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Background The COMMD (copper metabolism MURR1 domain-containing) protein family, characterized by a conserved COMM domain, plays crucial roles in metal ion transport and NF-κB signaling regulation in vertebrates. However, its functional roles in invertebrates, particularly crustaceans, remain poorly understood. This study aimed to identify and characterize COMMD family genes in red swamp crayfish ( Procambarus clarkii ) and to investigate their involvement in immune responses under environmental stress. Results Seven COMMD genes ( COMMD2 , COMMD3 , COMMD4 , COMMD5 , COMMD7 , COMMD8 , and COMMD10 ) were identified and analyzed in P. clarkii . These genes exhibited distinct genomic structures and tissue-specific expression profiles. COMMD2 , COMMD3 , COMMD8 , and COMMD10 were predominantly expressed in hemocytes and gills, whereas COMMD4 and COMMD7 showed higher expression in the hepatopancreas. Non-lethal heat shock (NLHS) significantly enhanced pathogen resistance in P. clarkii , which was accompanied by a rapid and strong induction of COMMD8. Transcriptome analysis and RNA interference experiments revealed that COMMD8 positively regulates the TLR/MyD88/NF-κB signaling pathway. Silencing of COMMD8 suppressed TLR1 and MyD88 expression and delayed NF-κB activation, indicating its role as a key modulator of immune signaling under stress conditions. Conclusions This study demonstrates that COMMD8 is a critical regulator of stress-induced immune responses in P. clarkii , mediating enhanced resistance through modulation of innate immune signaling. These findings offer new insights into the function of COMMD proteins in crustaceans and contribute to a better understanding of how environmental stress influences invertebrate immunity.

Each year from April to May, high mortality rates are reported in red swamp crayfish ( Procambarus clarkii ) cultured in Jiangsu and other regions, in China, and this phenomenon has come to be known as “Black May” disease (BMD). Therefore, in order to investigate the possible causes of this disease, this study gathered BMD-affected P. clarkii samples and performed transcriptome analysis on hepatopancreas, gill, and muscle tissues. A total of 19,995,164, 149,212,804, and 222,053,848 clean reads were respectively obtained from the gills, muscle, and hepatopancreas of BMD-affected P. clarkii , and 114,024 unigenes were identified. The number of differentially expressed genes (DEGs) in gill, muscle, and hepatopancreas was 1703, 964, and 476, respectively. GO and KEGG enrichment analyses of the DEGs were then conducted. Based on KEGG pathway enrichment analysis, the most significantly differentially expressed pathways were mainly those involved with metabolism, human disease, and cellular processes. Further analysis of the significantly DEGs revealed that they were mainly related to the mitochondrial-mediated apoptosis pathway and that the expression of these DEGs was mostly down-regulated. Moreover, the expression of genes related to immune and metabolism-related pathways was also significantly down-regulated, and these significantly-inhibited pathways were the likely causes of P. clarkii death. Therefore, our results provide a basis for the identification of BMD causes.

The aquaculture industry of red swamp crayfish (RSC), Procambarus clarkii, has grown significantly in recent decades due to increasing market demand. In China, low water temperatures, particularly during overwintering, pose a challenge, hindering the development of the RSC aquaculture industry in northern regions. Understanding the molecular mechanism of RSCs’ responses to cold stress could be beneficial for its aquaculture practices. In this study, we exposed RSCs to 4 °C (T4) and 22 °C (T22: control) for 96 h. Transcriptomic and metabolomic analyses of hepatopancreas tissues were performed to identify key genes and metabolites that participate in cold stress response. A total of 787 differentially expressed genes (DEGs) and 198 differentially expressed metabolites (DEMs) were identified between T4 and T22. DEGs were significantly enriched in KEGG pathways related to carbohydrate, lipid, amino acid, and nucleotide metabolism, immunity, and signaling, while DEMs were significantly enriched in pathways associated with lipid and amino acid metabolism and membrane transport. The results indicated that cold stress altered RSCs’ metabolism and their innate immune system. This study provides valuable information to increase our understanding of cold stress responses in RSCs.

