Aphanomyces astaci in Mexico: A new haplotype from dwarf crayfish Cambarellus montezumae.

The crayfish plague is an emerging infectious disease caused by the pathogen Aphanomyces astaci (Oomycota), which is responsible for the decimation of Eurasian freshwater crayfish. This pathogen can coexist with the North American crayfish. These are chronic carriers of the disease as consequence of an immune response that can contain the growth of the pathogen without killing it. The origin of A. astaci locates in the southeastern United States and coincides with the origin of the family Cambaridae. This diverse family of decapods is distributed in North America from southern Canada to Honduras. However, only the native crayfish species from Canada and the USA have been examined for the presence of A. astaci. In this study, we describe for the first time the presence of A. astaci in Mexico in a population of the native species Cambarellus montezumae. By analyzing the small (rrnS) and large (rrnL) mitochondrial ribosomal regions, we showed the presence of two haplotypes of A. astaci within the same population (d1-haplotype and, a novel haplotype that was named, mex1-haplotype). The finding of A. astaci in Mexico confirms the occurrence of this pathogen within the range of the family Cambaridae. The individuals of C. montezumae appear to be chronic carriers of A. astaci, indicated by the lack of documented crayfish plague outbreaks in this population, similar to the pattern observed in other North American species. Thus, the results are of special concern to susceptible species of southern regions of America, i.e., Parastacidae. Therefore, this work emphasizes the need to better understand the distribution and genetic diversity of A. astaci within the distribution range of the natural carriers, i.e., North American species, especially the unexplored area of the family Cambaridae.

Vibrio areninigrae as a pathogenic bacterium in a crustacean.

The occurrence of infectious diseases poses a significant threat to the aquaculture industry worldwide. Therefore, characterization of potentially harmful pathogens is one of the most important strategies to control disease outbreaks. In the present study, we investigated for the first time the pathogenicity of two Vibrio species, Vibrio metschnikovii, a foodborne pathogen that causes fatalities in humans, and Vibrio areninigrae, a bacteria isolated from black sand in Korea, using a crustacean model, the signal crayfish Pacifastacus leniusculus. Mortality challenges indicated that injection of V. metschnikovii (108 CFU/crayfish) has a mortality percentage of 22% in crayfish. In contrast, injection of P. leniusculus with 108 or 107 CFU of V. areninigrae resulted in 100% mortality within one and two days post-injection, respectively. V. areninigrae was successfully re-isolated from hepatopancreas of infected crayfish and caused 100% mortality when reinjected into new healthy crayfish. As a consequence of this infection, histopathological analysis revealed nodule formation in crayfish hepatopancreas, heart, and gills, as well as sloughed cells inside hepatopancreatic tubules and atrophy. Moreover, extracellular crude products (ECP's) were obtained from V. areninigrae in order to investigate putative virulence factors. In vivo challenges with ECP's caused >90% mortalities within the first 24 h. In vitro challenges with ECP's of hemocytes induced cytotoxicity of hemocytes within the first hour of exposure. These findings represent the first report that V. areninigrae is a highly pathogenic bacterium that can cause disease in crustaceans. On the contrary, V. metschnikovii could not represent a threat for freshwater crayfish.

Environmental concentrations of sulfamethoxazole increase crayfish Pacifastacus leniusculus susceptibility to White Spot Syndrome Virus.

Antibiotics used for humans and livestock are emerging as pollutants in aquatic environments. However, little is known about their effect on aquatic organisms, especially in crustaceans. In the present study, the freshwater crayfish Pacifastacus leniusculus was exposed during 21 days to environmental concentrations of sulfamethoxazole (SMX) (100 ng/L and 1 µg/L). Subsequently, the crayfish susceptibility to infection was evaluated by using White Spot Syndrome Virus (WSSV) challenge, a well-known crustacean pathogen. The median survival time of the infected crayfish exposed to 100 ng/L SMX was one day, whereas the control and the group exposed to 1 µg/L SMX survived for two and three days, respectively. In order to elucidate the effect of SMX upon the crayfish immune response, new sets of crayfish were exposed to the same SMX treatments to evaluate mRNA levels of immune-related genes which are expressed and present in hemocytes and intestine, and to perform total and differential hemocyte counts. These results show a significant down-regulation of the antimicrobial peptide (AMP) Crustin 3 in hemocytes from the 100 ng/L SMX group, as well as a significant up-regulation of the AMP Crustin 1 in intestines from the 1 µg/L SMX group. Semigranular and total hemocyte cell number were observed to be significantly lower after exposure to 100 ng/L SMX in comparison with the control group. The present study demonstrates that environmentally relevant SMX concentrations in the water at 100 ng/L led to an increased WSSV susceptibility, that may have been caused by a reduction of circulating hemocytes. Nevertheless, SMX concentrations of 1 µg/L could marginally and for a few days have an immunostimulatory effect.

Intestinal microbiome in crayfish: Its role upon growth and disease presentation.

