAGCTTGAGGTGGCA ATCTGCACTTGGACCGATGG CTTGCTTGCCACCATCTGC CACCTCGTTCATGGTAAAGAGC GCACAGCGTTATGTTGGTGAAG CGCCCTGCTCATTCATGTCATAT CACAGCCACATCACCCACGAA TTAACACAGTCCGAGGCCAG TTGGATGAAGTAGAGACG TCTGTCGTGGTTCCTTGTAGCCTT CACTGCTTTGGCTCATCCTTG CGGAACCTCTCATTGCCGA
97 105 188 90 107 114 124 121 120 94 91 95
60.0 60.5 60.3 59.7 59.8 59.6 59.0 59.5 60.2 61.0 60.3 60.8
LITAF: lipopolysaccharide induced TNF factor, CYP2: cytochrome P450, family 2, CYP3: cytochrome P450, family 3, CYP4: cytochrome P450, family 4, GST: glutathione -S-transferase, CncC, cap'n'collar transcription factor.
2.4. Growth performance, survival and hepatosomatic index
an increase in absorbance of 0.001/min.
At the end of the feeding trial, weight gain (WG), speciﬁc growth rate (SGR), survival rate (SR) and the hepatosomatic index (HSI) were calculated as follows:
2.6. RT-qPCR analysis in hepatopancreas tissues Total RNA of the hepatopancreas was isolated using Trizol (RN0101, Aidlab, China) following the manufacturer's instructions. The quantity and concentration of total RNA were measured by the Nanodrop 2000 (Thermo, USA). Then the cDNA was synthesized by the reverse transcription kit (KR116, Tiangen Biotech, China) and kept at −20 °C for real-time quantitative PCR (RT-qPCR). The RT-qPCR was performed in the CFX96TM Real-Time system (Bio-Rad, Hercules, CA, USA) with a SYBR kit (PC3302, Aidlab, China). The qPCR reactions were carried out in a ﬁnal volume of 20 μL, containing 10 μL SYBR mixture, 0.4 μL (10 μM) forward and reverse primers, 8.2 μL H2O and 1 μL cDNA template. The reaction programs were set at 95 °C for 2 min, 40 cycles of 95 °C for 5 s and 60 °C for 30 s with the melt curve ananlysis for 95 °C for 5 s and 0.5 °C per 5 s increment from 65 °C to 95 °C. Relative expression levels were calculated by the 2−ΔΔCT method as described  with β-actin as the housekeeping gene. Oligo nucleotide primers were designed using the Oligo 7 (Table 2).
Weight gain (%) = (ﬁnal weight – initial weight) × 100 / initial weight; Speciﬁc growth rate (%/d) = (Ln ﬁnal weight – Ln initial weight) × 100 /days; Survival rate (%) = Final crab number/ Initial crab number × 100; Hepatosomatic index (%) = (wet hepatopancreatic weight / wet body weight) × 100
2.5. Biochemical assay 2.5.1. Total hemocyte count (THC) measurement The hemocytes were counted on a phase contrast microscope (Nikon TS100, Japan) with a drop of hemolymph-anticoagulant mixture on the hemocytometer.
2.7. Histopathological analysis on the hepatopancreas The hepatopancreas tissue samples were immediately ﬁxed in 4% paraformaldehyde for 24 h, and then dehydrated in a graded series of ethanol (50%–95%) and embedded in paraﬃn. The 5-μm thickness sections were obtained using a microtome and stained with hematoxylin/eosin (H&E). Histopathological changes were observed on an Axioskop microscope (BX51, Olympus, Tokyo, Japan).
2.5.2. Respiratory burst activity Respiratory burst of the hemolymph was measured using the nitroblue-tetrazolium (NBT; Sangon Biotech Co., Ltd., China) assay following the method of Anderson and Siwicki . Brieﬂy, 100 μL anticoagulant and hemolymph mixture was mixed with 0.1 mL 0.2% NBT solution and incubated for 30 min at room temperature. The N, N-dimethyl formamide (DMF; Sangon Biotech Co., Ltd., China) was added into the mixture and the optical density (OD) of supernatant was measured using a microplate reader at 540 nm with DMF as the blank.
