An i-type lysozyme from a crustacean, Pacifastacus leniusculus, functions as a clot-destabilising 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 bonds 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 destabilising activity in crustacean blood.

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.

The stress-immunity axis in shellfish.

It is a difficult task to describe what constitutes a 'healthy' shellfish (e.g., crustacean, bivalve). Visible defects such as discolouration, missing limbs or spines, fouling, lesions, and exoskeletal fractures can be indicative of underlying issues, senescence, or a 'stressed' animal. The absence of such symptoms is not evidence of a disease-free or a stress-free state. Now, more than ever, aquatic invertebrates must cope with acute and chronic environmental perturbations, such as, heatwaves and cold shocks, xenobiotic contaminants, intoxication events, and promiscuous pathogens expanding their host and geographic ranges. With that in mind, how does one determine the extent to which shellfish become stressed in situ (natural) or in cultured (artificial) settings to enhance disease susceptibility? Many biomarkers - predominantly biochemical and cellular measures of shellfish blood (haemolymph) - are considered to gauge immunosuppression and immunocompetence. Such measures range from immune cell (haemocyte) counts to enzymic activities and metabolite quantitation. Stressed invertebrates often reflect degraded conditions of their ecosystems, referred to as environmental indicators. We audit briefly the broad immune functions of shellfish, how they are modulated by known and emerging stressors, and discuss these concepts with respect to neuroendocrinology and immunotoxicology. We assert that chronic stress, alone or in combination with microbial, chemical and abiotic factors, increases the risk of infectious disease in shellfish, exacerbates idiopathic morbidity, and reduces the likelihood of recovery. Acute stress events can lead to immunomodulation, but these effects are largely transient. Enhancing our understanding of shellfish health and immunity is imperative for tackling losses at each stage of the aquatic food cycle and disease outbreaks in the wild.

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.

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.

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.

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.

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.

Correction: Caspase-1-Like Regulation of the proPO-System and Role of ppA and Caspase-1-Like Cleaved Peptides from proPO in Innate Immunity.

[This corrects the article DOI: 10.1371/journal.ppat.1004059.].

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.

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.

Reactive Oxygen Species Affect Transglutaminase Activity and Regulate Hematopoiesis in a Crustacean.

Reactive oxygen species (ROS) serve as a prime signal in the commitment to hematopoiesis in both mammals and Drosophila In this study, the potential function of ROS during hematopoiesis in the crayfish Pacifastacus leniusculus was examined. The antioxidant N-acetylcysteine (NAC) was used to decrease ROS in both in vivo and in vitro experiments. An increase in ROS was observed in the anterior proliferation center (APC) after LPS injection. In the absence of NAC, the LPS-induced increase in ROS levels resulted in the rapid restoration of the circulating hemocyte number. In the presence of NAC, a delay in the recovery rate of the hemocyte number was observed. NAC treatment also blocked the spread of APC and other hematopoietic tissue (HPT) cells, maintaining these cells at an undifferentiated stage. Extracellular transglutaminase (TGase) has been shown previously to play a role in maintaining HPT cells in an undifferentiated form. In this study, we show that extracellular TGase activity increased when the ROS level in HPT or APC cells was reduced after NAC treatment. In addition, collagen, a major component of the extracellular matrix and a TGase substrate were co-localized on the HPT cell surface. Taken together, the results of this study show that ROS are involved in crayfish hematopoiesis, in which a low ROS level is required to maintain hematopoietic progenitor cells in the tissue and to reduce hemocyte release. The potential roles of TGase in this process are investigated and discussed.

A Pacifastacus leniusculus serine protease interacts with WSSV.

Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PlcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S) motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PlcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells.

Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity.

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.

Characterization of a hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus.

Hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus has a distinct role from that of its homolog PmHHAP in the shrimp Penaeus monodon. Knockdown of PlHHAP in vitro using double-stranded RNA (dsRNA) had no effect on the cell morphology of hematopoietic tissue (HPT) cells. The total hemocyte number and caspase activity were unchanged after PlHHAP knockdown in vivo, in contrast to the results found in shrimp. Moreover, suppression of PlHHAP both in vitro and in vivo did not change the mRNA levels of some genes involved in hematopoiesis and hemocyte homeostasis. Interestingly, bacterial count and scanning electron microscope revealed that depletion of PlHHAP in intestine by RNAi resulted in higher number of bacteria in the crayfish intestine. Together, these results suggest that PlHHAP is not involved in hemocyte homeostasis in the crayfish P. leniusculus but appears to affect the bacterial number in the intestine through an unknown mechanism. Since PlHHAP has different functions from PmHHAP, we therefore named it HHAP-like protein.

Astakine 2--the dark knight linking melatonin to circadian regulation in crustaceans.

Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.

Hijacking of host calreticulin is required for the white spot syndrome virus replication cycle.

We have previously shown that multifunctional calreticulin (CRT), which resides in the endoplasmic reticulum (ER) and is involved in ER-associated protein processing, responds to infection with white spot syndrome virus (WSSV) by increasing mRNA and protein expression and by forming a complex with gC1qR and thereby delaying apoptosis. Here, we show that CRT can directly interact with WSSV structural proteins, including VP15 and VP28, during an early stage of virus infection. The binding of VP28 with CRT does not promote WSSV entry, and CRT-VP15 interaction was detected in the viral genome in virally infected host cells and thus may have an effect on WSSV replication. Moreover, CRT was detected in the viral envelope of purified WSSV virions. CRT was also found to be of high importance for proper oligomerization of the viral structural proteins VP26 and VP28, and when CRT glycosylation was blocked with tunicamycin, a significant decrease in both viral replication and assembly was detected. Together, these findings suggest that CRT confers several advantages to WSSV, from the initial steps of WSSV infection to the assembly of virions. Therefore, CRT is required as a "vital factor" and is hijacked by WSSV for its replication cycle. Importance: White spot syndrome virus (WSSV) is a double-stranded DNA virus and the cause of a serious disease in a wide range of crustaceans that often leads to high mortality rates. We have previously shown that the protein calreticulin (CRT), which resides in the endoplasmic reticulum (ER) of the cell, is important in the host response to the virus. In this report, we show that the virus uses this host protein to enter the cell and to make the host produce new viral structural proteins. Through its interaction with two viral proteins, the virus "hijacks" host calreticulin and uses it for its own needs. These findings provide new insight into the interaction between a large DNA virus and the host protein CRT and may help in understanding the viral infection process in general.

Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.