Research Article |
Corresponding author: Agata Mrugała ( agata_mrugala@wp.pl ) Academic editor: Belinda Gallardo
© 2019 Agata Mrugała, Miloš Buřič, Adam Petrusek, Antonín Kouba.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Mrugała A, Buřič M, Petrusek A, Kouba A (2019) May atyid shrimps act as potential vectors of crayfish plague? NeoBiota 51: 65-80. https://doi.org/10.3897/neobiota.51.37718
|
The causative agent of crayfish plague, Aphanomyces astaci Schikora, was long considered to be a specialist pathogen whose host range is limited to freshwater crayfish. Recent studies, however, provided evidence that this parasite does not only grow within the tissues of freshwater-inhabiting crabs but can also be successfully transmitted by them to European crayfish species. The potential to act as alternative A. astaci hosts was also indicated for freshwater shrimps. We experimentally tested resistance of two freshwater atyid shrimps: Atyopsis moluccensis (De Haan, 1849) and Atya gabonensis Giebel, 1875. They were infected with the A. astaci strain associated with the globally widespread North American red swamp crayfish, Procambarus clarkii (Girard, 1852), the typical host of the A. astaci genotype group D. As popular ornamental species, both shrimps may get in contact with infected P. clarkii not only in the wild but also in the aquarium trade. We assessed the potential of shrimps to transmit A. astaci to susceptible crayfish by cohabiting A. gabonensis previously exposed to A. astaci zoospores with the European noble crayfish, Astacus astacus (Linnaeus, 1758). In both experiments, the presence of A. astaci infection was analysed with species-specific quantitative PCR. We detected A. astaci in bodies and exuviae of both shrimp species exposed to A. astaci zoospores, however, the intensity of infection differed between the species and analysed samples; it was higher in A. moluccensis and the exuviae of both species. A. astaci was also detected in one A. astacus individual in the transmission experiment. This finding reveals that freshwater shrimps may be able to transmit A. astaci to crayfish hosts; this is particularly important as even a single successful infection contributes to the spread of the disease. Moreover, our results indicate that the tested shrimp species may be capable of resisting A. astaci infection and reducing its intensity through moulting. Although their potential to act as prominent A. astaci vectors requires further research, it should not be ignored as these freshwater animals may then facilitate A. astaci spread to susceptible crayfish species in aquarium and aquaculture facilities as well as in the wild.
Aphanomyces astaci, aquatic invasion, Cherax destructor, disease transmission, oomycetes, species introduction
Invasive alien species (IAS) are considered one of the major threats to native biodiversity (
The emergence in Europe of the oomycete Aphanomyces astaci Schikora, the causative agent of crayfish plague, exemplifies the devastating impacts that a novel pathogen may impose on native fauna. Its spread across the continent caused irreversible declines of native European crayfish populations and still threatens their remaining stocks (
In Europe, the spread of A. astaci is mainly facilitated by its original hosts, North American crayfish species (
The releases and escapes from aquaculture and aquarium trade were assessed as the most important entry pathways of non-native freshwater species in Europe (
A. astaci was long considered to be a specialist pathogen whose host range is limited to freshwater crayfish (Decapoda: Astacoidea and Parastacoidea). Recent studies, however, confirmed assumptions of
Apart from their ecological significance, many freshwater shrimps and crabs are involved in intensive aquaculture and pet trade, and hence they have considerable socioeconomic importance. Their potential sensitivity towards the crayfish plague pathogen might thus have far-reaching consequences (
The present study focuses on interactions of freshwater shrimp species with A. astaci, and experimentally tests two hypotheses evaluating shrimps’ potential to act as its alternative vectors: 1) the chosen shrimp species may host A. astaci, and 2) they may transmit this parasite to susceptible crayfish. Two widespread filter-feeding atyid shrimps (Decapoda: Caridea) frequently traded for ornamental purposes were chosen: Atya gabonensis Giebel, 1875 originating from West Africa, and Atyopsis moluccensis (De Haan, 1849) from South-East Asia (
A. gabonensis is relatively abundant in West Africa, occurring from the Democratic Republic of Congo to Senegal. There are also reports of its presence in South America, however, these are probably erroneous and concern its congener, A. scabra (Leach, 1816) (
The experimental animals were exposed to zoospores of A. astaci strain belonging to the genotype group D (
The study consists of two subsequent experiments that were conducted in the facilities of the FFPW USB in Vodňany. The infection experiment lasted 120 days between March and July 2016, and was followed after 20 days by a transmission experiment that lasted a further 130 days until December 2016 (Fig.