2.8. Statistical analyses All data were statistically analyzed using SPSS statistics 20 (IBM, Armonk, NY, USA) with one-way analysis of variance (ANOVA) and the diﬀerences were analyzed among groups by Duncan's multiple-comparison test. A value of P < 0.05 was set as a signiﬁcant diﬀerence.
2.5.3. Enzymatic analysis Acid phosphatase (ACP), alkaline phosphatase (AKP), malondialdehyde (MDA), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), catalase (CAT) and glutathione peroxidase (GPx) activities were detected according to the manufacturer's instructions of commercial kits (Cat. No. A060–2, A059–2, A003–1, A001–1, A015–2, A007-1-1 and A005, Nanjing Jiancheng Bioengineering Institute, China). The phenoloxidase (PO) was evaluated spectrophotometrically using L-DOPA (A610407, Sangon Biotech Co., Ltd., China) as the substrate in a 96-well plate following the method described by Ashida (1971). The enzyme activity was measured at the absorbance of 490 nm wavelength using a microplate reader in an interval of 2 min for 20 min. The PO activity was deﬁned as the amount of enzyme yielding
3. Result 3.1. Growth performance and survival At the end of the 8-week feeding trial, weight gain and speciﬁc growth rate signiﬁcantly decreased in the T-2 toxin treatment groups (P < 0.05) (Fig. 1A and B) compared with those in the control. The lowest survival was observed in the T3 group with 5 mg/kg dietary T-2 toxin (P < 0.05) (Fig. 1E). No signiﬁcant diﬀerences were observed in feed intake and hepatosomatic index among all groups (P > 0.05) 595
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Fig. 1. Growth performance and survival rate of crabs fed diﬀerent concentrations of T-2 toxin for 56 days. A. Weight gain; B. Speciﬁc growth rate; C. Feed intake; D. Hepatosomatic index; E. Survival rate. Diﬀerent letters show signiﬁcant diﬀerences among treatments (P < 0.05).
(P < 0.05) (Fig. 3C and F). Dietary T-2 toxin signiﬁcantly downregulated the ACP and AKP activities in the hepatopancreas (P < 0.05) (Fig. 3G and H), while in the hemolymph, no signiﬁcant diﬀerence was found in ACP and AKP activities among all the groups (P > 0.05) (Fig. 3D and E).
(Fig. 1C and D). 3.2. Antioxidant status in hepatopancreas As shown in Fig. 2, the hepatopancreas MDA content, activities of TSOD, GPx and T-AOC and CncC (cap ‘n’ collar isoform-C) gene expression were signiﬁcantly aﬀected by dietary T-2 toxin in a dose-dependent manner after the 8-week feeding trial (P < 0.05). Compared with the control, a signiﬁcant increase in hepatopancreatic MDA and depression of hepatopancreatic T-SOD, GPx, T-AOC and CncC were observed in the T-2 groups (P < 0.05).
3.4. Expression of inﬂammation-related genes in hepatopancreas The expression of LITAF and relish were signiﬁcantly upregulated in T3 group compared with the control (P < 0.05) (Fig. 4A). Furthermore, the ALF1 gene expression in T1 group was signiﬁcantly higher than that in control (P < 0.05) (Fig. 4C). The mRNA level of ALF2 in groups T1 and T2 were markedly higher than those in the control (P < 0.05) (Fig. 4D).
3.3. Enzyme activities of non-speciﬁc immunity in hemolymph and hepatopancreas
3.5. Expression of genes associated with apoptosis
THC and respiratory burst activity in the hemolymph signiﬁcantly decreased compared to the control (P < 0.05) (Fig. 3A and B). Furthermore, the PO activity in the hemolymph and hepatopancreas signiﬁcantly decreased in the crabs fed T-2 toxin supplementation
Compared with the control, the expression of the caspase-3 gene was signiﬁcantly increased in the T1 and T3 groups (P < 0.05) Fig. 2. The content of malondialdehyde (MDA) (A), activities of total superoxide dismutase (TSOD) (B), glutathione peroxidase (GPx, GSH-Px) (C) and total antioxidant capacity (T-AOC) (D) and CncC (E) gene expression in the hepatopancreas of crabs fed diﬀerent concentrations of T-2 toxin for 56 days. Diﬀerent letters show signiﬁcant diﬀerences among treatments (P < 0.05).
