Ferritin Heavy-like subunit is involved in the innate immune defense of the red swamp crayfish Procambarus clarkii.

Iron-binding proteins, known as ferritins, play pivotal roles in immunological response, detoxification, and iron storage. Despite their significance to organisms, little is known about how they affect the immunological system of the red swamp crayfish (Procambarus clarkii). In our previous research, one ferritin subunit was completely discovered as an H-like subunit (PcFeH) from P. clarkii. The full-length cDNA of PcFerH is 1779 bp, including a 5'-UTR (untranslated region, UTR) of 89 bp, 3'-UTR (untranslated region, UTR) of 1180 bp and an ORF (open reading frame, ORF) of 510 bp encoding a polypeptide of 169 amino acids that contains a signal peptide and a Ferritin domain. The deduced PcFerH protein sequence has highly identity with other crayfish. PcFerH protein's estimated tertiary structure is quite comparable to animal structure. The PcFerH is close to Cherax quadricarinatus, according to phylogenetic analysis. All the organs examined showed widespread expression of PcFerH mRNA, with the ovary exhibiting the highest levels of expression. Additionally, in crayfish muscles, intestines, and gills, the mRNA transcript of PcFerH was noticeably up-regulated, after LPS and Poly I:C challenge. The expression of downstream genes in the immunological signaling system was suppressed when the PcFerH gene was knocked down. All of these findings suggested that PcFerH played a vital role in regulating the expression of downstream effectors in the immunological signaling pathway of crayfish.

Insights into chlorantraniliprole exposure via activating cytochrome P450-mediated xenobiotic metabolism pathway in the Procambarus clarkii: Identification of P450 genes involved in detoxification.

To investigate the impact of chlorantraniliprole on Procambarus clarkii, acute toxicity tests were performed. Results indicated that 96 h post-exposure to chlorantraniliprole (60 mg/L) led to the separation of the hepatopancreas basement membrane, causing cell swelling, rupture, and vacuolation. Moreover, acid phosphatase (ACP) and alkaline phosphatase (AKP) activities exhibited divergent trends across four concentrations of chlorantraniliprole (0, 30, 60, and 90 mg/L). Hydrogen peroxide (H2O2) and catalase (CAT) levels significantly increased, while total superoxide dismutase (T-SOD) and malonaldehyde (MDA) activities decreased, indicating oxidative stress in the hepatopancreas. A total of 276 differentially expressed genes (DEGs) were identified, with 204 up-regulated and 72 down-regulated. Out of these, 114 DEGs were successfully annotated and classified into 99 pathways, with a primary focus on the cytochrome P450-mediated xenobiotic metabolism pathway. The DEGs enriched in this pathway, along with transcriptome data, were validated using quantitative-polymerase chain reaction. This study enhances the transcriptome database of P. clarkii and provides fundamental insights into its immune defense and antioxidant mechanisms. Additionally, it lays a theoretical foundation for future research on disease prevention in P. clarkii within rice-shrimp culture systems.

Transcriptome Analysis Reveals Antioxidant Defense Mechanisms in the Silkworm Bombyx mori after Exposure to Lead.

Lead (Pb) is a major source of heavy metal contamination, and poses a threat to biodiversity and human health. Elevated levels of Pb can hinder insect growth and development, leading to apoptosis via mechanisms like oxidative damage. The midgut of silkworms is the main organ exposed to heavy metals. As an economically important lepidopteran model insect in China, heavy metal-induced stress on silkworms causes considerable losses in sericulture, thereby causing substantial economic damage. This study aimed to investigate Pb-induced detoxification-related genes in the midgut of silkworms using high-throughput sequencing methods to achieve a deeper comprehension of the genes' reactions to lead exposure. This study identified 11,567 unigenes and 14,978 transcripts. A total of 1265 differentially expressed genes (DEGs) were screened, comprising 907 upregulated and 358 downregulated genes. Subsequently, Gene Ontology (GO) classification analysis revealed that the 1265 DEGs were distributed across biological processes, cellular components, and molecular functions. This suggests that the silkworm midgut may affect various organelle functions and biological processes, providing crucial clues for further exploration of DEG function. Additionally, the expression levels of 12 selected detoxification-related DEGs were validated using qRT-PCR, which confirmed the reliability of the RNA-seq results. This study not only provides new insights into the detoxification defense mechanisms of silkworms after Pb exposure, but also establishes a valuable foundation for further investigation into the molecular detoxification mechanisms in silkworms.