Summary of the experimental design. The study consisted of two subsequent experiments: the infection experiment (120 days long) that was followed after 20 days by a transmission experiment (130 days long). Ten individuals of C. destructor, A. moluccensis and A. gabonensis were used in each of the three treatments: no A. astaci zoospores (negative control group) and an addition of one of the two spore doses differing in concentration by an order of magnitude. Six A. gabonensis individuals from each treatment were subsequently used in the transmission experiment, and each individual was placed separately with one A. astacus. To avoid physical interactions and predation by crayfish, A. gabonensis were placed under perforated plastic cages.
Both shrimp species and C. destructor were used in the infection experiment. C. destructor served as a sensitive control to evaluate A. astaci virulence. This crayfish species was reported to be susceptible to an A. astaci strain from the genotype group D (
The A. astaci zoospores were produced as described in
Due to a high mortality of A. moluccensis, only A. gabonensis individuals (six from either treatment, including two infection treatments and negative control group) were used in the transmission experiment. Each potentially infected A. gabonensis was kept individually for 20 days in a plastic container. Subsequently, one A. astacus individual was placed in each container. To avoid physical interactions and predation by crayfish, A. gabonensis were placed under perforated plastic cages. The animals were handled in the same way as during the infection experiment. Water temperature was 18.7±0.3 °C for first 100 days, followed by 23.5±0.2 °C for final 30 days to trigger shedding of the shrimp exoskeleton as zoospore concentrations were observed to increase during crayfish moulting (e.g.,
All experimental animals were tested for the presence of A. astaci DNA in their tissues, presumably indicating infection. Due to a limited number of available animals we did not test any additional individuals for the presence of A. astaci infection prior to the beginning of the experiment. The surfaces of all animals were thoroughly rinsed with tap water prior to DNA isolation to remove potentially attached cysts. The total body length of each specimen was measured, and each animal was also examined for any presence of melanised spots on its body, which may indicate a local presence of infection. However, it should be noted that melanisation is a common defence mechanism in crustaceans that can have various causes (
For the detection of A. astaci infection, we used the TaqMan minor groove binder real-time PCR assay targeting ITS1 region developed by
The data analyses were performed in R version 3.4.3 (
No presence of A. astaci DNA was detected in any shrimp or crayfish individual from the negative control groups. All C. destructor and A. gabonensis used in the control groups survived, whereas eight out of ten control A. moluccensis died before the end of the experimental trial.
Infection by A. astaci was detected in all C. destructor individuals from the two zoospore treatments. The infection reached moderate to very high agent levels in crayfish bodies (Table
Results of the qPCR analyses of crayfish and shrimp bodies after the experimental infection. N: number of individuals of each species exposed to zoospores. Semi-quantitative agent levels based on the estimated amounts of PCR-forming units (PFU) in the reaction (according to
Species | Zoospore dose (spores ml-1) | N | Agent level in infected animals (died during exp./survived exp. infection) | Survival rate (%) | |||||
---|---|---|---|---|---|---|---|---|---|
A2 | A3 | A4 | A5 | A6 | A7 | ||||
Cherax destructor | 10 | 10 | 3/2 | 5/0 | 20 | ||||
100 | 10 | 5/0 | 5/0 | 0 | |||||
Atya gabonensis | 100 | 4* | 0/1 | 100 | |||||
1000 | 4* | 0/1 | 100 | ||||||
Atyopsis moluccensis | 100 | 10 | 4/1 | 2/0 | 10 | ||||
1000 | 10 | 1/0 | 3/0 | 40 |
A. astaci DNA was detected in bodies or exuviae of all A. moluccensis and the majority of A. gabonensis exposed to A. astaci zoospores. The detected A. astaci agent levels in the zoospore treatments ranged from very low to low (Table
Among A. moluccensis, the mortality occurred 14–101 days post-infection (median: 23rd day, one surviving individual) in the low-dose treatment and 15–86 days post-infection (median: 32nd day, four surviving individuals) in the high-dose treatment. No statistical difference was found between these two treatments (χ2 = 0.6, df = 1, p = 0.439). The high mortality, however, was also observed among the control individuals, not differing significantly from either infected A. moluccensis group (χ2 = 0.6, df = 2, p = 0.737). Specifically, eight control A. moluccensis died 14–115 days after the experiment started (median: 29th day, two surviving individuals).
