An i-type lysozyme from a crustacean, Pacifastacus leniusculus, functions as a clot-destabilizing enzyme.

Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bond formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots. We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus, which was found to be upregulated at the protein level in response to an injection of the ß-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate L-γ-glutamine-p-nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilizing activity in crustacean blood.

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.

Blood cell formation in crustaceans.

In crustacean animals the hemocytes are key players in immunity and of crucial importance for the health of the animals. Hemocytes are mainly produced in the hematopoietic tissue and from there released into the circulation where they finally mature. In this review we summarize the latest findings about crustacean hemocyte formation. The role of the extracellular matrix and crosslinking enzyme transglutaminase is discussed. Moreover, important growth factors, transcriptional regulation and recent findings about inducers of hematopoiesis are covered. Finally, we discuss the use of different markers for classification of crustacean hemocytes.

Characterization of hemocytes and hematopoietic cells of a freshwater crayfish based on single-cell transcriptome analysis.

Crustaceans constitute a species-rich and ecologically important animal group, and their circulating blood cells (hemocytes) are of critical importance in immunity as key players in pathogen recognition, phagocytosis, melanization, and antimicrobial defense. To gain a better understanding of the immune responses to different pathogens, it is crucial that we identify different hemocyte subpopulations with different functions and gain a better understanding of how these cells are formed. Here, we performed single-cell RNA sequencing of isolated hematopoietic tissue (HPT) cells and hemocytes from the crayfish Pacifastacus leniusculus to identify hitherto undescribed hemocyte types in the circulation and show that the circulating cells are more diversified than previously recognized. In addition, we discovered cell populations in the HPT with clear precursor characteristics as well as cells involved in iron homeostasis, representing a previously undiscovered cell type. These findings may improve our understanding of hematopoietic stem cell regulation in crustaceans and other animals.

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.

Early Changes in Crayfish Hemocyte Proteins after Injection with a ß-1,3-glucan, Compared to Saline Injected and Naive Animals.

Early changes in hemocyte proteins in freshwater crayfish Pacifastacus leniusculus, in response to an injection with the fungal pattern recognition protein ß-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 ß-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.

Sexual conflict drives micro- and macroevolution of sexual dimorphism in immunity.

BACKGROUND: Sexual dimorphism in immunity is believed to reflect sex differences in reproductive strategies and trade-offs between competing life history demands. Sexual selection can have major effects on mating rates and sex-specific costs of mating and may thereby influence sex differences in immunity as well as associated host-pathogen dynamics. Yet, experimental evidence linking the mating system to evolved sexual dimorphism in immunity are scarce and the direct effects of mating rate on immunity are not well established. Here, we use transcriptomic analyses, experimental evolution and phylogenetic comparative methods to study the association between the mating system and sexual dimorphism in immunity in seed beetles, where mating causes internal injuries in females. RESULTS: We demonstrate that female phenoloxidase (PO) activity, involved in wound healing and defence against parasitic infections, is elevated relative to males. This difference is accompanied by concomitant sex differences in the expression of genes in the prophenoloxidase activating cascade. We document substantial phenotypic plasticity in female PO activity in response to mating and show that experimental evolution under enforced monogamy (resulting in low remating rates and reduced sexual conflict relative to natural polygamy) rapidly decreases female (but not male) PO activity. Moreover, monogamous females had evolved increased tolerance to bacterial infection unrelated to mating, implying that female responses to costly mating may trade off with other aspects of immune defence, an hypothesis which broadly accords with the documented sex differences in gene expression. Finally, female (but not male) PO activity shows correlated evolution with the perceived harmfulness of male genitalia across 12 species of seed beetles, suggesting that sexual conflict has a significant influence on sexual dimorphisms in immunity in this group of insects. CONCLUSIONS: Our study provides insights into the links between sexual conflict and sexual dimorphism in immunity and suggests that selection pressures moulded by mating interactions can lead to a sex-specific mosaic of immune responses with important implications for host-pathogen dynamics in sexually reproducing organisms.

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.

A transcription factor glial cell missing (Gcm) in the freshwater crayfish Pacifastacus leniusculus.

The transcription factor glial cell missing, Gcm, is known to be an important protein in the determination of glial cell fate as well as embryonic plasmatocyte differentiation in Drosophila melanogaster. So far, no function for Gcm in crustaceans has been reported. In this study, we show the cDNA sequence of a Gcm homologue in the freshwater crayfish Pacifastacus leniusculus. The P. leniusculus Gcm transcript is expressed exclusively in brain and nervous tissue, and by in situ hybridization we show that the expression is restricted to a small number of large cells with morphology similar to neurosecretory cells. Furthermore, we show that the expression of Gcm coincides with the expression of a Repo homologue, that is induced in expression by Gcm in Drosophila. Moreover, the Gcm transcript is increased shortly and transiently after injection of cystamine, a substance that inhibits transglutaminase and also strongly affects the movement behavior of crayfish. This finding of Gcm transcripts in a subpopulation of brain cells in very low numbers may enable more detailed studies about Gcm in adult crustaceans.

