Time-resolved, integrated analysis of clonally evolving genomes.

Clonal genome evolution is a key feature of asexually reproducing species and human cancer development. While many studies have described the landscapes of clonal genome evolution in cancer, few determine the underlying evolutionary parameters from molecular data, and even fewer integrate theory with data. We derived theoretical results linking mutation rate, time, expansion dynamics, and biological/clinical parameters. Subsequently, we inferred time-resolved estimates of evolutionary parameters from mutation accumulation, mutational signatures and selection. We then applied this framework to predict the time of speciation of the marbled crayfish, an enigmatic, globally invasive parthenogenetic freshwater crayfish. The results predict that speciation occurred between 1986 and 1990, which is consistent with biological records. We also used our framework to analyze whole-genome sequencing datasets from primary and relapsed glioblastoma, an aggressive brain tumor. The results identified evolutionary subgroups and showed that tumor cell survival could be inferred from genomic data that was generated during the resection of the primary tumor. In conclusion, our framework allowed a time-resolved, integrated analysis of key parameters in clonally evolving genomes, and provided novel insights into the evolutionary age of marbled crayfish and the progression of glioblastoma.

Salvia miltiorrhiza polysaccharide promotes the health of crayfish (Procambarus clarkii) by promoting hemocyte phagocytosis, protecting hepatopancreas and enhancing intestinal barrier function.

Plant polysaccharides as immunomodulators are considered one of the effective measures to reduce antibiotic therapy in aquaculture. The immunomodulatory function of Salvia miltiorrhiza polysaccharides (SMP) has been demonstrated and begun to be applied in vertebrates, but its potential effect on crustaceans is unclear. In this study, crayfish (Procambarus clarkii) was fed with 0 %, 0.3 %, 0.7 %, 1.1 %, and 1.5 % SMP for 4 weeks to investigate the effects of SMP on hemocytes phagocytosis, hepatopancreatic function, and intestinal barrier function. The results revealed that hemocyte phagocytic activity was increased in all SMP groups. During the process of hemocytes phagocytic recognition and formation of phagosomes and phagolysosomes, the mRNA expression levels of mas, hem, rab3, ctsb, and lamp-1 were up-regulated mainly in the 0.3 % SMP group. During the clearance phase of phagocytosis, respiratory burst activity, ROS level, T-SOD, CAT, GST, and LZM activities were mainly increased in the 1.5 % SMP group. Hepatopancreas AKP and GOT activity were no significant change in all SMP groups. ACP activity was significantly enhanced in the 1.1 % SMP group. The GPT activity of 0.3-0.7 % SMP group was significantly decreased. The 0.7 % SMP group had the highest intestinal fold height. The highest index values of OTUs, Ace, Chao, and Shannon were in the 0.3 % SMP group. The dietary addition of 0.3 % SMP led to a tendency of increased relative abundance of Firmicutes and Bacteroidota at the phylum level, while the relative abundance of Proteobacteria at the phylum level decreased. In conclusion, dietary SMP could promote crayfish health by enhancing phagocytosis, protecting hepatopancreas and enhancing intestinal barrier function. This study contributes to the theoretical foundation for exploring the potential application of plant polysaccharides in crustaceans.

Effects of replacing fishmeal with soybean meal on the immune and antioxidant capacity, and intestinal metabolic functions of red swamp crayfish Procambarus clarkii.

