One-year-old adult M. nipponense (weight, 4.4 ± 0.2 g) were bought from Huzhou fish farm (Zhejiang, China) and raised in 50 L glass aquaria with aerated water, a controlled temperature of 25 ± 2 °C, and the photoperiod was a 14 h:10 h light/dark cycle. Males were chosen to minimize possible interfering effects of sex. MC-LR (purity ≥ 98%) was purchased from Alexis Biochemicals (Lauseanne, Switzerland).

M. nipponense were exposed to MC-LR or a solvent control (0.05% methanol, v/v) in a 20 L glass tank for 12, 24, 48, 72 and 96 h (15 individuals per tank in triplicate and approximately 3.3 g shrimp/L). The MC-LR doses were 0.2, 1, 5, and 25 μg/L, according to environmental content of MCs. Half of the exposure solution was changed every day in order to guarantee the concentration of MC-LR and water quality. They were fed with commercial pellet food once a day. Water quality was regularly monitored and no differences were recorded between tanks during the entire experimental period. No crayfish died during the exposure period. M. nipponense in each group were immediately sacrificed following exposure, and tissues were dissected, frozen in liquid nitrogen, and maintained at −80 °C until use.

M. nipponense hepatopancreas, muscle, stomach, intestine, and gill tissues were assayed for cat and gst mRNAs. Tissue samples for cDNA cloning and quantitative real-time polymerase chain reaction (qRT-PCR) analysis were homogenized in TRIZOL reagent (Invitrogen, Carlsbad, CA, USA), total RNA was extracted as described by Wang et al. [21], and treated with RNase-free DNase I (Fermentas, Burlington, ON, Canada) to remove genomic DNA contamination. Total RNA concentrations were calculated at an absorbance of 260 nm. Total RNA quality was verified on 1% agarose gels by visual inspection of the 18 S and 28 S ribosomal RNA bands and by the A260 nm/A280 nm ratio (range 1.90–2.05) measured with a Nanodrop spectrophotometer (Thermo Electron Corp., Waltham, MA, USA). The cDNAs were synthesized from 5 μg total RNA with M-MLV reverse transcriptase (Invitrogen) and the oligo (dT)₁₈ primer in a 20 μL final volume.

Partial cDNA fragments for the two target genes in M. nipponense were amplified using primers designed from the conserved regions of their counterparts in other crustaceans (Table S1). PCR was performed with 2 μL of the cDNA template synthesized by RT-PCR in 20 μL containing 5 pmol of each specific primer and 1 unit of Taq DNA polymerase (TaKaRa Bio, Shiga, Japan). The purified PCR products of these cDNA fragments were inserted into the pMD₁₈-T plasmid using a TA cloning kit (TaKaRa Bio) following the manufacturer’s instructions. Three confirmed recombinant plasmid clones for each gene were sequenced separately by Genscript Corp. (Nanjing, China), using the ABI 3730 automated DNA sequencer (BigDye Terminator Chemistry) and the BigDye Terminator Cycle Sequencing Kit (PE Biosystems, Foster City, CA, USA). The nucleotide sequences were determined for both sense and antisense strands of the plasmid inserts. The 3′ and 5′ ends of both cDNAs were obtained with several pairs of gene-specific primers designed for the target genes to overlap the sense or antisense regions of their amplified partial fragments sequenced above. The 5′ and 3′ rapid amplification of cDNA ends (RACE) procedures were performed with the RACE cDNA Amplification Kit (Invitrogen), using total RNA and following the manufacturer’s instructions, respectively. The nested 5′- and 3′-RACE PCR products of the expected size were processed and sequenced as described above. The cat and gst full-length cDNAs were assembled by aligning the partial cDNA fragments and the 5′- and 3′-RACE fragments using SeqMan in Lasergene software (DNASTAR, Madison, WI, USA).

