Immune properties of invertebrate phenoloxidases.

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.

Crayfish immunity - Recent findings.

Freshwater crayfish is an important commodity as well as a successful model for studies on crustacean immunity. Due to the ease with which they are kept and the available methods for hemocyte separation and culture they have proven to be very useful. Here, recent progress regarding pattern recognition, immune effector production and antiviral mechanisms are discussed.

Variable immune molecules in invertebrates.

Recently it has become evident that invertebrates may mount a highly variable immune response that is dependent on which pathogen is involved. The molecular mechanisms behind this diversity are beginning to be unravelled and in several invertebrate taxa immune proteins exhibiting a broad range of diversity have been found. In some cases, evidence has been gathered suggesting that this molecular diversity translates into the ability of an affected invertebrate to mount a defence that is specifically aimed at a particular pathogen.

Coagulation in invertebrates.

In most animals there is a need to quickly prevent the loss of blood or equivalent fluids through inflicted injuries. In invertebrates with an open circulatory system (and sometimes a hydroskeleton as well) these losses may otherwise soon be fatal. Also, there is a need to prevent microbes that have gained access to the body through the wound from disseminating throughout the open circulatory system.

Proteinase inhibitory activities of two two-domain Kazal proteinase inhibitors from the freshwater crayfish Pacifastacus leniusculus and the importance of the P(2) position in proteinase inhibitory activity.

Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors (KPIs), KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P.

Proteolytic cascades and their involvement in invertebrate immunity.

Bacteria and other potential pathogens are cleared rapidly from the body fluids of invertebrates by the immediate response of the innate immune system. Proteolytic cascades, following their initiation by pattern recognition proteins, control several such reactions, notably coagulation, melanisation, activation of the Toll receptor and complement-like reactions. However, there is considerable variation among invertebrates and these cascades, although widespread, are not present in all phyla.

Crustacean immunity.

This chapter provides are view of recent progress in the elucidation of innate immune mechanisms in crustaceans. Mainly due to the importance of crustacean aquaculture interest in this field is large and the subject for extensive research efforts. Here, we provide detailed data on the molecular characterisation of lectins, antiviral reactions, hemocyte formation and differentiation and on the regulation of innate immune pathways.

In vitro effects on bacterial growth of phenoloxidase reaction products.

An active phenoloxidase preparation from the freshwater crayfish Pacifastacus leniusculus exhibited a strong antibacterial effect in vitro on the bacteria Aeromonas hydrophila, Escherichia coli, Streptococcus pneumoniae whereas a weaker but still significant effect against Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus. In most cases reduction of bacterial growth was stronger when dopamine was used as substrate as compared to L-dopa. The effect on bacteria was abolished if no substrate was available for the phenoloxidase or in the presence of the phenoloxidase inhibitor phenylthiourea.

High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans.

Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly.

Phylogenetic relationships among plant and animal parasites, and saprotrophs in Aphanomyces (Oomycetes).

Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) were analyzed based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group.

The proPO-system: pros and cons for its role in invertebrate immunity.

Melanisation is an important immune response in many invertebrates. Recent evidence also strongly implies that the melanisation (prophenoloxidase activating) cascade is intimately associated with the appearance of factors stimulating cellular defence by aiding phagocytosis and encapsulation reactions. However, some controversy exists in the field, and at least in flies and mosquitoes, the successful combat of some pathogens does not seem to be dependent on phenoloxidase activity.

Phenoloxidase is an important component of the defense against Aeromonas hydrophila Infection in a crustacean, Pacifastacus leniusculus.

The melanization cascade, in which phenoloxidase is the terminal enzyme, appears to play a key role in recognition of and defense against microbial infections in invertebrates. Here, we show that phenoloxidase activity and melanization are important for the immune defense toward a highly pathogenic bacterium, Aeromonas hydrophila, in the freshwater crayfish, Pacifastacus leniusculus. RNA interference-mediated depletion of crayfish prophenoloxidase leads to increased bacterial growth, lower phagocytosis, lower phenoloxidase activity, lower nodule formation, and higher mortality when infected with this bacterium.

Re-evaluation of the enigmatic species complex Saprolegnia diclina-Saprolegnia parasitica based on morphological, physiological and molecular data.

