Research Article |
Corresponding author: Sandra Hudina ( sandra.hudina@biol.pmf.hr ) Academic editor: Eric Larson
© 2022 Ana Dobrović, Sunčana Geček, Tin Klanjšček, Ines Haberle, Paula Dragičević, Dora Pavić, Ana Petelinec, Ljudevit Luka Boštjančić, Lena Bonassin, Kathrin Theissinger, Sandra Hudina.
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:
Dobrović A, Geček S, Klanjšček T, Haberle I, Dragičević P, Pavić D, Petelinec A, Boštjančić LL, Bonassin L, Theissinger K, Hudina S (2022) Recurring infection by crayfish plague pathogen only marginally affects survival and growth of marbled crayfish. NeoBiota 77: 155-177. https://doi.org/10.3897/neobiota.77.87474
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Invasive alien crayfish threaten the diversity of freshwater ecosystems and native crayfish fauna. In Europe, this is largely due to transmission of the crayfish plague to susceptible native crayfish. Many invasive species tolerate crayfish plague, but the infection still has the potential to reduce the fitness of a tolerant host due to energy trade-offs between immune response maintenance and life-history traits, such as growth and reproduction. In combination with other unfavourable conditions, such a response could alter further invasion success of an otherwise successful crayfish invader. We examined whether repeated infection with one of the most virulent haplogroups of crayfish plague agent (Aphanomyces astaci) affects growth or survival of the juvenile marbled crayfish (Procambarus virginalis). Juveniles were exposed to i) two levels of pathogen concentrations, and ii) two different feeding regimes under the higher pathogen concentration. In all performed trials, repeated infection reduced growth rates, while the combination of recurring infection and food limitation significantly increased mortality. The average energy cost of the immune response was estimated at 12.07 J/day for individuals weighing 0.3 grams. Since infections were frequent and pathogen concentrations high, results suggest that marbled crayfish is resistant to A. astaci pathogen and its survival is only affected by adding the stress of food limitation. The survival of almost half of the individuals exposed to high pathogen loads and extreme food limitation indicates that chronic infection by crayfish plague is unlikely to be an important factor impeding invasion success of the marbled crayfish, even under harsh conditions. Our results add to the growing body of evidence that marbled crayfish has potential to become one of the most successful freshwater invaders.
food limitation, freshwater, immunity cost, infection, invasive species, trade-off
Invasive alien species drive biodiversity loss and impair ecosystem services worldwide (
North American crayfish species tolerate the crayfish plague due to their rapid immune reaction to the pathogen. The fast activation of the prophenoloxidase (ProPO) system encapsulates the pathogen’s hyphae in melanin (
We analyse whether chronic exposure to A. astaci may lead to significant sublethal or lethal effects in the marbled crayfish (Procambarus virginalis
Juvenile marbled crayfish were taken from an adult female transferred from Šoderica Lake in northern Croatia (46°14'20.9"N, 16°54'33.6"E) to the laboratory of the Department of Biology at the Faculty of Science in Zagreb. Adult marbled crayfish were kept in the laboratory for five months prior to oviposition, when juveniles for the experiments were obtained. For marbled crayfish keeping and research, we obtained a licence from the responsible Ministry of Environment and Energy, Croatia (Licence number: UP/I-612-07/19-43/01; 517-05-1-1-20-6), in accordance with the EU Regulation on Invasive Alien Species (EU Regulation No. 1143/2014).