The putative amino acid sequences of the M. nipponense cat and gst genes were aligned with their counterparts from other species using the Megalign program in Lasergene software. We aligned diverse invertebrate cat and gst genes at the amino acid level using the ClustalX (1.83) sequence alignment program to establish phylogenetic trees for the two M. nipponense genes [22]. The neighbor-joining algorithm method [23] in the Mega 4.0 program was used to construct the phylogenetic trees [24]. Bootstrap analyses were conducted using 1000 replicates.

The qRT-PCR analysis was performed on a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) thermocycler with the SYBR Premix ExTaq II kit (TaKaRa Bio). The qRT-PCR reactions were carried out in a final volume of 25 μL using 1 × SYBR Premix Ex Taq, 0.4 μM of each primer, and 2.5 μL RT reaction solution. The reactions were denatured at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s. To assess the specificity of each amplicon, a melting curve analysis was performed at the end of each PCR thermal profile. CFX Manager software (Bio-Rad) was used to analyze SYBR Green I density and to determine the threshold cycle (Ct) value. All samples were run in triplicate. qRT-PCR efficiency (E) was calculated from the given slopes using CFX Manager software and a 10-fold diluted cDNA sample series with five dilution points measured in triplicate. E was determined using the equation: E = 10 − 1 slope [25].

qRT-PCR was conducted with male M. nipponense hepatopancreas, intestine, gill, stomach, and muscle tissues to analyze tissue distribution of the cat and gst transcripts, with the β-actin gene as the endogenous control [26]. The qRT-PCR primers are listed in Table S1. The primers were tested during normal PCR amplification, and the PCR products were visualized on a 1.5% agarose gel before qRT-PCR to verify primer specificity. The relative changes in mRNA levels of the two genes in different tissues were calculated using the 2^−ΔΔCt method [27]. The analysis was performed on tissues from nine males. All data are expressed as mean ± standard deviation (SD).

Nine hepatopancreatic samples from each group were defrosted twice and homogenized on ice with 10 volumes of cold buffer (250 mM sucrose, 5 mM Tris-HCL, and 0.1 mM edetic acid-2Na; pH 7.5). The homogenate was centrifuged at 13,000 g and 4 °C for 10 min to obtain the supernatant for the assays. Protein concentration was determined using a commercial protein assay kit (Nanjing Jiancheng Bioengineering Institute). GST, SOD, CAT, and GPx activities were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). One unit (U) of GST, CAT, and GPx activity was defined as the amount of enzyme consuming 1 μmol of substrate or generating 1 μmol of product/min/mg soluble protein (U/mg prot). One U of SOD activity was defined as the amount of enzyme required to inhibit the oxidation reaction by 50% and was also expressed as U/mg prot.

The gene expression patterns of hepatopancreatic gst, Cu/Zn-sod, cat, and gpx were detected by qRT-PCR in M. nipponense after exposure to 0.2–25 μg/L MC-LR for 96 h. Each transcript was analyzed in 9 individuals. The β-actin gene was used as an endogenous control. Relative mRNA levels were calculated using the 2^−ΔΔCt method and the formula F = 2^−ΔΔCt, ΔΔCt = (C_{t, target gene} − C_{t, β-actin}) _MC-LR − (C_{t, target gene} − C_{t, β-actin}) _control [27]. Data are expressed as mean ± SD. Statistical differences were tested with analysis of variance and the least significant difference test.

The full-length cDNA sequences of cat (GenBank accession No. KC485002) and gst (GenBank accession No. KC485003) genes were determined by the RT-PCR and RACE methods. The cat cDNA was 1699 bp and contained a 1554 bp open reading frame (ORF) encoding a 518 amino acid polypeptide with a theoretical pI of 6.66 and a calculated molecular mass of 58.57 kDa and 135 bp 5′-untranslated region (UTR) (Figure S1). Meanwhile, the gst cDNA was 1122 bp and contained a 651 bp ORF encoding a 217 amino acid polypeptide with a theoretical pI of 5.35 and a calculated molecular weight of 25.45 kDa, 173 bp 5′-UTR and 298 bp 3′-UTRs with a polyadenylation signal (AATAAA) (Figure S2).

