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The crayfish plague is caused by the oomycete Aphanomyces astaci with North American crayfish (for example Pacifastacus leniusculus and Procambarus clarkii) serving as carriers and vectors for this pathogen. This poses a constant threat to native crayfish in Europe, Asia, South America and Australia, which all are highly susceptible to this pathogen. In this study we now show how the symbiotic balance between the pathogen and its host are maintained at the molecular level. The host factors involved in this balance between the pathogen, A. astaci and the host, P. leniusculus, are one glycine-rich antimicrobial peptide (GRP) that is specifically active against A. astaci, but not to other microorganisms and two Kazal proteinase inhibitors (KPI2 and KPILA) inhibit secreted A. astaci proteases by binding to subtilisin enzymes from the pathogen. Accordingly, the expression of GRP, KPI2, KPILA, as well as proPO mRNAs increases following A. astaci infection. Silencing GRP, or KPI2 + KPILA mRNAs results in death of the crayfish from infection. Over time, this host-pathogen relationship has evolved to allow resistant crayfish to coexist with A. astaci in their cuticle for life, provided critical components remain unaltered by environmental changes or other pathogens. It is unclear whether a similar relationship could develop between currently susceptible crayfish and A. astaci.

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

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 isopeptidase 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 epsilon-(gamma-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 (3-1,3-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-gamma-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.

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.

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-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 hemocyte proteins in freshwater crayfish Pacifastacus leniusculus, in response to an injection with the fungal pattern recognition protein beta-1,3-glucan (laminarin) were investigated, as well as changes after saline (vehicle) injection and in naive 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 beta-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.

Melanin production from different types of phenoloxidases (POs) confers immunity from a variety of pathogens ranging from viruses and microorganisms to parasites. The arthropod proPO expresses a variety of activities including cytokine, opsonin and microbiocidal activities independent of and even without melanin production. Proteolytic processing of proPO and its activating enzyme gives rise to several peptide fragments with a variety of separate activities in a process reminiscent of vertebrate complement system activation although proPO bears no sequence similarity to vertebrate complement factors. Pathogens influence proPO activation and thereby what types of immune effects that will be produced. An increasing number of specialised pathogens - from parasites to viruses - have been identified who can synthesise compounds specifically aimed at the proPO-system. In invertebrates outside the arthropods phylogenetically unrelated POs are participating in melanization reactions obviously aimed at intruders and/or aberrant tissues.