Excess utilization of plant protein sources in animal feed has been found to adversely affect the antioxidant properties and immunity of animals. While the role of gut microbes in plant protein-induced inflammation has been identified in various models, the specific mechanisms regulating gut microbes in crustaceans remain unclear. Accordingly, this study was designed to investigate the effects of replacing fishmeal with soybean meal (SM) on the hepatopancreas antioxidant and immune capacities, and gut microbial functions of crayfish, as well as the potential microbial regulatory mechanisms. 750 crayfish (4.00 g) were randomly divided into five groups: SS0, SS25, SS50, SS75, and SS100, and fed diets with different levels of soybean meal substituted for fishmeal for six weeks. High SM supplementation proved detrimental to maintaining hepatopancreas health, as indicated by an increase in hemolymph MDA content, GPT, and GOT activities, the observed rupture of hepatopancreas cell basement membranes, along with the decreased number of hepatopancreatic F cells. Moreover, crayfish subjected to high SM diets experienced obvious inflammation in hepatopancreas, together with up-regulated mRNA expression levels of nfkb, alf, and tlr (p<0.05), whereas the lzm mRNA expression level exhibited the highest value in the SS25 group. Furthermore, hepatopancreas antioxidant properties highly attenuated by the level of dietary SM substitution levels, as evidenced by the observed increase in MDA content (p<0.05), decrease in GSH content (p<0.05), and inhabitation of SOD, CAT, GPx, and GST activities (p<0.05), along with down-regulated hepatopancreas cat, gpx, gst, and mmnsod mRNA expression levels via inhibiting nrf2/keap1 pathway. Functional genes contributing to metabolism identified that high SM diets feeding significantly activated lipopolysaccharide biosynthesis, revealing gut dysfunction acted as the cause of inflammation. The global microbial co-occurrence network further indicated that the microbes contributing more to serum indicators and immunity were in module eigengene 17 (ME17). A structural equation model revealed that the genes related to alf directly drove the serum enzyme activities through microbes in ME17, with OTU399 and OTU533 identified as major biomarkers and classified into Proteobacteria that secrete endotoxins. To conclude, SM could replace 25 % of fishmeal in crayfish diets without negatively affecting immunity, and antioxidant capacity. Excessive SM levels contributed to gut dysfunction and weakened the innate immune system of crayfish.

Genetic prospective of a local invader: the strange pattern of Pontastacus leptodactylus population structure in Greece and Turkey based on microsatellite DNA.

BACKGROUND: The combination of the increasing demand of freshwater crayfish exports, the reduced population sizes due to overfishing, the crayfish plague epidemics and the habitat degradation, have led to unrecorded translocations of Pontastacus leptodactylus in Greek lakes. METHODS AND RESULTS: In the present study, the genetics of five narrow clawed crayfish (P. leptodactylus) populations were studied, namely three translocated populations inhabiting in Northern Greece, one native Greek population from Evros river and one potential progeny source population from Turkey. Nine microsatellite loci previously designed for the specific species were investigated, in order to assess the levels of genetic diversity and further to confirm the origin of these translocated populations some decades after the translocation events. Our results confirmed that the source population for the translocated Greek population is the Turkish lake Egirdir. Further, despite the low values of the number of alleles, heterozygosity, and FST the populations were generally diverse, providing evidence for local adaptation. CONCLUSIONS: The low values of FIS for the translocated populations in combination with the high values of gene flow, possibly indicate the existence of re-introducing events. Apart from the translocated populations, high levels of genetic diversity and heterozygosity were observed in Evros population, suggesting it as a possible unit for future conservation purposes both as a donor population for reintroduction purposes as well as a unique gene pool protection source. To the best of our knowledge this is the first study dealing with the genetic composition of Greek P. leptodactylus populations from Nothern Greece, operating as a first step towards the development of proper management practices for restocking events and monitoring of translocated populations.

Effects of Aminomethylphosphonic Acid on the Transcriptome and Metabolome of Red Swamp Crayfish, Procambarus clarkii.