The putative CAT of M. nipponense contains NADPH binding site, proximal heme-ligand signature, and proximal active site signature (Figure S3). The amino acid sequence identities of M. nipponense CAT with its counterparts from other crustaceans were more than 80%. The M. nipponense GST protein sequence showed 53.3%–72.1% similarity with the GST mu class from different organisms and had several conserved GSH binding sites and substrate binding sites (Figure S4).

Two phylogenetic trees were constructed from the amino acid sequence alignments for CAT and GST in crustaceans, fish, and mammals using the neighbor-joining method to better understand the positions of M. nipponense CAT and GST in the evolutionary history of the respective proteins. M. nipponense CAT was more similar to its counterpart in Macrobrachium rosenbergii (Figure 1). GST was more similar to its counterpart in Lepeophtheirus salmonis than those of other crustaceans and fish (Figure 2).

The cat and gst tissue distributions in M. nipponense are shown in Figure 3. Both cat and gst mRNAs were expressed predominantly in the hepatopancreas. Meanwhile, cat and gst mRNAs were expressed at low level in other tissues with decreasing order of stomach, muscle, intestines and gill. The cat and gst mRNA levels in hepatopancreas were 80.1-fold and 16.8-fold higher than in intestine, respectively.

MC-LR had no effect on GST activity after the 12- and 24-h exposures (Figure 4A), but GST activity increased significantly following MC-LR exposure at four concentrations for 48–96 h, except MC-LR exposure at 1 μg/L for 48 h and 0.2 μg/L for 96 h (Figure 4A).

MC-LR at 5 and 25 μg/L caused a significant increase in SOD activity after the 12-h exposure, compared with control (p < 0.05, Figure 4B). At the same time, 0.2–25 μg/L MC-LR all significantly stimulated SOD activity in the 24- and 48-h exposures (1.06–1.38 times, p < 0.05, Figure 4B). Amounts of 0.2 and 5 μg/L MC-LR also significantly increased SOD activity in the 72-h exposure (p < 0.05, Figure 4B), but 1 and 25 μg/L MC-LR did not increase SOD activity. However, in the 96-h exposure, 25 μg/L MC-LR significantly inhibited SOD activity, while MC-LR at 0.2–5 μg/L had no significant effect (p < 0.05, Figure 4B).

In 12-h exposure, only 25 μg/L MC-LR significantly stimulated CAT activity, while 0.2–5 μg/L MC-LR had no significant effect (Figure 4C). In 24-h exposure, MC-LR at 0.2–25 μg/L all significantly increased CAT activity (Figure 4C). In 48-h exposure, 0.2 and 1 μg/L MC-LR caused 1.8- and 1.4-fold significant increases in CAT activity, respectively (p < 0.05, Figure 4C), while 5 and 25 μg/L MC-LR had no significant effect. For 72-h exposure, all MC-LR treatments did not affect CAT activity. MC-LR at 1 and 25 μg/L significantly suppressed CAT activity after the 96-h exposure, whereas 0.2 and 5 μg/L MC-LR had no significant effect (Figure 4C).

It was interesting that none of the MC-LR concentrations had a significant effect on GPx activity in the 12- and 24-h exposures (Figure 4D). MC-LR at 0.2 and 5 μg/L significantly enhanced GPx activity after the 48-h exposure, whereas 1 and 25 μg/L MC-LR did not change GPx activity. In the 72- and 96-h exposures, MC-LR at 0.2–25 μg/L all significantly stimulated GPx activity (p < 0.05, Figure 4D).

