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Freshwater crayfish and the crayfish plague fungus: Disease diagnosis and effects of fungal infection on immunity and reproduction.

Objective

1. Designing molecular tools to detect the crayfish plague fungus.
2. Design tools to identify, follow and trace origin of infection of crayfish parasites primarily crayfish plague.
3. Distinguishing between resistant and non-resistant populations and individuals with the aim of breeding resistant native European crayfish.
4. Design tools which can be used to determine health status (immune status) of farmed and wild crayfish so that stress conditions can be avoided.
5. Provide knowledge to possibly enhance the efficiency of the immune system.
6. Determine the association between the immune system and reproduction to aid in the development of a better broadstock.
The possibility to produce a specific probe to detect the crayfish plague fungus on crayfishes is at hand, but still needs extensive additional work before it can be evaluated for its suitability as a molecular probe to detect this pathogenic fungus. Epidemiological studies have been carried out using a RAPD-PCR technique and thus partner 1 and 2 have shown that signal crayfish has caused crayfish plague outbreaks in both Germany and Finland. Using this method we have developed a simple tool to use for such epidemiological studies since we have characterised about 30 different A.astaci strains and found that they can be placed in 4 different genotypes and this allows us to determine the origin of the crayfish plague fungus from any infected lake or waterbody. Two papers in international journals have been published. One lipoprotein involved in clotting has been cloned and characterised and found to belong to the vitellogenin super family of proteins which is of considerable interest since this shows that reproduction and an innate immune reaction are linked. A manuscript on the characterisation of this clotting protein is in press in Proc. Natl Acad. Sci.USA. The work on studying changes in gene expression during a fungal infection has so far shown that some transcripts are upregulated and some are downregulated. These transcripts are presently being cloned and sequenced and we anticipate that during the second year we will have these genes characterised. Partner 3 has also successfully characterised the lipid classes and fatty acid distribution in two different crayfish species and an interesting finding was that triacyIglycerols are only present in females.

FUTURE ACTIONS

Partner 2 in collaboration with partner 1 will continue with their work on designing and testing a molecular probe to be used to detect the crayfish plague fungus on different crayfish species. Partner 2 will continue to characterise the genes which are up- and downregulated and also with the new method of RN'ase protection analysis study if more genes are affected by a fungal infection during different periods after infection. Partner 3 and 2 will continue their work on the study of changes in amount of lipids and lipoproteins during a fungal infection.
DESCRIPTION OF THE WORK AND PROGRESS

The work to design molecular tools to allow identification of the crayfish plague fungus is performed in laboratory of the associated partner in Munchen with continuous contacts with Uppsala University and is well under way. By using information about the internal transcribed regions surrounding the 5.8S nuclear ribosomal gene it seems to be possible to obtain a sequence within the M region which appears to be specific for crayfish plague strains as judged from several fungi and crayfish plague strains tested. Still however more A.astaci strains need to be tested as well as more closely related species to A. astaci than tested so far to ascertain that the intended probe will be specific for A.astaci strains. However, it seems so far that the results are very promising and that it seems likely to produce a crayfish plague specific molecular probe, which can be used to detect crayfish plague fungus on crayfishes. The spread of the crayfish plague fungus has been followed in some countries in Europe by using molecular techniques. Thus partner 1 in Uppsala and University in Munchen have been able to show that two recent crayfish plague outbreaks in Germany are caused by fungal strains originating from signal crayfish. By examining DNA from two crayfish plague strains which were isolated from plague outbreaks in two Finnish lakes we found that one was caused by a strain from signal crayfish suggesting that this crayfish carrying the plague fungus may have been illegally implanted in this Finnish lake. We have also obtained isolates of crayfish plague outbreaks from two different localities in Spain through the courtesy of Dr. Javier Dieguez-Uribeondo and we have now shown that also these crayfish plague epidemics are due to implanted signal crayfish. Thus by using this RAPD-PCR technique it is possible determine to which genotype the fungal strains and hence which alien crayfish the crayfish plague fungus originates from.
The work on identifying different immune factors which may be involved in mediating resistance towards pathogenic fungi in crayfish has been dealt with by utilizing two completely different techniques. One is based on the fact that the immune genes which have been cloned and characterised from the more resistant crayfish, Pacifastacus leniusculus, are similar to those in Astacus astacus and that the use of cDNA probes and DNA/RNA hybridizations, is possible. We first choose to test whether probes for the two immune genes prophenoloxidase and the glucan binding protein, BGBP, hybridised with RNA from the noble crayfish, A. astacus. In both cases we found that the probes developed in signal crayfish recognised corresponding RNA in the noble crayfish. However since individual variation in gene expression of the tested genes during an infection was observed we instead turned over to use RN:ase protection analysis of the transcripts, which is a more sensitive assay than a Northern analysis and permits analysis of individual animals. This methodology is presently being optimised in the laboratory of Partner 1
During an infection with the crayfish plague fungus in noble crayfish a rather dramatic effect on the hemocyte behaviour was observed, since the hemocytes were shown to be strongly activated at early stages of infection and at later stages the hemocyte number was lowered quite significantly. This means that the animal has few or small options to combat the ongoing infection and will die as a result of this experimental fungal infection. The other technique to identify changes in immune gene expression and to possibly identify new genes which are affected in their expression during an infection is by using a differential display RT-PCR technique. For this purpose RNA has been isolated from crayfish plague-infected and healthy Astacus astacus, respectively. In signal crayfish total RNA was isolated from crayfish previously injected with bacteria and from non-injected signal crayfish. The display from the experiments with signal crayfish gave two transcripts which were induced following injection with bacteria and two which were down regulated. The resulting PCR products were sub cloned but since the PCR products were located in the UTR, in the so-called untranslated region, no sequence comparisons could be made and instead we concentrated our experiments on the noble crayfish. In this crayfish 2 bands were found to be induced and 2 were decreased in their expression following a fungal infection. These products are now being subclconed and sequenced in laboratory of partner 1. Partner 3 and 1 have analysed the effect of crayfish plague fungus on lipoproteins, lipid transport and lipid accumulation in eggs during reproduction in crayfish. Two experiments were performed to provide initial and detailed data on hemolymph lipids, lipid classes, fatty acid distribution in two crayfish species before and after experimentally induced fungal infections. The data obtained from pooled samples in Experiment 1 showed that phospholipids (PL) are the most abundant lipids in the hemolymph and that triacy1glycerols (TAG) were detected only in females. Fungal infection reduced the levels of lipid classes [PL, diacy1glycerols (DAG)and cholesterol] but had no effect on the distribution of fatty acid in samples collected from male and female crayfish of the two tested species (Astacus astacus and Pacifastacus leniusculus). Similar results were found in the HDL and VHDI, fractions obtained from the hemolymph. Preliminary results indicated some effect on the integrity of the hemolymph lipoproteins after fungal infection as revealed by polyacrylamide gel electrophoresis. Moreover, the effects in A. astacus were more pronounced than those for the more resistant P. leniusculus. In order to verify and estimate the individual variation and assess the effect of fungal infection during the reproductive season in vitellogenic females, a second experiment was performed using A. astacus. Results from this experiment indicated that the ellect afrepeated bleeding of crayfish was large-r than the effect obtained following fungal infection probably as a result of the relatively high variation between samples within each treatment group.

Coordinator

Uppsala University
Address
Villavagen 6
751 36 Uppsala
Sweden

Participants (2)

Israel Oceanographic and Limnological Research Ltd.
Israel
Address

31080 Haifa
Ludwig-Maximilians-Universitat Munchen
Germany
Address
Kaulbachstr. 37
80539 Munchen