Transglutaminase 1 and 2 are localized in different blood cells in the freshwater crayfish Pacifastacus leniusculus.

In the present study we show that hemocytes in the freshwater crayfish Pacifastacus leniusculus express two different transglutaminases. We describe the sequence of a previously unknown TGase (Pl_TGase1) and named this as Pl_TGase2 and compared this sequence with similar sequences from other crustaceans. The catalytic core domain is similar to the previously described TGase in P. leniusculus, but Pl_TGase2 has significant differences in the N-terminal and C-terminal domains. Further, we show conclusive evidences that these different transglutaminases are specific for different hemocyte types so that Pl_TGase1 is expressed in the hematopoietic tissue and in the cytoplasm of semigranular hemocytes, while Pl_TGase2 is expressed in vesicles in the granular hemocytes. By in situ hybridization we show that both Pl_TGase1 and Pl_TGase2 mRNA are present only in a subset of the respective hemocyte population. This observation indicates that there may be different subtypes of semigranular as well as granular hemocytes which may have different specific functions.

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.

The N-terminal peptide generated after activation of prophenoloxidase affects crayfish hematopoiesis.

The circulating hemocytes of invertebrates are important mediators of immunity, and hemocyte homeostasis is of high importance for survival and health of crustaceans. The prophenoloxidase (proPO)-activating system is one of the most essential immune reactions, which can be activated by pattern recognition proteins from microorganisms. Activation of proPO by the proPO activating enzyme generates an N-terminal peptide, with cleavage site after Arg176, as well as the active enzyme phenoloxidase, which is the key enzyme for melanization. In the present study we demonstrate a role for the N-terminal proPO-peptide in hematopoiesis. Injection of this proPO-peptide increased the number of circulating hemocytes and especially granular hemocytes. We also show that the reactive oxygen species (ROS) production in the anterior proliferative center was enhanced after proPO peptide injection, which is a prerequisite for rapid hemocyte release from the hematopoietic tissue. Moreover, this peptide had an effect on ROS production in in vitro cultured hematopoietic cells and induced spreading of these cells within 72 h. Taken together, our findings show a role of the N-terminal proPO peptide in stimulation of hematopoiesis in crayfish, Pacifastacus leniusculus.

Astakine1 forms protein complex in plasma.

Astakine 1 is a small cytokine-like peptide which is directly involved in hematopoiesis in crustaceans. Astakines are present in many different invertebrate groups primarily in arthropods. In this study we found that astakine1 was present as a high molecular weight (HMW) complex in plasma. It is known that calcium concentration are fluctuating in several crustaceans especially during the molting process. This HMW-complex was formed under low calcium concentrations in plasma and could be partially reversed provided calcium was added. The biological function of the naïve astakine1 and that in the HMW complex was about the same, but if the protein is to be isolated or studied for its function it is important to know about this property of astakine1 which may previously have hampered isolation and functional studies in other animals than freshwater crayfish.

A comparative global proteomic analysis of the hematopoietic lineages in the crustacean Pacifastacus leniusculus.

In crustaceans as in other arthropods, the circulating hemocytes are vital for protecting the animal against attacking microorganisms. As many hemocytes are destroyed early during an infection, new hemocytes must fast get in place to prevent disperse of a pathogenic microbe, In order to understand the hematopoietic process in more detail we here report a complete proteomic analysis from purified cell types from the APC of the hematopoietic tissue, via the remaining parts of the HPT to the mature semigranular and granular hemocytes. Several possible cell type specific proteins are detected and new putative biomarkers within the crayfish hematopoietic lineage that can be used to increase the understanding of how the differentiation process is regulated is described.

Transglutaminase inhibition stimulates hematopoiesis and reduces aggressive behavior of crayfish, Pacifastacus leniusculus.

Transglutaminase (TGase) is a Ca2+-dependent cross-linking enzyme, which has both enzymatic and nonenzymatic properties. TGase is involved in several cellular activities, including adhesion, migration, survival, apoptosis, and extracellular matrix (ECM) organization. In this study, we focused on the role of the TGase enzyme in controlling hematopoiesis in the crayfish, Pacifastacus leniusculus We hypothesized that a high TGase activity could mediate an interaction of progenitor cells with the ECM to maintain cells in an undifferentiated stage in the hematopoietic tissue (HPT). We found here that the reversible inhibitor cystamine decreases the enzymatic activity of TGase from crayfish HPT, as well as from guinea pig, in a concentration-dependent manner. Cystamine injection decreased TGase activity in HPT without affecting production of reactive oxygen species. Moreover, the decrease in TGase activity in the HPT increased the number of circulating hemocytes. Interestingly the cystamine-mediated TGase inhibition reduced aggressive behavior and movement in crayfish. In conclusion, we show that cystamine-mediated TGase inhibition directly releases HPT progenitor cells from the HPT into the peripheral circulation in the hemolymph and strongly reduces aggressive behavior in crayfish.