The analyses of the complete mitochondrial genomes of three crabs revealed novel gene rearrangements and phylogenetic relationships of Brachyura.

BACKGROUND: Brachyura crab is the largest branch of Decapoda crustacean. Phylogenetic relationships within Brachyura remain controversial to be investigated. The mitochondrial genome (mitogenome) is an important molecular marker for studying the phylogenetic relationships of Brachyura. METHODS AND RESULTS: To understand the phylogeny of Brachyura, the three complete mitogenomes from Charybdis annulata, Leptodius exaratus, and Spider crab were sequenced and annotated. Their full length was 15,747, 15,716, and 16,608 bp long, respectively. The first two crabs both contained 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. However, Spider crab contained 13 PCGs, two rRNA genes, 25 tRNA genes and a control region. The mitogenomes of each of the three crabs exhibited high AT content (67.8%, 69.1%, and 70.8%), with negative AT skews (-0.014, - 0.028, and - 0.017) and GC skews (-0.269, - 0.286, and - 0.341). The gene order of C. annulata was identical to the ancestor of Brachyura. Compared with the ancestor of Brachyura, L. exaratus exhibited the gene rearrangements of Val (V)-rrnS-control region, and Spider crab had the four copies of Lys (K). Phylogenetic analyses indicated that C. annulata belonged to Portunidae family, Portunoidea superfamilies, L. exaratus belonged to Xanthidae family, Xanthoidea superfamilies, and Spider crab belonged to Mithracidae family, Majoidea superfamilies. Phylogenetic analyses showed that the two species (Somanniathelphusa boyangensis and Huananpotamon lichuanense) belonging to the Potamoidea were sister groups to the Thoracotremata, thus supporting the conclusion that Heterotremata is polyphyletic. CONCLUSION: The results of this study enriched the crab mitogenome database and enabled us to better understand the phylogenetic relationships of Brachyura.

Complete mitochondrial genome of Parasa sinica: New insights into the phylogeny of Limacodidae.

In this study, the whole mitochondrial genome (mitogenome) of Parasa sinica was sequenced. It contains 15,306 base pairs (bp), 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and one non-coding regulatory area (CR), all of which are shared by other lepidopterans. It follows the same gene order as ordinary lepidopterans. Further, out of these 37 genes, 23 are present on the heavy strand whereas the remaining 14 are located on the light strand. The A + T composition of the mitogenome is relatively high. Although P. sinica has a negative AT-skew and GC-skew, the GC-skew value is significantly lower than the AT-skew value. All PCGs, with the exception of CO1, carry the same start codon (ATN). All tRNAs exhibit the usual cloverleaf secondary structure. We identified the conserved motif "ATAGA + poly-T″ found in other lepidopteran insects at the beginning of the CR. We collected the concatenated PCGs sequences in the mitochondrial genome of 15 species of Zygaenoidea, with the sequences of Geometridae as outgroups, including P. sinica, and constructed phylogenetic trees using Bayesian inference (BI) and maximum likelihood (ML) methods. The monolineage of each superfamily is usually well supported. Based on phylogenetic analysis, P. sinica is a member of family Limacodidae, strongly supporting the monophyly of the Zygaenoidea groups.

Integrins in the Immunity of Insects: A Review.

Integrins are a large group of cell-surface proteins that are classified as transmembrane proteins. Integrins are classified into different types based on sequence variations, leading to structural and functional diversity. They are broadly distributed in animals and have a wide range of biological functions such as cell-to-cell communication, intracellular cytoskeleton organization, cellular signaling, immune responses, etc. Integrins are among the most abundant cell surface proteins in insects, exhibiting their indispensability in insect physiology. Because of their critical biological involvement in physiological processes, they appear to be a novel target for designing effective pest control strategies. In the current literature review, we first discuss the discovery and expression responses of integrins against various types of pathogens. Secondly, we examine the specific biological roles of integrins in controlling microbial pathogens, such as phagocytosis, encapsulation, nodulation, immune signaling, and so on. Finally, we describe the possible uses of integrins to control agricultural insect pests.