Juvenile marbled crayfish were used in all experiments, since they moult often, allowing faster assessment of effects of exposure to A. astaci pathogen on growth and since juvenile growth is linear (
Juvenile marbled crayfish were kept individually in 100 ml of aerated tap water in 250 ml plastic containers with a diameter of 90 mm. Containers were placed in cooled incubators (ST 6 COMF, POL-EKO-APARATURA, Poland) 16–18 days before the start of the experiments to allow the juveniles to adapt to housing conditions (temperature regime 20 °C, photoperiod 12:12 light:dark). To avoid cross-contamination, each experimental group (described below) was housed in a separate incubator. The selected temperature results in the lowest mortality in the laboratory (
Haplogroup B strain of A. astaci, associated with the signal crayfish (
A. astaci PEC8 isolate (haplogroup B) was obtained from Prof. Frédéric Grandjean from University of Poitiers, France. The isolate was grown on PG1 agar (
Prior to the infection experiment, virulence of the PEC8 isolate and sublethal load was tested in a pilot experiment to determine two sublethal concentrations of A. astaci zoospores and frequency of the repeated infections (
During the experiments, crayfish from all experimental groups (control and infected groups) were photographed and weighed i) directly before each infection, ii) two weeks after the last infection (Experiment 1 and 2), and iii) six weeks after the last infection (Experiment 1). Due to the small size of the juveniles, their total length (TL; from the top of the rostrum to the end of the telson, in mm) was measured from photographs using the image processing programme, ImageJ (
A total of 55 juveniles were used in this experiment. For the trial, juvenile marbled crayfish were randomly divided into three experimental groups: 1) control group (15 individuals, non-infected), 2) group 7500 (20 individuals infected with 7500 zoospores/ml), and 3) group 15000 (20 individuals infected with 15000 zoospores/ml). The infection experiment was conducted for 18 weeks, with A. astaci infection performed every other week, six infections in total. In one case, the third infection was performed four weeks after the second infection due to unsuccessful A. astaci zoospore production at the time. Surviving crayfish were euthanised six weeks after the last infection and the cuticle of all crayfish was tested for presence of A. astaci (those that died during the experiment and those that survived until the end of the experiment; crayfish plague detection described in Suppl. material
A total of 60 individuals from a separate batch were used to test the interaction of chronic exposure to A. astaci with food availability. While food availability is unlikely to be a limiting factor in nature because crayfish are omnivores (
In this experiment, crayfish were adapted to laboratory conditions for 16 days during which all individuals were fed twice a week. Then, four experimental groups of 15 individuals were used: 1) control - fed five times a week, non-infected, 2) control, food-restricted - fed once a week, non-infected, 3) infected - fed five times a week, and 4) infected, food-restricted - fed once a week. This experiment was conducted for 12 weeks, also with A. astaci infection intervals every other week (five in total).
Surviving crayfish were euthanised two weeks after the last infection and cuticle samples of all crayfish were tested for A. astaci, as in Experiment 1 (crayfish plague detection described in Suppl. material
We estimated energy cost of immunity response to chronic infection based on the differences in weight of non-infected and infected food-restricted groups in Experiment 2. Neither of the food-restricted groups grew appreciably in length, so differences in weight change between the two groups stem from differences in energy reserve dynamic: if size is the same, lighter individuals have smaller energy reserves. The reduction in energy reserves can be explained either by smaller energy input (e.g. reduction in food intake due to infection) or a higher basal metabolic rate related to the infection. Food intake was equal for both groups because all food had been eaten, so reduction in energy reserves must have been caused by increased metabolism due to the infection. Therefore, the energy content corresponding to the difference in weight represents the cost of the infection. In crayfish, changes in energy reserves are primarily reflected in change of hepatosomatic index, i.e. hepatopancreas weight (
E = −0.336M + 54.189. (1)
Average moisture content of hepatopancreas of marbled crayfish from our sampling site was reported to be 62% (
The effects of repeated infections using two A. astaci concentrations and interactive effects of repeated infections and food availability were examined through three major endpoints: 1) total growth (i.e. weight/length gain throughout the total duration of both experiments), 2) rate of growth (i.e. weight/length increment over time, in each experimental week, presented in Suppl. material
Robust ANOVAs based on trimmed means (20% of trimming level;
Growth rate analysis was performed to analyse questions such as: (a) what average growth trajectory best describes the rate of growth over time for all crayfish, (b) what is the variability in growth rates across crayfish, and finally, (c) does A. astaci infection and food availability explain variability in growth rates? Growth rate analysis, unlike total growth analysis, is based on complete longitudinal measurements of weight (and/or length) rather than just initial and final values. As the measurements at specific time points are clustered (nested) within individual crayfish, they imply a hierarchical structure of the data and intra-individual correlation. Therefore, the multilevel modelling (MLM) technique was used for data analysis (
Fisher’s exact tests were used to determine the association between exposure to A. astaci and mortality in both experiments. Pairwise (post-hoc) Fisher test from the ‘rstatix’ package in R (
The Kruskal-Wallis test was used to determine whether the expression of immune and metabolic genes differed significantly between the groups in Experiment 2. Dunn’s test from the ‘FSA’ package in R (
Data are permanently deposited in an open repository (Dryad: https://doi.org/10.5061/dryad.3xsj3txkp).