Red swamp crayfish, Procambarus clarkii (P. clarkii), is an important model crustacean organism used in many types of research. However, the effects of different doses of aminomethylphosphonic acid (AMAP) on the transcriptome and metabolites of P. clarkii have not been explored. Thus, this study investigated the molecular and metabolic mechanisms activated at the different exposure dosages of AMAP in P. clarkii to provide new insights into the strategies of P. clarkii in response to the high concentrations of AMAP in the environment. In the present study, the P. clarkii were divided into three groups (control group; low-dosage AMAP exposure; high-dosage AMAP exposure), and hepatopancreatic tissue samples were dependently taken from the three groups. The response mechanisms at the different dosages of AMAP were investigated based on the transcriptome and metabolome data of P. clarkii. Differentially expressed genes and differentially abundant metabolites were identified in the distinct AMAP dosage exposure groups. The genes related to ribosome cell components were significantly up-regulated, suggesting that ribosomes play an essential role in responding to AMAP stress. The metabolite taurine, involved in the taurine and hypotaurine metabolism pathway, was significantly down-regulated. P. clarkii may provide feedback to counteract different dosages of AMAP via the upregulation of ribosome-related genes and multiple metabolic pathways. These key genes and metabolites play an important role in the response to AMAP stress to better prepare for survival in high AMAP concentrations.

De Novo Cloning and Functional Characterization of a Mechanosensitive Piezo-Like Ion Channel in the Crayfish.

BACKGROUND/AIMS: Mechanosensitive ion channels are the principal elements in the transduction of mechanical force to neural activity. To date, considerably fewer studies have been published about the molecular and structural properties of mechanosensitive channels. Piezo channels are the only ion channel family in eukaryotes which is selectively gated by the membrane tension. Piezo channels have been described in mammals and some other eukaryotes. However, not much information is available for the crustaceans. METHODS: Conventional cloning methods were used to clone the putative PIEZO channel mRNA in crayfish ganglia samples. HEK293T cells were transfected by the plasmid of the cloned gene for functional studies. The CDS of the mRNA translated into the protein sequence and three-dimensional structure of the channel has been calculated. RESULTS: An mRNA, 9378 bp, was firstly cloned from crayfish which codes a 2674 residues protein. The cloned sequence is similar to the piezo channel mRNAs reported in the other species. The sequence of the coded protein has been analyzed, and some functional domains have been identified. A three-dimensional structure of the coded protein was successfully calculated in reference to mouse piezo 1 channel protein data. A plasmid with a fluorescent protein indicator was synthesized for heterologous expression in HEK293T cells. The evoked calcium response to mechanical stimulation was not different from those observed in the control cells. However, the transfected cells were more sensitive to the gating modifier YODA-1. CONCLUSION: Based on the apparent similarity in sequence, structure and functional properties to other known piezo channels, it has been proposed that cloned mRNA may code a piezo-like ion channel in crayfish.

Genomic analysis reveals strong population structure in the Giant Sydney Crayfish (Euastacus spinifer (Heller, 1865)).

Australia is home to over 140 species of freshwater crayfish (Decapoda: Parastacidae), representing a centre of diversity for this group in the Southern Hemisphere. Species delimitation in freshwater crayfish is difficult because many species show significant variation in colouration and morphology. This is particularly evident in the genus Euastacus, which exhibits large variations in colour and spination throughout its putative range. To understand this variation, we investigated the genetic diversity, population structure, phylogeny, and evolutionary timescale of the Giant Sydney Crayfish (Euastacus spinifer (Heller, 1865)). Our data set is sampled from over 70 individuals from across the ∼600 km range of the species, and includes a combination of two mitochondrial markers and more than 7000 single-nucleotide polymorphisms (SNPs) from the nuclear genome. Data were also obtained for representatives of the close relative, Euastacus vesper McCormack and Ahyong, 2017. Genomic SNP analyses revealed strong population structure, with multiple distinct populations showing little evidence of gene flow or migration. Phylogenetic analyses of mitochondrial data revealed similar structure between populations. Taken together, our analyses suggest that E. spinifer, as currently understood, represents a species complex, of which E. vesper is a member. Molecular clock estimates place the divergences within this group during the Pleistocene. The isolated and highly fragmented populations identified in our analyses probably represent relict populations of a previously widespread ancestral species. Periodic flooding events during the Pleistocene are likely to have facilitated the movement of these otherwise restricted freshwater crayfish within and between drainage basins, including the Murray-Darling and South East Coast Drainages. We present evidence supporting the recognition of populations in the southern parts of the range of E. spinifer as one or two separate species, which would raise the number of species within the E. spinifer complex to at least three. Our results add to the growing body of evidence that many freshwater crayfish exhibit highly fragmented, range-restricted distributions. In combination with the life-history traits of these species, the restricted distributions exacerbate the threats already placed on freshwater crayfish, which are among the five most endangered animal groups globally.