The intestine-associated microbiota in crustaceans are considered a key element for maintaining homeostasis and health within the organisms. Recently, efforts have been made to characterize bacterial communities of freshwater crustaceans, including crayfish, and their interplay with the host's physiology and the aquatic environments. As a result, it has become evident that crayfish intestinal microbial communities display high plasticity, which is strongly influenced by both the diet, especially in aquaculture, and the environment. Moreover, studies regarding the characterization and distribution of the microbiota along the gut portions led to the discovery of bacteria with probiotic potential. The addition of these microorganisms to their food has shown a limited positive correlation with the growth and development of crayfish freshwater species. Finally, there is evidence that infections, particularly those from viral etiology, lead to low diversity and abundance of the intestinal microbial communities. In the present article, we have reviewed data on the crayfish' intestinal microbiota, highlighting the most frequently observed taxa and emphasizing the dominance of phylum within this community. In addition, we have also searched for evidence of microbiome manipulation and its potential impact on productive parameters, and discussed the role of the microbiome in the regulation of diseases presentation, and environmental perturbations.

Gut microbiome alterations in the crustacean Pacifastacus leniusculus exposed to environmental concentrations of antibiotics and effects on susceptibility to bacteria challenges.

Gut-associated microbiota in crustaceans are recognized as a key element for maintaining homeostasis and health in the animal. Since the richness of these microbial communities is strongly influenced by the local environment, especially in aquatic organisms, it is important to address to what extent environmental variations can affect these communities. In the present study, we used high-throughput 16S rRNA sequencing technology to study the composition of gut-associated microbiota of the crayfish Pacifastacus leniusculus after exposure to environmentally-relevant concentrations of an antibiotic, namely sulfamethoxazole. Also, we examined if alterations of microbiota caused by environmentally-relevant concentrations of this antibiotic affected the host susceptibility to bacterial diseases, including Vibrio species. As a result, we found high individual variability of bacterial abundance and composition in the intestinal microbiome of crayfish, in both antibiotic-exposed and antibiotic-free crayfish. However, an increase of chitinolytic bacteria including Vibrio spp. was detected in some animals exposed to the antibiotic. Moreover, when crayfish susceptibility to bacterial infections was tested, the antibiotic-exposed crayfish survived longer than the control crayfish group. This study represents the first approach for investigating the interplay between crayfish and intestinal bacteria during antibiotic-pollution scenarios. Results herein should be considered by scientists before planning experiments under laboratory conditions, especially to study environmental effects on aquatic animals' intestinal health and immune status.

iTRAQ-Based Proteomic Profile Analysis of the Hepatopancreas of Caribbean Spiny Lobsters Infected With Panulirus argus Virus 1: Metabolic and Physiological Implications.

The Caribbean spiny lobster Panulirus argus (Latreille, 1084) sustains economically valuable fisheries throughout the wider Caribbean region. This species is currently affected by the pathogenic virus Panulirus argus Virus 1 (PaV1) that causes a systemic and chronic-degenerative infection in juvenile spiny lobsters P. argus. To date, there is no available information regarding the host alterations induced by this pathogen at the molecular level. In the present study, comparative proteomic analyses of the changes in the hepatopancreas between infected and non-infected juvenile lobsters were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) coupled to synchronous precursor selection (SPS)-based MS3. We identified a total of 636 proteins, being 68 down-regulated and 71 up-regulated proteins. Among the down-regulated proteins, we identified several enzymes involved in the metabolism of hormones and lipids, digestive proteases and glycosidases, while proteins associated with the histone core, protein synthesis, immune response and RNA regulation were up-regulated. Several misregulated enzymes involved in the regulation of neuromodulators were also identified. RT-qPCR assays were used to validate the expression of transcripts encoding for selected differential proteins that were in concordance to proteomic data, as well as the tendency observed in the enzymatic activities of trypsin, chymotrypsin, and glycosidase. In a similar way, we observed glycogen reduction in muscle, and an increase in plasma acylglycerides and glucose, which may be explained by proteomic data. This study provides the first insight into the molecular changes in the hepatopancreas of Caribbean spiny lobsters associated to PaV1 infection. Data provided herein would help to clarify the origin of the molecular misregulations observed at macroscopic level in this host-pathogen interaction.

Proteomic profiling of the white shrimp Litopenaeus vannamei (Boone, 1931) hemocytes infected with white spot syndrome virus reveals the induction of allergy-related proteins.

To elucidate the proteomic responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection at the proteome level, a quantitative shotgun proteomic analysis was performed to detect differentially synthesized proteins in infected hemocytes of white shrimp (Litopenaeus vannamei). We identified 1528 proteins associated to 203 gene ontology (GO) categories. The most representative GO categories were regulation of cellular processes, organic substance metabolic processes and nitrogen compound metabolic processes. Most of the 83 detected up-regulated proteins are involved in DNA regulation and organization and cell signaling. In contrast, most of the 40 down-regulated proteins were related to immune defense processes, protein folding, and development. Differentially induced proteins were further analyzed at the transcript level by RT-qPCR to validate the results. This work provides new insights into the alterations of L. vannamei hemocytes at the protein level at 12 h post-infection with WSSV. Interestingly, several of the up-regulated proteins are allergy-related proteins in humans. Based on our results, we suggest a deeper analysis of the effects of this interaction on the regulation of allergy related-proteins as their up-regulation during WSSV could represent a threat to human health.