In both experiments, all crayfish that died (marked in red colour in Fig.
Detection of A. astaci in the cuticle of marbled crayfish in A Experiment 1 and B Experiment 2. In Experiment 1, surviving crayfish were euthanised six weeks after the last infection; in Experiment 2, they were euthanised after two weeks. PCR+ = A. astaci detected, PCR - = A. astaci not detected.
Repeated exposure to the crayfish plague pathogen (Fig.
Effects of repeated infection of marbled crayfish juveniles using two A. astaci zoospore concentrations (7500 and 15000 zoospore/ml) on A total weight gain (total growth) B rate of weight increment (rate of growth) and C survival probability of individuals. Different letters in panel A denote significant differences, error bars represent 95% confidence intervals (CIs) around the mean. Results for total length gain and rates of length increment are presented in Suppl. material
Due to the complexity of the MLM model, a detailed description of the results on individual and group growth rates is given in Suppl. material
The effect of exposure to pathogens on mortality was not significant (p = 0.26, Fisher’s exact test). A similar result, i.e. insignificant effect of infection, was also inferred from the overlapping confidence intervals of the Kaplan-Meier survival curves (Fig.
Food availability (i.e. different feeding regimes) significantly influenced crayfish growth (Fig.
Effects of repeated infection of marbled crayfish juveniles with 15000 zoospore/ml of A. astaci under two feeding regimes (once a week and five times a week) on A total weight gain (total growth) B rate of weight increment (rate of growth) and C survival probability of individuals. Different letters in panel A denote significant differences, error bars represent 95% confidence intervals (CIs) around the mean. Results for total length gain and rates of length increment are presented in Suppl. material
A detailed analysis of the results of the MLM model can be found in Suppl. material S3.3 and S5, Figs S5.2, S5.3, Tables S5.2, S5.3); here, a summary of the main results (fixed effects) is given. Both food restriction and infection with A. astaci, as well as their interactive effect, significantly reduced growth rate of weight (and length). The average growth rate of uninfected crayfish fed five times per week was 30.8 mg/wk, compared with an average growth rate of 21.0 mg/wk for the infected group under the same feeding regime, and an average growth rate of 2.5 mg/wk for the food-restricted control group. The average growth rate of the infected crayfish with food restriction was only 0.2 mg/wk. The infection-induced decrease in growth rate was greater when food was abundant than when food was restricted.
Food limitation and A. astaci infection increased individual mortality. Mortality differed significantly amongst groups in Experiment 2 (p < 10−3, Fisher’s exact test), with the highest mortality occurring in the infected group fed once a week (8 deaths, i.e. 53% in 10 weeks; Fig.
Repeated exposure to A. astaci and different food availability significantly affected the expression of the metabolic genes CS (H (3) = 15.34, p = 0.002) and GAPDH (H (3) = 9.72, p = 0.021), and the immune gene C/EBP-β (H (3) = 14.54, p = 0.002), with no significant effects on ProPO expression (Fig.
Differential expression of immune and metabolic genes in Experiment 2 analysed using A inferential comparison between groups and B principal component analysis (PCA). Different letters in panel A denote significant differences.
Relationships amongst gene expression variables were further analysed using the principal component analysis. Two principal components captured most of the variability in the dataset (87.6%, Fig.
The food-restricted infected group of Experiment 2 had to activate and maintain immune response to the pathogen, inducing additional energy costs in contrast to the respective control (non-infected) group. Based on the average moisture content of the hepatopancreas, crayfish had 33.357 kJ/g in reserve. The comparison of total weight change between the two groups showed a total difference of 0.025 g, corresponding to 12.07 J/day of energy reserves used for immune response maintenance in infected individuals weighing on average 0.3 g.