The Role of Biogeography in Shaping Intestinal Flora and Influence on Fatty Acid Composition in Red Swamp Crayfish (Procambarus clarkii).

Intestinal microbiota plays an important role in promoting digestion, metabolism, and immunity. Intestinal microbiota and fatty acids are important indicators to evaluate the health and nutritional composition of Procambarus clarkii. They have been shown to be strongly influence by environmental and genetic factors. However, it is not clear whether environmental factors have a greater impact on the intestinal microbiota and fatty acid composition of crayfish. The link between the intestinal microbial communities and fatty acid (FA) compositions of red swamp crayfish from different geographical has not yet been studied. Thus, the current paper focuses on the influence of different environments on the fatty acids in muscles of crayfish and the possible existence between gut microbiota and fatty acids. Therefore, in this study, we compared the fatty acid compositions and intestinal microbiota of five crayfish populations from different geographical locations. The results were further analyzed to determine whether there is a relationship between geographical location, fatty acid compositions and intestinal microbiota. The gut microbial communities of the crayfish populations were characterized using 16S rRNA high-throughput gene sequencing. The results showed that there were significant differences in FA compositions of crayfish populations from different geographical locations. A similar trend was observed in the gut microbiome, which also varied significantly according to geographic location. Interestingly, the analysis revealed that there was a relationship between fatty acid compositions and intestinal microbes, revealed by alpha diversity analysis and cluster analysis. However, further studies of the interactions between the P. clarkii gut microbiota and biochemical composition are needed, which will ultimately reveal the complexity of microbial ecosystems with potential applications in aquaculture and species conservation.

Dietary melatonin positively impacts the immune system of crayfish, Cherax destructor, as revealed by comparative proteomics analysis.

Melatonin, an indoleamine with various biological activities, is being used increasingly in the aquaculture industry for its broad immune effects. Cherax destructor is an emerging economically cultured crayfish that faces many problems in the breeding process. Previous work found that dietary melatonin has positive effects on the growth and immunity of C. destructor, but the specific mechanism involved remained unclear. In this study, proteomics was used to determine the mechanism of action of melatonin in C. destructor. Results showed that dietary melatonin resulted in decreased levels of hydrogen peroxide, alanine aminotransferase, and aspartate aminotransferase, but increased levels of glutathione peroxidase, acid phosphatase, and glutathione S-transferases. In total, 608 proteins were differentially expressed (418 upregulated and 190 downregulated), and were enriched in three main categories: innate immunity (B cell receptor signaling pathway and natural killer cell-mediated cytotoxicity), glucose metabolism (pentose phosphate pathway, pentose and glucuronate interconversions, and propionate metabolism), and amino acid metabolism (valine, leucine, and isoleucine degradation, and cysteine and methionine metabolism). In addition, dietary melatonin was also involved in the regulation of the mTOR signaling pathway, and upregulated the expression of genes encoding key factors, such as Ras-related GTP-binding protein A/B, eukaryotic initiation factor 4E, eukaryotic initiation factor 4E-binding protein, and p70 ribosomal S6 kinase. Overall, this study demonstrates the role of melatonin in the physiological regulation of C. destructor, laying the foundation for the development of melatonin as a feed additive in the aquaculture of this species.

New insights into the regulation mechanism of red claw crayfish (Cherax quadricarinatus) hepatopancreas under air exposure using transcriptome analysis.