Differentially expressed genes in head kidney of Pelteobagrus fulvidraco following Vibrio cholerae challenge.

The yellow catfish (Pelteobagrus fulvidraco) is a freshwater fish with high economic value in eastern China. Nevertheless, pathogens causing bacterial diseases in P. fulvidraco have brought about huge economic loss and high mortality in artificial aquaculture. For disease control, it is critical to further understand the immune system of yellow catfish and immune-related genes with which they respond to pathogenic infections. In this study, high-throughput sequencing methods were used to analyze the transcriptomic spectrum of the head kidney from P. fulvidraco challenged by Vibrio cholera. A total of 45,544 unique transcript fragments (unigenes) were acquired after assembly and annotation, with an average length of 1,373 bp. Additionally, 674 differentially expressed genes (DEGs) were identified after stimulation with V. cholerae, 353 and 321 genes were identified as remarkably up- or downregulated, respectively. To further study the immune-related DEGs, we performed KEGG enrichment and GO enrichment. The results showed gene regulation of response to stimulus, immune response, immune system progress, response to external stimuli and cellular response to stimuli. Analysis of KEGG enrichment is important to identify chief immune related pathways. Real-time quantitative reverse transcription-PCR (qRT-PCR) results indicated 10 immune response genes that were found to be upregulated compared to a control group after 6 h of V. cholerae challenging. In summary, the results of our study are helpful to determine the defense mechanisms and immune system responses of yellow catfish in reaction to bacterial challenges.

Transcriptome analysis reveals antioxidant defense mechanisms in the red swamp crayfish Procambarus clarkia after exposure to chromium.

Chromium (Cr) as a chromate anion has a strong redox capacity that seriously threatens the ecological environment and human health. Cr can contaminate water and impart toxicity to aquatic species. Procambarus clarkii is an important food source that once represented a large proportion of the aquaculture industry due to its rapid reproduction and high economic value. However, there have been reports on the death of P. clarkii due to heavy metal pollution. The underlying mechanism regarding heavy metal toxicity was studied in this paper. The transcriptome data of hemocytes extracted from P. clarkii injected with Cr were analyzed by high-throughput sequencing and compared to the control group. In total, 48,128,748 clean reads were obtained in the treatment group and 56,480,556 clean reads were obtained in the control group. The reads were assembled using Trinity and the identified unigenes were then annotated. Then, 421 differentially-expressed genes (DEGs) were found, 170 of which were upregulated and 251 downregulated. Many of these genes were found to be related to glutathione metabolism and transportation. The glutathione metabolic pathway of P. clarkii was thus activated by Cr exposure to detoxify and maintain body function. Validation of DEGs with quantitative real-time PCR confirms the changes in gene expression. Thus, this study provides data supporting a glutathione-focused response of P. clarkii to exposure to heavy metals.

Transcriptome analysis of differentially expressed genes in the red swamp crayfish Procambarus clarkii challenged with Aeromonas hydrophila.

As an important economic species in China, aquaculture of the crayfish Procambarus clarkii has suffered huge losses due to infection by pathogenic bacteria, mainly by Aeromonas hydrophila, which leads to high mortality and huge economic loss. To better understand the immune response of crayfish against bacterial infection, we compared and analyzed transcriptome data of hepatopancreatic tissue from P. clarkii that were either challenged with A. hydrophila or treated with PBS. After assembly and annotation of the data, 32,041 unigenes with an average length of 1512 base pairs were identified. Compared to control group, Differential gene expression (DEG) analysis revealed 608 DEGs were obtained, of which 274 unigenes were upregulated and 334 were downregulated in the A. hydrophila group. Furthermore, the expression levels of eight selected immune-related DEGs were validated by qRT-PCR, substantiating the reliability of RNA-seq results. This study not only provides effective data support for immune defense strategies of P. clarkii in response to bacterial infections, but also provides new information about the P. clarkii immune system and defense mechanisms, and a valuable basis for further studies to elucidate the molecular immune mechanisms of this species.