The red swamp crayfish ( Procambarus clarkii ) is one of the important freshwater aquaculture species in China, but its growth and development are greatly affected by temperature, which makes it difficult to expand its aquaculture range to the northern regions of China. The composition of gut microbes plays a vital role in resisting environmental pressure, and is also an important driving factor for amino acid metabolism in the body. However, little is known about the relationship between microorganisms, metabolism, and cold-resistance ability of P. clarkii . In this study, we performed the cold-resistance and antioxidant ability test, gut microbiota diversity analysis, quantitative analysis of histamine, and bioinformatics analysis of histamine receptor (HR) family on P. clarkii . The results showed that the cold-resistance crayfish exhibited high antioxidant ability and low gut microbiota diversity after acute cold stress. Next, we also found that there was significant correlation between the Lactobacilli genus and histamine abundance, indicating that the excellent cold tolerance ability of crayfish may stem from the degradation of histamine by Lactobacilli . Finally, it was revealed that HR genes had considerable quantity of gene copies, conservative evolution in crustacean lineages and expression differences in low-temperature tolerant populations. These results suggested that the diversity of Lactobacillus mediated changes in histamine metabolism affect antioxidant capacity, which is one of the reasons why P. clarkii exhibits cold resistance ability. This finding provided a theoretical basis for understanding the microorganism-histamine regulation mechanism of red swamp crayfish under cold stress, promoting the breeding and healthy culture of cold-resistance strain.

Freshwater crayfish Pontastacus leptodactylus is an indigenous European species of considerable ecological and commercial significance. However, it is highly susceptible to the oomycete Aphanomyces astaci , the causative agent of crayfish plague. Although its importance is widely recognized, little information exists on how seasonality affects the physiology and welfare of this species. The present study was designed to (i) investigate seasonal effects on the physiological responses of P. leptodactylus in relation to abiotic factors and crayfish plague outbreak, and (ii) identify the most suitable period for restocking in temperate regions. To achieve this, we characterized for the first time the mRNA expression of hsp70, hsp90, hif-1α, ubiquitin, and gapdh (as endogenous control gene), alongside protein levels of Hsp70, Hsp90, hydroxylated Hif-1α, ubiquitin conjugates, cleaved caspases, Bax/Bcl-2 ratio, and interleukin-6 (IL-6). Our findings revealed that cold stress triggered both the heat shock response and apoptosis. Interestingly, despite the activation of apoptosis, which generally suppresses inflammation, IL-6 levels increased, suggesting a possible association with A. astaci infection. Moreover, elevated Hif-1α levels indicated hypoxic stress during warmer months, likely linked to reduced oxygen availability and turbidity. Overall results suggest that crayfish plague epidemics may intensify with rising temperatures, whereas winter appears to be the optimal period for restocking. Interestingly, in addition, freshwater crayfish in temperate latitudes are organisms that face both cold and heat stress seasonally in the same ecosystem.

Early changes in hemocyte proteins in freshwater crayfish Pacifastacus leniusculus, in response to an injection with the fungal pattern recognition protein β-1,3-glucan (laminarin) were investigated, as well as changes after saline (vehicle) injection and in naïve animals. Injection of saline resulted in rapid recruitment of granular hemocytes from surrounding tissues, whereas laminarin injection on the other hand induced an initial dramatic drop of hemocytes. At six hours after injection, the hemocyte populations therefore were of different composition. The results show that mature granular hemocytes increase in number after saline injection as indicated by the high abundance of proteins present in granular cell vesicles, such as a vitelline membrane outer layer protein 1 homolog, mannose-binding lectin, masquerade, crustin 1 and serine protease homolog 1. After injection with the β-1,3-glucan, only three proteins were enhanced in expression, in comparison with saline-injected animals and uninjected controls. All of them may be associated with immune responses, such as a new and previously undescribed Kazal proteinase inhibitor. One interesting observation was that the clotting protein was increased dramatically in most of the animals injected with laminarin. The number of significantly affected proteins was very few after a laminarin injection when compared to uninjected and saline-injected crayfish. This finding may demonstrate some problematic issues with gene and protein expression studies from other crustaceans receiving injections with pathogens or pattern recognition proteins. If no uninjected controls are included and no information about hemocyte count (total or differential) is given, expressions data for proteins or mRNAs are very difficult to properly interpret.