Red claw crayfish (Cherax quadricarinatus) is an important freshwater shrimp species worldwide with enormous economic value. Waterless transportation is an inherent feature of red claw crayfish transportation. However, the high mortality of red claw crayfish is a severe problem in the aquaculture of crayfish after waterless transportation. In this study, we investigated the responses of the hepatopancreas from the red claw crayfish undergoing air exposure stress and normal conditions on transcriptome levels. We used Illumina-based RNA sequencing (RNA-Seq) to perform a transcriptome analysis from the hepatopancreas of red claw crayfish challenged by air exposure. An average of 57,148,800 clean reads per library was obtained, and 33,567 unigenes could be predicted and classified according to their homology with matches in the National Center for Biotechnology Information (NCBI) non-redundant protein sequences (Nr), Gene Ontology (GO), a manually annotated and reviewed protein sequence database (Swiss-Prot), protein families (Pfam), Clusters of Orthologous Groups (COG) of proteins, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. 690 and 3407 differentially expressed genes (DEGs) were identified between the two stress stages of the red claw crayfish. More DEGs were identified in 12 h, indicating that gene expressions were largely changed at 12 h. Some immune-related pathways and genes were identified according to KEGG and GO enrichment analysis. A total of 12 DEGs involved in immune response and trehalose mechanism were verified by quantitative real-time-polymerase chain reaction (qRT-PCR). The results indicated that the red claw crayfish might counteract the stress of air exposure at the transcriptomic level by increasing expression levels of antioxidant-, immune-, and trehalose metabolism-related genes. These transcriptome results from the hepatopancreas provide significant insights into the influence mechanism of air exposure to the trehalose mechanism and immune response in the red claw crayfish.

Molecular cloning and functional characterization of peroxiredoxin 4 (prx 4) in freshwater crayfish, Procambarus clarkii.

Peroxiredoxin (Prx), which is a newly discovered member of the antioxidant protein family, performs important biological functions in intracellular signal transduction. In the present study, a peroxiredoxin 4 gene was cloned from crayfish for the first time and named Pc-prx 4. According to the amino acid sequence signature, Pc-Prx 4 was identified as the typical 2-Cys Prx molecule, which possessed two conserved cysteines (Cys98 and Cys219). Time-course expression patterns post V. harveyi infection revealed that Pc-prx 4 was likely related to crayfish innate immune defense responses. In particular, the highest fold upregulation of the Pc-prx 4 mRNA transcript reached approximately 170 post V. harveyi infection in the crayfish hepatopancreas. The results of the mixed functional oxidase assay showed that rPc-Prx 4△ could resist the damaging effect of reactive oxygen species generated from the thiol/Fe3+/O2- reaction system to some extent. In addition, the results of the RNAi assay revealed that the crayfish survival rate was obviously increased post injection of V. harveyi when Pc-prx 4 was knocked down. Further study revealed that both hemolymph melanization and PO activity were strengthened to different degrees in the RNAi assay. Therefore, we speculated that the increase in the crayfish survival rate was likely due to the increase in hemolymph melanization. The obviously reinforced hemolymph melanization was directly caused by the upregulation of hemolymph PO activity, which was induced by the knockdown of Pc-prx 4. However, further studies are still indispensable for illuminating the molecular mechanism of Pc-prx 4 in the crayfish innate immune defense system.

Molecular cloning and functional characterization of peroxinectin from red swamp crayfish, Procambarus clarkii.

Peroxinectin, which has both peroxidase and cell adhesion activities, is crucial for invertebrate innate immune responses. In this study, we first cloned the full-length cDNA of Procambarus clarkii Peroxinectin (denoted as Pc-Px) and evaluated its immune roles. The Pc-Px cDNA had 2460 base pairs (bp) and 819 amino acid residues, including peroxidase domain and a putative integrin-binding motif. Pc-Px tissue expression was found to be ubiquitous in all examined tissues under normal physiological conditions. Pc-Px mRNA levels were highest in hemocytes, followed by gills and heart, and were lowest in the gut. The LPS, PGN, and Poly I:C treatment significantly up-regulated the transcript level of Pc-Px gene, but the expression trends were different after the microbials component treatments. Pc-Px knockdown using double-stranded RNA altered the transcription profiles of various immune-related genes in hepatopancreas of P. clarkii. Taken together, Pc-Px is an important component of immune system that likely to modulate immune function of P. clarkii via regulating immune-associated genes.