Similarly to the infection experiment, no A. astaci DNA was detected in the control A. astacus and A. gabonensis. The shrimp individuals were exposed to A. astaci spores prior to the transmission experiment and their infection status was confirmed only after its termination. In the low-dose treatment, A. astaci DNA was detected in two shrimps and in exuviae of another individual, whereas in the high-dose treatment A. astaci DNA was detected in all shrimps, either in their bodies or exuviae (Table
Results of the qPCR analyses of Atya gabonensis and Astacus astacus from the transmission experiment. Semi-quantitative agent levels based on the estimated amounts of PCR-forming units (PFU) in the reaction (according to
Treatment of A. gabonensis (spore ml-1) | Aquarium number | Agent level | |||
---|---|---|---|---|---|
Bodies | Exuviae | ||||
A. gabonensis | A. astacus | A. gabonensis | A. astacus | ||
100 | 1 | A0 | A0 | A0 | |
2 | A2 | A0 | A0 | ||
3 | A0 | A2 | A1 | ||
4 | A2 | A0 | |||
5 | A0 | A0 | A0 | ||
6 | A0 | A0 | A2 | ||
1000 | 1 | A2 | A0 | A0 | |
2 | A0 | A0 | A2 | ||
3 | A0 | A0 | A2 | A0 | |
4 | A0 | A0 | A3 | ||
5 | A0 | A0 | A4 | ||
6 | A2 | A0 | A0 | A0 |
Four individuals of A. gabonensis were partially eaten by the A. astacus, which managed to reach shrimps despite the attempted physical separation. Three A. astacus died during the experiment, however, no A. astaci DNA was detected in their tissues. However, a very low agent level was detected in one A. astacus individual at the end of the treatment. The cohabiting A. gabonensis individual moulted after the increase in temperature and trace amounts of A. astaci DNA were detected in its exuviae (Table
It was assumed for decades that crayfish are the only hosts of A. astaci. Unfortunately, recent studies provided evidence that A. astaci does not only grow within the tissues of freshwater-inhabiting crabs (
The elevated resistance of North American crayfish hosts to A. astaci has been attributed to the rapid response of their immune system that efficiently limits parasite growth in their cuticles. This defence mechanism is an outcome of long co-evolutionary history between A. astaci and its North American crayfish hosts (
The progress and success of A. astaci infection may be also influenced by the frequent moulting of its hosts, especially those exhibiting increased resistance (
The growth of A. astaci in host bodies and the subsequent production of motile zoospores is a prerequisite for its successful transmission to the next host. The horizontal transmission of A. astaci between different crayfish species has been widely documented in the experimental settings, aquarium facilities as well as from the wild (e.g.,
A recent experimental study confirmed an elevated resistance to A. astaci also in the Australian C. destructor (
Among all commercially used crayfish species, the red swamp crayfish P. clarkii (the typical host of A. astaci genotype group D) has become the most cosmopolitan crayfish introduced to almost all continents, except Australia and Antarctica, thanks to its intensive use for aquaculture, stocking purposes and as an ornamental species (
We thank Satu Viljamaa-Dirks for the axenic culture of A. astaci strain (Evira10823/13). Belinda Gallardo, Lucian Pârvulescu and an anonymous reviewer provided valuable comments on previous versions of this manuscript.
This study was supported by the Czech Science Foundation (No. 19-04431S).
Aphanomyces astaci infection levels in bodies and exuviae of animals that moulted during both experiments. Semi-quantitative agent levels based on the estimated amounts of PCR-forming units (PFU) in the reaction (according to
Species | Concentration (spore ml-1) | Animal | Agent level in animal body | Agent level in exuviae | ||
---|---|---|---|---|---|---|
Moulting 1 | Moulting 2 | Moulting 3 | ||||
Cherax destructor | 10 | 1 | A4 | A6 | ||
2 | A6 | A7 | ||||
3 | A6 | A7 | ||||
4 | A4 | A6 | ||||
5 | A6 | A6 | ||||
6 | A4 | A6 | ||||
7 | A4 | A6 | ||||
8 | A4 | A4 | A6 | |||
100* | 1 | A6 | A4 | |||
Atyopsis moluccensis | 100 | 1 | A0 | A2 | ||
2 | A2 | A3 | ||||
3 | A0 | A2 | ||||
4 | A0 | A3 | ||||
5 | A2 | A3 | ||||
1000 | 1 | A0 | A3 | A0 | A3 | |
2 | A0 | A4 | A0 | |||
3 | A0 | A3 | ||||
4 | A0 | A2 | ||||
5 | A0 | A4 | A0 | |||
6 | A0 | A3 | A0 | |||
Atya gabonensis | 100 | 1T* | A0 | A1 | ||
2T | A0 | A2 | ||||
1000 | 1T* | A0 | A2 | |||
2T* | A0 | A2 | ||||
3T* | A0 | A3 | ||||
4T* | A0 | A4 | ||||
5 | A0 | A4 | ||||
6 | A0 | A2 | ||||
7 | A0 | A4 |