Chronic infection of invasive marbled crayfish juveniles with the A. astaci pathogen leads to trade-offs in energy use that reduces growth. Although this is the first such finding for crayfish, this is consistent with previous research on other invertebrates (i.e. arthropods:
Growth was reduced under both pathogen concentrations in Experiment 1 and under both feeding regimes in Experiment 2. Even though not statistically significant in some scenarios, the omnipresence of effects on growth suggest that exposure to pathogens increases allocation of energy to the immune system. Alternatively, the observed slower growth of infected groups could be the consequence of suppressed food intake suggested to occur during immune challenges (
A larger effect size of repeated infections on weight was observed in both experiments and became especially evident in Experiment 2. Here, the extreme food limitation (feeding once a week) almost completely ceased growth in both control (non-infected) and infected groups. The miniscule (if any) weight increase of the control food-restricted group suggests that those individuals were very near or at the point of starvation. Additional need to fuel the immune response of infected individuals further depleted energy reserves, thus increasing the severity of starvation resulting in increased mortality of the infected group fed once a week.
Starvation, as well as the difference in severity of starvation, was reflected in the down-regulation of genes involved in the central metabolic pathways of glycolysis and citrate cycle in both infected and non-infected group fed once a week. This is in line with previous studies on freshwater species which have shown that the expression of both CS and GAPDH decrease during starvation and fasting (
Food limitation and the resulting starvation induced up-regulation of the C/EBP-β transcription factor, belonging to the CCAAT/enhancer-binding protein (C/EBP) family, which are involved in the regulation of the metabolism, cell growth, differentiation, death, immune and inflammatory processes (
Lower mortality in the group fed five times a week indicates that these individuals were more able to cover the additional energy costs of fighting the disease, including the potential reparation of damage, as opposed to the group fed once a week. The scale of the cost is well represented by the differences in weight gain between the infected and non-infected food-restricted groups: the ’missing’ weight can be solely attributed to energy costs of fighting the infection (immune response maintenance costs) and related consequences.
Chronic exposure to the A. astaci pathogen is unlikely to have a long-term effect on marbled crayfish populations, except under extreme limitation of food availability, which was used in this study as a proxy for density-dependent effects. In all experimental groups, infected individuals grew more slowly than non-infected. In nature, the slower growth could translate into slower maturation rates and/or lower fecundity of infected individuals due to size-dependence of these traits (
Furthermore, a lower proportion (40 - 55%) of individuals tested positive for A. astaci in Experiment 1 (analyses performed six weeks after last infection) compared to Experiment 2 (analyses performed two weeks after the last infection), where almost all individuals (87 - 100%) were A. astaci positive. This either means that: i) infections were less successful in Experiment 1 or that ii) some individuals from Experiment 1 were able to contain the infection during the six weeks since the last A. astaci infection, unlike in Experiment 2 where A. astaci detection was performed two weeks after the last infection. We consider the former unlikely, as the procedures were the same in both experimental trials and number of infections was higher in Experiment 1 (6 repeated infections in Experiment 1 vs. 5 in Experiment 2). If the latter is true, this suggests that at least some proportion of marbled crayfish that survived repeated infections could potentially efficiently contain the pathogen and minimise further trade-offs with growth.
Our results add to the growing knowledge regarding the high tolerance of marbled crayfish to multiple single and combined stressors (
This research has been supported by Croatian Science Foundation (HRZZ) [installation grant HRZZ-UIP-2017-05-1720 to SH, PD and TK, DOK-2018-09-4671 to IH and DOK-09-2018 to AD]. We thank Ana Bielen, Anđela Miljanović and Filip Ložek for their help in design of experimental procedures and help in crayfish plague cultivation. Additionally, we thank all students (Mara Vukelić, Dominik Guzalić, Marina Malnar, Jana Zekirovski, Mihaela Šurina, Iva Žulj) and Mirjana Hudina for their help with crayfish maintenance and help in the laboratory during infection trials. We also thank two reviewers and the editor (Eric R. Larson) for their helpful comments and suggestions.
Protocols, primers, statistical support data, images
Data type: pdf
Explanation note: The file contains addtional detailed descriptions of used protocols and applied statistical analyses, additional statistical support data and images.