Peroxiredoxin 1 of Procambarus clarkii govern immune responses during pathogen infection.

Members of the peroxiredoxin family are involved in a wide variety of physiological processes, including the ability to combat the effects of oxidative stress and immune responses, among others. Here, we cloned the cDNA of Procambarus clarkii Peroxiredoxin 1 (designated as PcPrx-1) and investigated its biological role in immune system functions in relation to microbial pathogens. The PcPrx-1 cDNA had 744 base pairs in an open reading frame that encoded 247 amino acid residues and contained a PRX_Typ2cys domain. The analysis of tissue specific expression patterns revealed that PcPrx-1 expression was ubiquitous in all tissues. In addition, the mRNA transcript of PcPrx-1 was found to be highest in the hepatopancreas. There was a significant upregulation of PcPrx-1 gene transcripts after exposure to LPS, PGN, and Poly I:C, but the transcription patterns were different after pathogen challenge. Double-stranded RNA was used to knockdown PcPrx-1, which resulted in a striking change in the expression of all the tested P. clarkii immune-associated genes, including lectin, Toll, cactus, chitinase, phospholipase, and sptzale. On the whole, these results suggest that PcPrx-1 is important to confer innate immunity against pathogens by governing the expression of critical transcripts that encode immune-associated genes.

Comparative transcriptome analysis of differentially expressed genes and pathways in Procambarus clarkii (Louisiana crawfish) at different acute temperature stress.

Procambarus clarkii is an important economic species in China, and exhibit heat and cold tolerance in the main culture regions. To understand the mechanisms, we analyzed the hepatopancreas transcriptome of P. clarkii treated at 10 °C, 25 °C, and 30 °C, then 2092 DEGs and 6929 DEGs were found in 30 °C stress group and 10 °C stress group, respectively. KEGG pathway enrichment results showed that immune pathway is the main stress pathway for 10 °C treatment and metabolic pathway is the main response pathway for 30 °C treatment, which implies low temperature stress induces the damage of the immune system and increases the susceptibility of bacteria while the body response to high temperature stress through metabolic adjustment. In addition, flow cytometry proved that both high and low temperature stress caused different degrees of apoptosis of hemocytes, and dynamic transcription heat map analysis also identified the differential expression of HSPs family genes and apoptosis pathway genes under different heat stresses. This indicates that preventing damaged protein misfolding and accelerating cell apoptosis are necessary mechanisms for P. clarkii to cope with high and low temperature stress. Our research has deepened our understanding of the complex molecular mechanisms of P. clarkii in response to acute temperature stress, and provided a potential strategy for aquatic animals to relieve environmental duress.

Complete Mitochondrial Genome Sequence and Phylogenetic Analysis of Procambarus clarkii and Cambaroides dauricus from China.

To enhance the management and protection of crayfish genetic diversity and germplasm resources in Cambaroides dauricus (C. dauricus), a common species of Procambarus clarkii (P. clarkii) was used as a control group to compare the whole mitochondrial genome sequence using Illumina sequencing technology. This study found that the mitochondrial genome of C. dauricus is 15580 bp in length, with a base composition of A (31.84%), G (17.66%), C (9.42%), and T (41.08%) and a C + G content of 27.08%. The C + G in the D-loop is rich in 17.06%, indicating a significant preference. The mitochondrial genome of C. dauricus contains 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes, with most of the genes labeled in the negative direction, except for a few genes that are labeled in the positive direction. The start codons of the ten coding sequences are ATG, and the quintessential TAA and TAG are the stop codons. This study also found that the Ka/Ks ratios of most protein-coding genes in the mitochondria of both shrimps are lower than 1, indicating weak natural selection, except for nad 2, nad 5, and cox 1. The Ka/Ks ratio of cox 3 is the lowest (less than 0.1), indicating that this protein-coding gene bears strong natural selection pressure and functional constraint in the process of mitochondrial genetic evolution of both shrimps. Furthermore, we constructed phylogenetic analyses based on the entire sequence, which effectively distinguishes the high body from other shrimp species of the genus based on the mitochondrial genome. This study provides molecular genetic data for the diversity investigation and protection of fishery resources with Chinese characteristics and a scientific reference for the evolutionary study of Procambarus.