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Fig. 3. Total hemocyte count (THC) (A), respiratory burst (B), phenoloxidase (PO) (C), acid phosphatase (ACP) (D) and alkaline phosphatase (AKP) (E) in hemolymph and phenoloxidase (PO) (F), acid phosphatase (ACP) (G) and alkaline phosphatase (AKP) (H) in hepatopancreas of crabs exposed to diﬀerent concentrations of T-2 toxin for 56 days. Diﬀerent letters show signiﬁcant diﬀerences among treatments (P < 0.05).
3.7. Hepatopancreas morphology Compared to the control, the volume of B cell increased, and the number of the R cell decreased in the T-2 toxin group. With the increase of dietary T-2 toxin, the basal membrane of hepatopancreatic tubules was seriously detached and the lumen space was enlarged in the T3 group (Fig. 7).
4. Discussion In the present study, dietary T-2 toxin depressed the growth of juvenile E. sinensis, which is consistent with previous studies on Litopenaeus vannamei [8,37]. The low survival of crabs fed 4.6 mg/kg T2 toxin indicates that the health status of crabs is aﬀected by T-2 toxin. Similarly, a marked reduction in survival was also reported in Paciﬁc white shrimp  and rainbow trout . A previous study suggests that reactive oxygen species (ROS) can intensify the toxicity induced by T-2 toxin . The excessive ROS reacts with biological molecules such as lipids, causing lipid peroxidation, and this process can be measured by the content of MDA . According to the present result, the MDA content in hepatopancreas increased when the crabs were fed with T-2 toxin, suggesting that lipid peroxidation is a response in E. sinensis to dietary T-2 toxin. At the same time, T-2 toxin decreased antioxidant enzyme activities (T-SOD, GPx and T-AOC), suggesting that T-2 toxin could attenuate antioxidant capacity in crabs. The T-SOD and GPx are both critical anti-oxidative enzymes with the function of scavenging ROS, and T-AOC is an important index to evaluate the antioxidative status . Moreover, antioxidases could be regulated by the key nucleus transcription factor, nuclear erythroid 2-related factor (Nrf2) to ameliorate oxidative stress in vertebrates . In invertebrates, the CncC gene is orthologous to the vertebrate Nrf2 gene . In this study, the CncC gene expression in the hepatopancreas decreased in the crab fed T-2, suggesting that T-2 toxin could suppress the Nrf2 signal pathway. Furthermore, the activities of antioxidases were positively correlated with Nrf2 gene expression , which conﬁrms that T-2 toxin can suppress the antioxidases by inhibiting the Nrf2 signal pathway and suggest that T-2 toxin can impair the anti-oxidative ability and cause oxidative damage in the hepatopancreas of juvenile crabs. Crustaceans lack adaptive immunity and depend solely on the non-
Fig. 4. Relative Lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) (A), relish (B), Anti-lipopolysaccharide factor 1 (ALF1) (C) and Antilipopolysaccharide factor 2 (ALF2) (D) mRNA expression in the hepatopancreas of crabs exposed to diﬀerent concentrations of T-2 toxin for 56 days. Diﬀerent letters show signiﬁcant diﬀerences among treatments (P < 0.05).
(Fig. 5A). The expressions of the caspase-8 gene and Bax were signiﬁcantly increased in T3 group (P < 0.05) (Fig. 5B and C), while expression of Bcl-2 gene was signiﬁcantly increased in group T1 (P < 0.05) (Fig. 5D).
3.6. Detoxiﬁcation The mRNA expressions of CYP2, CYP3 and CYP4 in T1 group were higher than in other groups (Fig. 6A, B and 6C). The mRNA level of GST in T2 group was signiﬁcantly higher than that in the control and T1 or T3 group (P < 0.05) (Fig. 6D).