The phylogenetic relationships among isolates of the Saprolegnia diclina-Saprolegnia parasitica complex were investigated based on ITS rDNA sequences, and correlated with morphological and physiological characters. The isolates studied belong to five phylogenetically separate clades. The majority of presumed parasitic isolates, mostly isolated from fish lesions, fell within a clade that comprises isolates which has been variously named as S.

Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Pacifastacus leniusculus.

In a study of genes expressed differentially in the freshwater crayfish Pacifastacus leniusculus infected experimentally with the white spot syndrome virus (WSSV), one protein, known as antilipopolysaccharide factor (ALF), was chosen, among those whose transcript levels increased upon viral infection, for further studies. ALF RNA interference (RNAi) experiments in whole animals and in cell cultures indicated that ALF can protect against WSSV infection, since knockdown of ALF by RNAi specifically resulted in higher rates of viral propagation. In a cell culture of hematopoietic tissue (Hpt) from P.

Purification of properoxinectin, a myeloperoxidase homologue and its activation to a cell adhesion molecule.

Peroxidases are important mediators of innate immune reactions throughout the animal kingdom. In many arthropods a myeloperoxidase homologue, peroxinectin, is known to function as a cell adhesion factor and an opsonin. Here, we report in the freshwater crayfish Pacifastacus leniusculus the isolation of properoxinectin, inactive in cell adhesion, and we also show that properoxinectin is produced in the mature blood cells whereas the hematopoietic tissue contains very little of this protein.

Physiological and genetic characterisation of some new Aphanomyces strains isolated from freshwater crayfish.

Five Aphanomyces strains were isolated during suspected outbreaks of crayfish disease in Spain and Italy. Genetic and physiological evidence show that the strains isolated from the freshwater crayfish Procambarus clarkii and Pacifastacus leniusculus, do not fit into any previously identified group of Aphanomyces astaci and are not capable of killing crayfish following standardised experimental infection. RAPD-PCR and ITS sequencing analysis show a high degree of similarity between the new isolates, while they are clearly different from the A.

The prophenoloxidase-activating system in invertebrates.

A major innate defense system in invertebrates is the melanization of pathogens and damaged tissues. This important process is controlled by the enzyme phenoloxidase (PO) that in turn is regulated in a highly elaborate manner for avoiding unnecessary production of highly toxic and reactive compounds. Recent progress, especially in arthropods, in the elucidation of mechanisms controlling the activation of zymogenic proPO into active PO by a cascade of serine proteinases and other factors is reviewed.

Host prophenoloxidase expression in freshwater crayfish is linked to increased resistance to the crayfish plague fungus, Aphanomyces astaci.

The crayfish plague (Aphanomyces astaci) susceptible freshwater crayfish Astacus astacus and the resistant species Pacifastacus leniusculus were compared with respect to differential haemocyte count and expression of prophenoloxidase and peroxinectin. A major difference found was that resistant crayfish continuously produced high levels of prophenoloxidase (proPO) transcripts and that these levels could not be further increased, whereas in susceptible crayfish proPO transcript levels and resistance were augmented by immunostimulants. In As.

Prevalence of a single fish-pathogenic Saprolegnia sp. clone in Finland and Sweden.

Thirty-one isolates of Saprolegnia sp., most originating from infected salmon or trout, were characterised genetically and physiologically. The majority (6 of 31) of the isolates from several widely separated geographical locations was found to be genetically almost identical as assessed by RAPD-PCR.

Pumilio homologue from saprolegnia parasitica specifically expressed in undifferentiated spore cysts.

The expression of spore-specific marker transcripts at different stages of the asexual life cycle of Saprolegnia parasitica was analyzed. One of the markers, designated puf1, was found to be expressed transiently upon each of several cycles of zoospore encystment and reemergence. The transcript is induced immediately upon zoospore encystment and is rapidly lost when a cyst is triggered to germinate.

Analysis of chitinase expression in the crayfish plague fungus Aphanomyces astaci.

Chitinase, as determined by enzymatic activity in the growth medium and by transcription of the chitinase gene AaChi1, is expressed at a high level during vegetative growth of the crayfish pathogen Aphanomyces astaci and expression is not further stimulated by chitin. Expression is not detected in zoospores and it does not increase to high levels until late during germination. Upon sporulation, chitinase expression increases to levels comparable with those seen in fast-growing mycelium.