Differentially expressed genes involved in immune pathways from yellowhead catfish (Tachysurus fulvidraco) after poly (I:C) challenge.

Yellowhead catfish (Tachysurus fulvidraco) is an important aquaculture fish species in China with a high market value. Infectious diseases pose serious threats in farmed fish species, and although vaccines can prevent certain infections, they rely on potent adjuvants. In this study, we analyzed the transcriptomic profiles of spleens from poly (I:C)-treated T. fulvidraco. We obtained 46,362,922 reads corresponding to 490,926 transcripts and 318,059 genes. Gene annotation using different databases and subsequent differential gene expression analyses led to the identification of 5587 differentially expressed genes (DEGs), of which 2473 were up-regulated and 3114 were down-regulated in poly (I:C)-treated fish. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs revealed the significant dysregulation of immune- and cancer-related genes in the spleens of poly (I:C)-treated fish. Notably, several components of JAK-STAT, MAPK, and p53 signaling pathways were significantly dysregulated in response to poly (I:C) treatment. Quantitative real-time PCR (qRT-PCR) analysis of 11 randomly selected immune response genes confirmed the reliability of our findings. In conclusion, our findings provide novel insight into the immune responses of T. fulvidraco and suggest that poly (I:C) may represent a promising adjuvant of fish vaccines.

Transcriptome analysis of immune-related genes in Sesarmops sinensis hepatopancreas in reaction to peptidoglycan challenge.

Sesarmops sinensis is a dominant omnivorous crab species, which plays an important ecological function in salt marsh ecosystems. To better understand its immune system and immune related genes under pathogen infection, the transcriptome was analyzed by comparing the data of S. sinensis hepatopancreas stimulated by PBS and PGN. A set of assembly and annotation identified 39,039 unigenes with an average length of 1105 bp, obtaining 1300 differentially expressed genes (DEGs) in all, which included 466 remarkably up-regulated unigenes and 834 remarkably down-regulated unigenes. In addition, based on mensurable real time-polymerase chain reaction and high-throughput sequencing, several immune responsive genes were found to be markedly up-regulated under PGN stimulation. In conclusion, in addition to enriching the existing transcriptome data of S. sinensis, this study also clarified the immune response of S. sinensis to PGN stimulation, which will help us to further understand the crustacean's immune system.

A complement factor I (CFI) gene mediates innate immune responses in yellow catfish Pelteobagrus fulvidraco.

This study isolated CFI gene from Pelteobagrus fulvidraco and named it PfCFI. The cDNA of PfCFI is 2374 bp long, including a 52 bp 5' untranslated sequence, a 222 bp 3' untranslated sequence, and an open reading frame (ORF) of 2100 bp encoding polypeptide consisting of 699 amino acids. Phylogenetic analysis revealed that the PfCFI was closely related to CFI of Ictalurus punctatus. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis indicate that there is the PfCFI gene which expressed in all the rest of tested tissues in varied levels, and mainly distributed in liver and least in heart. The reseachers induce the expressions level of PfCFI gene in liver, spleen, head kidney and blood at different points in time after challenged with lipopolysaccharide (LPS), and polyriboinosinic polyribocytidylic acid (poly I:C), respectively. Together these results suggested that CFI gene plays an important role in resistance to pathogens in yellow catfish immunity.

Comparative Mitochondrial Genome Analyses of Sesarmid and Other Brachyuran Crabs Reveal Gene Rearrangements and Phylogeny.

Mitochondrial genomes (mitogenomes) are important for understanding molecular evolution and phylogenetic relationships. The complete mitogenome of Perisesarma bidens was determined, which is 15,641 bp in length. The A + T content of P. bidens mitogenome was 74.81%. The AT skew was slightly negative (-0.021). The 22 tRNAs ranged from 65 to 73 bp and were highly A + T biased. All tRNA genes had typical cloverleaf structures, except for the trnS1 gene, which lacked a dihydrouridine (DHU) arm. The gene order within the P. bidens mitogenome was identical to the pancrustacean ground pattern, except for the translocation of the trnH. Additionally, the gene order of trnI-trnQ-trnM in pancrustacean ground pattern became trnQ-trnI-trnM in P. bidens. Phylogenetic analyses supported the inclusion of P. bidens in Sesarmidae and the promotion of Sesarminae to Sesarmidae. The results will help us to better understand the status and evolutionary history of Grapsoidea crabs.