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Fig. 5. Relative caspase-3 (A), caspase-8 (B), Bax (C) and Bcl-2 (D) mRNA expression and the ratio of Bax/Bcl-2 in the hepatopancreas of crabs exposed to diﬀerent concentrations of T-2 toxin for 56 days. Diﬀerent letters show signiﬁcant diﬀerences among treatments (P < 0.05).
and AKP activities of hepatopancreas and PO activities of hepatopancreas and hemolymph in all T-2 toxin additive groups were signiﬁcantly decreased compared to the control, indicating that dietary T-2 toxin can disable the response to xenobiotics or pathogen. Therefore, these results reveal that dietary T-2 toxin might be detrimental to the innate immune system of E. sinensis. The immune function is also associated with the inﬂammatory response related to pro-inﬂammatory cytokines modulated by the transcription factors LITAF and NF-κB [51,52]. In crustaceans, relish is an important member of the NF-κB family, and can induce pro-inﬂammatory cytokines expression . Moreover, LITAF, as a transcription factor, has a similar function as NF-κB to regulate the expression of TNF-α, a mediator of inﬂammation . In the present study, the expression of LITAF and relish genes was induced by T-2 toxin in T3 group, indicating that a high concentration of T-2 toxin may induce inﬂammation in the hepatopancreas of crabs. In addition, ALF1 and ALF2 are important antimicrobial polypeptides and can modulate inﬂammatory responses by downregulating the release of pro-inﬂammatory cytokines . After T-2 toxin exposure, ALF1 and ALF2 gene expressions in the hepatopancreas were induced in T1 group and then decreased in T2 and T3 groups with the dose increase of T-2 toxin in the diet. These results suggest that crabs have an adaptability to T-2 toxin stress initially, but the high concentrations T-2 toxin impede the recovery of homoeostasis. The changes of ALF1 and ALF2 gene expressions further prove the occurrence of inﬂammation in the hepatopancreas induced by T-2 toxin. The oxidative damage can contribute to cell apoptosis in grass carp . This study demonstrates that T-2 toxin resulted in oxidative stress in the hepatopancreas of crabs, suggesting that T-2 toxin may cause cell apoptosis in the hepatopancreas. Other studies show that apoptosis can be initiated mainly by the death receptor pathway (caspase-8) and mitochondrial apoptosis pathway (caspase-3) in mammalian regulated by up-regulating the transcription of pro-apoptotic genes (such as Bax) and anti-apoptotic genes (such as Bcl-2) [57,58]. Bax promotes apoptosis by homodimerizing or heterodimerizing Bcl-2 and the ratio of Bax/Bcl-2 to determine the progress of cell apoptosis . In the present study, apoptosis was characterized by activation of caspase-3, caspase-8 and Bax gene expressions, and inhibition of Bcl-2 gene expression induced by T-2 toxin even at the lowest T-2 concentration (0.5 mg/kg) in the hepatopancreas. These results suggest that apoptosis in the hepatopancreas after T-2 toxin intake is proceeded via both
Fig. 6. Relative CYP2 (A), CYP3 (B), CYP4 (C) and GST (D) mRNA expression in hepatopancreas of crabs exposed to diﬀerent concentrations of T-2 toxin for 56 days. Diﬀerent letters show signiﬁcant diﬀerences among treatments (P < 0.05).
speciﬁc immune system including against invading microbes . Hematological parameters such as hemolymph cell counts and respiratory burst activity are important for evaluating immune functions . The hemolymph cell count includes semi-granular cells involved in phagocytosis and large granular cells participated in the storage and release of PO, which is a sensitive indicator to reﬂect the immune status of invertebrates [46,47]. The respiratory burst activity is connected with the mechanism that pathogens and parasites are eliminated following phagocytosis . In this study, the decrease of total hemocyte counts (THCs) and respiratory burst activity following T-2 toxin exposure may be due to the inhibition of mobilization or apoptosis of hemocytes caused by T-2 toxin. Moreover, ACP, AKP, PO activities can reﬂect the innate immune capacity with important functions of elimination of extracellular invaders [49,50]. In the present study, the ACP 598
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Fig. 7. Hepatopancreas histopathology of crabs exposed to T-2 toxin for 56 days (H&E 20 x). A-D represent the control, group T1, group T2 and group T3. B: B cells (secretory cells); R: R cell (storage cells). Two-head arrows indicate separation between the myoepithelial layer and the epithelium. Scale bar = 50 μm.