The changes in gst, Cu/Zn-sod, cat and gpx mRNA expression are shown in Figure 5. The gst mRNA expression was significantly up-regulated 1.41-fold by 25 μg/L MC-LR after the 12-h exposure (p < 0.05) (Figure 5A), whereas MC-LR at the lower concentrations (0.2–5 μg/L) had no significant effect. In 24-h exposure, MC-LR at 5 and 25 μg/L significantly elevated gst transcription 1.62 and 1.73-fold, respectively (Figure 5A), while 0.2 and 1 μg/L MC-LR did not significantly affect it. In 48-h exposure, MC-LR at 1–25 μg/L caused 1.51–1.98-fold significant increases in gst transcription. In 72- and 96-h exposures, MC-LR at 0.2–25 μg/L all significantly up-regulated gst expression (1.25–2.06-fold, 1.39–2.07-fold, respectively, p < 0.05, Figure 5A).

In 12-h exposure, MC-LR at 1–25 μg/L caused 1.57–1.84-fold significant increase in Cu/Zn-sod transcript (p < 0.05), while 0.2 μg/L MC-LR had no significant effect (Figure 5B). After 24-h exposure, MC-LR at 0.2, 5, and 25 μg/L significantly elevated Cu/Zn-sod transcription (1.33–2.69-fold, p < 0.05), whereas 1 μg/L MC-LR had no significant effect. In 48-h exposure, Cu/Zn-sod expression was significantly up-regulated for 2.46 and 1.3 fold by 0.2 and 1 μg/L MC-LR, respectively, whereas MC-LR at higher concentrations (5 and 25 μg/L) had no significant effect on Cu/Zn-sod transcription. In 72-h exposure, Cu/Zn-sod was significantly up-regulated and down-regulated by 0.2 and 25 μg/L MC-LR, respectively. However, 1 and 5 μg/L MC-LR had no effect. Both 1 and 25 μg/L MC-LR significantly down-regulated Cu/Zn-sod expression after the 96-h exposure and MC-LR at 0.2 and 5 μg/L tended to inhibit Cu/Zn-sod transcription (Figure 5B).

After 12-h exposure, cat expression was significantly up-regulated by the 1 μg/L MC-LR for 1.57-fold (p < 0.01, Figure 5C), while MC-LR at 0.2, 5 and 25 μg/L all had no significant effect. In 24-h exposure, MC-LR at 0.2 and 1 μg/L significantly stimulated cat mRNA expression (1.4 and 1.2-fold, p < 0.05, respectively), whereas MC-LR at higher concentrations (5 and 25 μg/L) tended to inhibit its expression. In 72-h treatment, cat expression was significantly down-regulated by 25 μg/L MC-LR (0.8-fold, p < 0.05), and there was inhibitory tendency for cat expression in MC-LR at lower concentrations (0.2–5 μg/L). After 96-h exposure, MC-LR at 1 and 25 μg/L resulted in 0.82- and 0.72-fold decreases of cat transcription, respectively (p < 0.05, Figure 5C), while MC-LR at 1 and 5 μg/L tended to inhibit its expression. However, 48-h MC-LR at 4 concentrations did not affect cat expression.

After 12-h exposure, MC-LR at 5 and 25 μg/L significantly increased gpx mRNA expression for 1.48- and 1.29-fold, respectively (p < 0.05, Figure 5D), while MC-LR at lower concentrations had no effect. During 24-h exposure, MC-LR at 0.2–25 μg/L all significantly up-regulated gpx mRNA expression (1.24–1.85-fold, p < 0.05). After 48-h exposure, 1–25 μg/L MC-LR significantly stimulated gpx expression 1.85–2.38-fold, and 0.2 μg/L MC-LR tended to increase gpx expression. During 72-h exposure, MC-LR at 0.2–25 μg/L caused 1.87–2.5-fold, significant increases in gpx transcript (p < 0.01). MC-LR at 1–25 μg/L significantly increased gpx transcript 1.4–2.6 fold after 96-h exposure (p < 0.01) (Figure 5D), while 0.2 μg/L MC-LR did not affect its expression.