Characterization of the complete mitochondrial genome of Helice latimera and its phylogenetic implications in Brachyura.

Mitochondrial genomes (mitogenomes) help advance our learning of molecular evolution and phylogenetic relationships. The mitogenome of H. latimera is 16,246 bp in length, which typically contains 37 animal mitogenome genes consisting of 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes, as well as a control region. The AT content of H. latimera is 69.1%. The A + T skew of the mitogenome of H. latimera was slightly negative (-0.017). The size of Thirteen PCGs is from 162 bp to 1731 bp. Twenty-two tRNA genes ranged from 62 to 73 bp and were highly A + T biased. All tRNA genes owed a typical cloverleaf structure, not including the trnS1 gene lacking a dihydroxyuridine arm. One PCG, two rRNAs, and 12 of the tRNAs were rearranged compared to the pancrustacean gene order. Phylogenetic analysis revealed the locationt of H. latimera among the Varunidae family.

Proteomic analysis of differentially expressed proteins in the lipopolysaccharide-stimulated hepatopancreas of the freshwater crayfish Procambarus clarkii.

Procambarus clarkii is one of the most important aquatic invertebrates in China and has high commercial value. However, aquaculture has suffered great economic loss due to outbreaks of infectious diseases in P. clarkii. To identify red swamp crayfish related proteins involved in the response to bacterial infection, we analysed immune-related proteins following lipopolysaccharide (LPS) stimulation by quantitative proteomics. The proteome of the hepatopancreas of P. clarkii challenged with LPS and phosphate-buffered saline was analysed to evaluate the immune response. Based on liquid chromatography coupled with tandem mass spectrometry, 16 upregulated and 29 downregulated proteins were identified. A Gene Ontology analysis demonstrated 5 biological process, 11 cellular component, and 6 molecular function subcategories. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the identified proteins were mainly involved in metabolism, phagosome, and ribosome. Real-time quantitative reverse transcription-PCR revealed that eight immune-related genes were upregulated after LPS stimulation compared to the control. Taken together, the data enhance our understanding of the immune response of crayfish to LPS.

A non-mammalian Toll-like receptor 26 (TLR26)gene mediates innate immune responses in yellow catfish Pelteobagrus fulvidraco.

In this study, we identified a fish-specific Toll-like receptor (TLR) in Pelteobagrus fulvidraco, an economically important freshwater fish in China. This TLR, PfTLR26, was shown to be encoded by a 3084 bp open reading frame (ORF), producing a polypeptide 1027 amino acids in length. The PfTLR26 protein contains a signal peptide, eight leucine-rich repeat (LRR) domains, two LRR_TYP domains in the extracellular region, and a Toll/interleukin (IL)-1 receptor (TIR) domain in the cytoplasmic region, consistent with the characteristic TLR domain architecture. This predicted 117.1 kDa protein was highly homologous to those of other fish, with phylogenetic analysis revealing the closest relation to TLR26 of Ictalurus punctatus. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the PfTLR26 gene was expressed in all tissues tested, with the highest expression levels seen in the head kidney and blood, and the lowest seen in muscle. PfTLR26 exhibited significant upregulation in liver, spleen, head kidney, and blood at different time points following challenge with the common TLR agonists lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C). Taken together, these results suggest that PfTLR26 may be an important component of the P. fulvidraco innate immune system, participating in the transduction of TLR signaling under pathogen stimulation.

Characterization and expression analysis of immune-related genes in the red swamp crayfish, Procambarus clarkii in response to lipopolysaccharide challenge.