The effect of temperature on bacteria-host interactions in the freshwater crayfish, Pacifastacus leniusculus.

Water temperature is known to affect many aspects of aquatic life including immune responses and susceptibility to diseases. In this context, we studied the effect of temperature on the defense system of the freshwater crayfish Pacifastacus leniusculus. Animals were challenged with two pathogenic Gram-negative bacteria, Aeromonas hydrophila and Pseudomonas gessardii, as well as the bacterial cell wall component lipopolysaccharide (LPS) at two different temperatures, cold (6 °C) and room temperature (22 °C). The immune responses were compared by means of differences in mortality, phagocytosis, bacterial clearance, and the melanization reaction of the hemolymph at these two temperatures. We observed that crayfish survival was higher at cold temperature. The mortality rate was zero at 6 °C following A. hydrophila or LPS injections. Furthermore, the bacteria were completely cleared from crayfish after they had been held at 6 °C for more than 9 days. We also observed a strong melanization reaction of hemolymph at 22 °C when stimulated with LPS, as well as with bacteria. Taken together, our results suggest that the cellular immunity is more effective at low temperature in this cold-adapted animal and pathogens are efficiently removed from the body by mean of phagocytosis.

Characterization of a cold-active transglutaminase from a crayfish, Pacifastacus leniusculus.

Transglutaminase (TGase) from signal crayfish (Pacifastacus leniusculus) and its activity at low temperatures was studied. TGase is an abundant protein in the hematopoietic (HPT) cells and this tissue was used for TGase enzyme preparation. The optimal temperature and pH for the activity of crayfish TGase were determined. We found that TGase activity at 4 °C showed nearly the same activity as at a temperature of 22 °C. TGase activity from crayfish was compared with guinea pig liver TGase activity at 4 °C and the crayfish TGase displayed a higher activity while guinea pig liver TGase had a very low activity at this low temperature. By comparing kinetic parameters to guinea pig liver TGase, the results showed that a high activity of crayfish TGase was due to a decreasing Km value for pentylamine as a substrate, while it did not affect the kcat value (at 22 °C). The amino acid sequences of a krill and a crayfish TGase, which both are cold adapted, do not give any clue to why these two enzymes are cold-adapted. These results demonstrate that crayfish TGase is adapted to have significant activity at low temperatures and since crayfish are living in quite cold waters this is an interesting adaptation of this enzyme.

Transfection of crayfish hematopoietic tissue cells.

Transfection is a powerful tool useful for studying gene function. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. The crayfish hematopoietic tissue (Hpt) cell cultures have been proven to be suitable for studies on immunity and cell differentiation in crustaceans including shrimps, but no efficient gene transfer and expression method is available for these cells. Here we report a novel and highly efficient DNA transfection system based on electroporation. This method depends on a recombinant plasmid with the promoter from white spot syndrome virus immediate-early gene wsv249. This plasmid could be introduced into primary cells and efficiently express foreign genes by electroporation. By optimizing different electroporation parameters, more than 30% transfection efficiency could be achieved with the relative viability of cells around 50%. This is the first report of gene introduction to crayfish Hpt cells and will be useful for the expanding our research on crustacean immunity.

Clotting protein - An extracellular matrix (ECM) protein involved in crustacean hematopoiesis.

Hematopoietic progenitor cells in crustaceans are organized in lobule-like structures surrounded by different types of cells and extracellular matrix (ECM) proteins in a Hematopoietic tissue (HPT). Here we show that the clotting protein (CP) is part of the ECM in HPT and is secreted during HPT cell culture. The formation of a filamentous network of CP was observed in HPT cell culture. A high amount of CP protein was detected at the surfaces of undifferentiated cells (round-shaped) compared with migrating cells (spindle shaped). Co-localization of the CP protein and TGase activity was observed on the cell surface and filamentous network between cells. A role for CP together with collagen was revealed in a 3D culture in which a collagen-I matrix was immobilized with CP or supplemented with CP. The results showed possible functions of CP, collagen, TGase and the cytokine Ast1 in the regulation of HPT progenitor cell behavior. This is the first study to provide insight into the role of CP, which probably not only participates in clot formation but also functions as an ECM component protein controlling hematopoietic stem cell behavior.