Procambarus virginalis (marbled crayfish) is a parthenogenetically reproducing invasive crayfish species. Its global population is monoclonal, which raises questions about the mechanisms promoting their invasiveness. Here we show that environmental changes downregulate the highly conserved Dnmt1 DNA methyltransferase in marbled crayfish. When phenocopying this effect through a dsRNA-based in vivo knockdown, we observe enhanced invasiveness-related behavioral traits. Image cytometry and single-cell RNA sequencing reveal an expansion of mature granular immune cells and depletion of hemocyte-derived neuronal precursors, which support adult neurogenesis. Whole-genome bisulfite sequencing shows that these phenotypes coincide with a global loss of gene body DNA methylation and dysregulation of nervous and immune system genes. Additionally, we observe nucleosome destabilization to be associated with transcriptional changes after methylation loss. Taken together, our findings identify Dnmt1 as a potential canalizer of cellular and organismal phenotypes, thus providing a framework for how epigenetic mechanisms modulate invasiveness. The role of epigenetics in rapid adaptation remains poorly understood. This study shows that reduced Dnmt1-mediated DNA methylation in the clonal marbled crayfish alters chromatin organization, cell differentiation, and increases invasive behaviours.

Background The introduction of non-native species is a primary driver of biodiversity loss in freshwater ecosystems. The redclaw crayfish ( Cherax quadricarinatus ) is a freshwater species that exhibits tolerance to hypoxic stresses, fluctuating temperatures, high ammonia concentration. These hardy physiological characteristics make C. quadricarinatus a popular aquaculture species and a potential invasive species that can negatively impact tropical and subtropical ecosystems. Investigating the genomic basis of environmental tolerances and immune adaptation in C. quadricarinatus will facilitate the development of management strategies of this potential invasive species. Results We constructed a chromosome-level genome of C. quadricarinatus by integrating Nanopore and PacBio techniques. Comparative genomic analysis suggested that transposable elements and tandem repeats drove genome size evolution in decapod crustaceans. The expansion of nine immune-related gene families contributed to the disease resistance of C. quadricarinatus . Three hypoxia-related genes ( KDM3A , KDM5A , HMOX2 ) were identified as being subjected to positive selection in C. quadricarinatus . Additionally, in vivo analysis revealed that upregulating KDM5A was crucial for hypoxic response in C. quadricarinatus . Knockdown of KDM5A impaired hypoxia tolerance in this species. Conclusions Our results provide the genomic basis for hypoxic tolerance and immune adaptation in C. quadricarinatus , facilitating the management of this potential invasive species. Additionally, in vivo analysis in C. quadricarinatus suggests that the role of KDM5A in the hypoxic response of animals is complex.

Nuclear mitochondrial pseudogenes (numts) are nonfunctional copies of mtDNA in the nucleus that have been found in major clades of eukaryotic organisms. They can be easily coamplified with orthologous mtDNA by using conserved universal primers; however, this is especially problematic for DNA barcoding, which attempts to characterize all living organisms by using a short fragment of the mitochondrial cytochrome c oxidase I (COI) gene. Here, we study the effect of numts on DNA barcoding based on phylogenetic and barcoding analyses of numt and mtDNA sequences in two divergent lineages of arthropods: grasshoppers and crayfish. Single individuals from both organisms have numts of the COI gene, many of which are highly divergent from orthologous mtDNA sequences, and DNA barcoding analysis incorrectly overestimates the number of unique species based on the standard metric of 3% sequence divergence. Removal of numts based on a careful examination of sequence characteristics, including indels, in-frame stop codons, and nucleotide composition, drastically reduces the incorrect inferences of the number of unique species, but even such rigorous quality control measures fail to identify certain numts. We also show that the distribution of numts is lineage-specific and the presence of numts cannot be known a priori. Whereas DNA barcoding strives for rapid and inexpensive generation of molecular species tags, we demonstrate that the presence of COI numts makes this goal difficult to achieve when numts are prevalent and can introduce serious ambiguity into DNA barcoding.

The hematopoietic tissue (HPT) and anterior proliferation center (APC) are the main hemocyte-producing organs of the freshwater crayfish, Pacifastacus leniusculus . To deepen our understanding of immune responses to various pathogens, it is essential to identify distinct hemocyte subpopulations with specific functions and to further explore how these cells are generated. Here we provide an in-depth histological study of the HPT and APC in order to localize cell types in different developmental stages, and to provide some information regarding the hemocyte differentiation in the crayfish. We localized mRNA expression of previously identified genes in the HPT/APC and hemocytes by RNA-FISH. The expression of hemolectin and transglutaminase 1 was shown to be co-localized in a high number of the HPT cells, while transglutaminase 2 was expressed in different cell types mainly associated with epithelium or endothelium. Furthermore, by double RNA-FISH for hemolectin and a previously unidentified PDGF-like factor, combined with immunostaining for prophenoloxidase, we could identify several different subtypes of hemocytes, indicating that the immune function of hemocytes in crayfish is more diversified and complex than previously appreciated.