similar observation was reported in a previous study in which diclofenac could activate CYP450 gene expression . This indicates that CYP450 genes may play an important role in catalyzing detoxiﬁcation of T-2 toxin. In addition, the phase I metabolites are further metabolized by phase II enzyme glutathione S transferase (GST), and play important roles in the detoxiﬁcation of toxic endogenous substrates in almost all living organisms to produce water-soluble conjugates and make them easier to excrete [67–69]. The most signiﬁcantly increased hepatopancreatic GST gene expression in T2 group was observed in our study, which probably represents a detoxiﬁcation response to T-2 exposure. These results are in accordance with a study conducted by Wang et al. , who observed an increase at the low dose and a decrease at the high dose of GST activity after exposing Daphnia magna to ibuprofen. Presumably, the upregulations of GST and CYP450 in T1 and T2 may be due to the defense response of crabs and prompt to protecting themselves at a low T-2 dose. The biotransformation system was impaired at high T-2 doses.
mitochondrial apoptotic pathway and death receptor pathway regulated by the Bax/Bcl-2 ratio. The hepatopancreas in crustacean functions as the liver, pancreas, and intestine in vertebrates and plays a critical role in metabolism, nutrient absorption, and immune function . Therefore, it serves as a critical target organ when crabs are exposed to T-2 toxin . Thereby, histopathological alternations of the hepatopancreas could be used to evaluate T-2 toxin toxicity. In the current study, the T-2 toxin at different doses caused considerable histological damage of the hepatopancreas, including B cell dilatation, degenerated R cells and detached basal membrane of hepatopancreatic tubules. Similar histological variations in the hepatopancreas of L. vannamei were also reported by Wang et al.  after exposure to aﬂatoxin B1. This suggests that T-2 toxin can potentially cause an injury and aﬀect physiological functions of the hepatopancreas at the tissue and organ levels. In general, hepatopancreas is the main detoxifying organ for xenobiotics . When animals are exposed to xenobiotic, the biotransformation process in the hepatopancreas is activated for detoxiﬁcation mediated by the phase I and phase II biotransformation system . In phase I system, xenobiotic or toxin could be metabolized by facilitating bioactivation of the multi-enzymatic system cytochrome P450 (CYP450) gene superfamily. The CYP450 has been identiﬁed as a key enzyme in the metabolism of T-2 toxin, and can transform T-2 toxin into 3′–OH–T-2 toxin highly expressed in the liver . Moreover, The CYP2, CYP3 and CYP4 subfamilies have important roles in xenobiotic metabolism in crustaceans . In the present study, the expression of CYP450 homologous genes was signiﬁcantly induced in the T1 group, and then gradually decreased with the increase of T-2 toxin concentration, displaying a dose-dependent responding relationship. A
5. Conclusion In the present study, the toxic eﬀects of T-2 toxin on E. sinensis was evaluated for the ﬁrst time. Our data show that the decrease in growth performance and impairment of immune function induced by T-2 toxin might partially be due to (1) oxidative damage caused by ROS and reduction of antioxidase activity associated with the inhibition of CncC gene expression; (2) inﬂammation, cell apoptosis and hepatopancreatic injury; and (3) reduction in detoxiﬁcation capacity including the phase I and phase II biotransformation system. These data provide a better insight into the understanding of molecular toxic mechanisms of T-2 599
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toxin. Our results alert to the awareness of the ecological risks of T-2 toxin in aquatic feed.
Declaration of competing interest 
The authors declare no conﬂict of interest. Acknowledgements
This work was supported by Shanghai Sailing Program (Grant No. 18YF1406500), “Chenguang Program” supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (Grant No. 17CG26), the National Natural Science Foundation of China (Grant No. 31572629), Agriculture Research System of Shanghai, China (Grant No. 201804) and China Agriculture Research System-48 (CARS-48).
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