Host-pathogen coevolution drives innate immune response to Aphanomyces astaci infection in freshwater crayfish: transcriptomic evidence.

BACKGROUND: For over a century, scientists have studied host-pathogen interactions between the crayfish plague disease agent Aphanomyces astaci and freshwater crayfish. It has been hypothesised that North American crayfish hosts are disease-resistant due to the long-lasting coevolution with the pathogen. Similarly, the increasing number of latent infections reported in the historically sensitive European crayfish hosts seems to indicate that similar coevolutionary processes are occurring between European crayfish and A. astaci. Our current understanding of these host-pathogen interactions is largely focused on the innate immunity processes in the crayfish haemolymph and cuticle, but the molecular basis of the observed disease-resistance and susceptibility remain unclear. To understand how coevolution is shaping the host's molecular response to the pathogen, susceptible native European noble crayfish and invasive disease-resistant marbled crayfish were challenged with two A. astaci strains of different origin: a haplogroup A strain (introduced to Europe at least 50 years ago, low virulence) and a haplogroup B strain (signal crayfish in lake Tahoe, USA, high virulence). Here, we compare the gene expression profiles of the hepatopancreas, an integrated organ of crayfish immunity and metabolism. RESULTS: We characterised several novel innate immune-related gene groups in both crayfish species. Across all challenge groups, we detected 412 differentially expressed genes (DEGs) in the noble crayfish, and 257 DEGs in the marbled crayfish. In the noble crayfish, a clear immune response was detected to the haplogroup B strain, but not to the haplogroup A strain. In contrast, in the marbled crayfish we detected an immune response to the haplogroup A strain, but not to the haplogroup B strain. CONCLUSIONS: We highlight the hepatopancreas as an important hub for the synthesis of immune molecules in the response to A. astaci. A clear distinction between the innate immune response in the marbled crayfish and the noble crayfish is the capability of the marbled crayfish to mobilise a higher variety of innate immune response effectors. With this study we outline that the type and strength of the host immune response to the pathogen is strongly influenced by the coevolutionary history of the crayfish with specific A. astaci strains.

Genomic scale data shows that Parastacus nicoleti encompasses more than one species of burrowing continental crayfishes and that lineage divergence occurred with and without gene flow.

Delimiting species is a challenge, especially in scenarios of diversification with gene flow and when species are now allopatric where reproductive isolation cannot be directly tested. Continental burrowing crayfishes of the genus Parastacus present a disjoint distribution in southern South America. One of the species is P. nicoleti, which lives in underground waters in swampy and wooded areas of southern Chile. A previous assessment based on mitochondrial DNA sequences suggest that the taxon may represent a species complex. Here, using thousands of nuclear genomic single-nucleotide polymorphisms obtained via RADSeq from 81 specimens collected at 27 localities throughout the distributional range of the species, we apply an integrative species delimitation approach to test species boundaries and to investigate some aspects of the speciation process. Our analyses corroborate previous results; a scenario that we favor suggests that the P. nicoleti encompasses seven distinct species. Additionally, demographic analyses show that the distinct species have followed distinct trajectories in size change during the last 17.5 million years and that speciation in this group occurred both in strict isolation as well as in the presence of gene flow.

Activation of p53 in anoxic freshwater crayfish, Faxonius virilis.