To learn more about red swamp crayfish related genes in response to bacterial infections, we investigated immune-related genes induced by lipopolysaccharide (LPS) in the hepatopancreas using high-throughput sequencing method. In present the study, a total of 55,107 unigenes were identified, with an average length of 678 bp. A total of 2215 differentially expressed genes (DEGs) were found, including 669 up-regulated genes and 1546 down-regulated genes. The result of Gene ontology (GO) analysis revealed that 3017 DEGs were enriched in 19 biological process subcategories, 17 cellular component subcategories and 15 molecular function subcategories. The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that "ribosome" was the most abundant group, which had 34 DEGs. KEGG enrichment analysis identified several immune response pathways. Real-time quantitative reverse transcription-PCR (qRT-PCR) results exhibited that several immune responsive genes were greatly up-regulated following LPS stimulation as observed in the results of high-throughput sequencing. Overall, this study provides new insight into the immune defense mechanisms of P. clarkii against LPS infection.

The complete mitochondrial genome of Sinna extrema (Lepidoptera: Nolidae) and its implications for the phylogenetic relationships of Noctuoidea species.

The mitochondrial genome (mitogenome) can provide information for phylogenetic analysis and evolutionary biology. Here, we determined the complete mitogenome sequence of Sinna extrema (Noctuoidea: Nolidae). The newly determined mitogenome was 15,345 bp in length and included 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes and a control region (CR) similar to the known mitogenome of other lepidopterans. The entire mitogenome was biased towards using A and T, with an A + T content of 90.6%. Both the AT and GC skews in the mitogenome of S. extrema were negative. All PCGs were initiated with the standard start codon ATN, while cox1 began with CGA. All tRNA genes had the typical cloverleaf structure, except for the trnS1, which lacked a dihydroxyuridine arm. A gene rearrangement (trnM) was also found in the S. extrema mitogenome. To understand evolutionary relationships, we performed phylogenetic analysis using Bayesian inference (BI) and Maximum likelihood (ML) methods. Our phylogenetic analysis indicated that S. extrema and Gabala argentata cluster together with high nodal support values, and G. argentata is a member of the family Nolidae, which suggests that S. extrema belongs to Nolidae. Our results provide reference for further phylogenetic research of Noctuoidea species.

New insight into the molecular basis of Fe (III) stress responses of Procambarus clarkii by transcriptome analysis.

Iron in excess can have toxic effects on living organisms. In China, the freshwater crayfish Procambarus clarkii is a source of aquatic food with high-quality protein and has significant commercial value. P. clarkii shows oxidative stress on exposure to heavy metals, and antioxidant enzymes, such as ubiquitination enzymes and proteasomes, play important roles in oxidative stress. To understand the antioxidant defense system of P. clarkii, we analyzed the hepatopancreas transcriptomes of P. clarkii after stimulation with FeCl3. In total, 5199 differentially expressed genes (DEGs) were identified (2747 upregulated and 2452 downregulated). GO analysis revealed that these DEGs belonged to 16 cellular component, 16 molecular function, and 19 biological process subcategories. A total of 1069 DEGs were classified into 25 categories by using COG. Some antioxidant defense pathways, such as "Ubiquitin mediated proteolysis" and "Glutathione metabolism," were identified using KEGG. In addition, quantitative real time-PCR (qRT-PCR) substantiated the up-regulation of a random selection of DEGs including antioxidant and immune defense genes. We obtained information for P. clarkii transcriptome databases and new insights into the responses of P. clarkii hepatopancreas to heavy metals.

Transcriptome-wide identification of differentially expressed genes in Procambarus clarkii in response to chromium challenge.

Because of the high protein content and rich meat quality of crayfish Procambarus clarkii, it has become widely popular in China in recent years and has a high economic value. When P. clarkii is stimulated by heavy metals, it reacts to oxidation. P. clarkii has evolved antioxidant defense systems, including antioxidant enzymes such as catalase (CAT). The hexavalent form of Cr (VI) is a pathogenic factor that is of particular concern in aqueous systems because of its great toxicity to living organisms. In this study, we characterized the transcriptome of P. clarkii using a RNA sequencing method and performed a comparison between K2Cr2O7-treated samples and controls. In total, 34,237 unigenes were annotated. We identified 5098 significantly differentially expressed genes (DEGs), including 2536 and 2562 were significantly up-regulated and down-regulated, respectively. In addition, quantitative real time-PCR (qRT-PCR) confirmed the up-regulation of a random selection of DEGs. Our results contribute to a more comprehensive understanding of the antioxidant defense system used by P. clarkii in response to heavy metal stress.