Tumor suppressing transcription factor p53 regulates multiple pathways including DNA repair, cell survival, apoptosis and autophagy. Here, we studied the stress-induced activation of p53 in anoxic crayfish (Faxonius virilis). Relative levels of target proteins and mRNAs involved in the DNA damage response were measured in normoxic control and anoxic hepatopancreas and tail muscle. Phosphorylation levels of p53 were assessed using immunoblotting at sites known to be phosphorylated (serine 15 and 37) in response to DNA damage or reduced oxygen signaling. The capacity for DNA binding by phosphorylated p53 (p-p53) was also measured, followed by transcript analysis of a potentially pro-apoptotic downstream target, the etoposide induced (ei24) gene. Following this, both inhibitor (MDM2) and activator (p19-ARF) protein levels in response to low-oxygen stress were studied. The results showed an increase in p-p53 levels during anoxia in both hepatopancreas and tail muscle. Increased transcript levels of ei24 support the activation of p53 under anoxic stress. Cytoplasmic accumulation of Ser15 phosphorylated p53 was observed during anoxia when proteins from cytoplasmic and nuclear fractions were measured. Increased cytoplasmic concentration is known to initiate an apoptotic response, which can be assumed as a preparatory step to prevent autophagy. The results suggest that p53 might play a protective role in crayfish defense against low-oxygen stress. Understanding how anoxia-tolerant organisms are able to protect themselves against DNA damage could provide important clues towards survival under metabolic rate depression and preparation for recovery to minimize damage.

Studying phenotypic variation and DNA methylation across development, ecology and evolution in the clonal marbled crayfish: a paradigm for investigating epigenotype-phenotype relationships in macro-invertebrates.

Animals can produce different phenotypes from the same genome during development, environmental adaptation and evolution, which is mediated by epigenetic mechanisms including DNA methylation. The obligatory parthenogenetic marbled crayfish, Procambarus virginalis, whose genome and methylome are fully established, proved very suitable to study this issue in detail. Comparison between developmental stages and DNA methylation revealed low expression of Dnmt methylation and Tet demethylation enzymes from the spawned oocyte to the 256 cell embryo and considerably increased expression thereafter. The global 5-methylcytosine level was 2.78% at mid-embryonic development and decreased slightly to 2.41% in 2-year-old adults. Genetically identical clutch-mates raised in the same uniform laboratory setting showed broad variation in morphological, behavioural and life history traits and differences in DNA methylation. The invasion of diverse habitats in tropical to cold-temperate biomes in the last 20 years by the marbled crayfish was associated with the expression of significantly different phenotypic traits and DNA methylation patterns, despite extremely low genetic variation on the whole genome scale, suggesting the establishment of epigenetic ecotypes. The evolution of marbled crayfish from its parent species Procambarus fallax by autotriploidy a few decades ago was accompanied by a significant increase in body size, fertility and life span, a 20% reduction of global DNA methylation and alteration of methylation in hundreds of genes, suggesting that epigenetic mechanisms were involved in speciation and fitness enhancement. The combined analysis of phenotypic traits and DNA methylation across multiple biological contexts in the laboratory and field in marbled crayfish may serve as a blueprint for uncovering the role of epigenetic mechanisms in shaping of phenotypes in macro-invertebrates.

Robust strategy for disease resistance and increasing production breeding in red swamp crayfish (Procambarus clarkii).

Red swamp crayfish (Procambarus clarkii) is an important aquaculture species in China. With increasing crayfish culture, a number of outbreaks of various diseases have been identified in crayfish. Despite this, there are no reports on the application of disease resistance genes in the molecular breeding of crayfish. In this study, transcriptome analysis was performed to explore the disease resistance genes in crayfish, with a focus on investigating the genetic variations in the open reading frames of these genes, for subsequent haplotype analysis. Furthermore, pathogen-challenge experiments were carried out in the crayfish, to identify the favoured haplotypes. A novel disease resistance gene, R (Resistance), was identified by means of transcriptome analysis. In total, two, four, and five haplotypes of the three disease resistance genes, ALF, R, and crustin2, respectively, were detected. ALF1, R1, and Cru1 were the favoured haplotypes of ALF, R, and crustin2, respectively. Subsequently, the favoured haplotype combinations of the different genes were obtained, and a series of molecular markers were developed to identify them. Finally, we propose a molecular breeding strategy to enhance the disease resistance of crayfish, and thus, improve its production.