Research Article |
Corresponding author: Cybèle Prigot-Maurice ( cybele.prigot@gmail.com ) Academic editor: Spyros Sfenthourakis
© 2022 Cybèle Prigot-Maurice, Charlotte Depeux, Hélène Paulhac, Christine Braquart-Varnier, Sophie Beltran-Bech.
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:
Prigot-Maurice C, Depeux C, Paulhac H, Braquart-Varnier C, Beltran-Bech S (2022) Immune priming in Armadillidium vulgare against Salmonella enterica: direct or indirect costs on life history traits? In: De Smedt P, Taiti S, Sfenthourakis S, Campos-Filho IS (Eds) Facets of terrestrial isopod biology. ZooKeys 1101: 131-158. https://doi.org/10.3897/zookeys.1101.77216
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Invertebrate immune priming is defined as an enhanced protection against secondary pathogenic infections when individuals have been previously exposed to the same or a different pathogen. Immune priming can be energetically costly for individuals, thus impacting trade-offs between life-history traits, like reproduction, growth, and lifetime. Here, the reproductive cost(s) and senescence patterns of immune priming against S. enterica in the common woodlouse A. vulgare (Crustacea, Isopoda) were investigated. Four different groups of females were used that either (1) have never been injected (control), (2) were injected twice with S. enterica (7 days between infections), (3) were firstly injected with LB-broth, then with S. enterica, and (4) females injected only once with S. enterica. All females were allowed to breed with one non-infected male and were observed for eight months. Then, the number of clutches produced, the time taken to produce the clutch(es), the number of offspring in each clutch, the senescence biomarkers of females, and parameters of their haemocytes were compared. The result was that immune priming did not significantly impact reproductive abilities, senescence patterns, and haemocyte parameters of female A. vulgare, but had an indirect effect through body weight. The lighter immune primed females took less time to produce the first clutch, which contained less offspring, but they were more likely to produce a second clutch. The opposite effects were observed in the heavier immune primed females. By highlighting that immune priming was not as costly as expected in A. vulgare, these results provide new insights into the adaptive nature of this immune process.
Crustacea, haemocytes, immune priming, isopod, reproduction, senescence, survival, trade-off
Because fighting pathogens is a real challenge for all living organisms, they have developed an important and complex biological process, the immune system (
Although immune priming is advantageous in terms of survival ability, its expression could be costly for individuals, particularly when it relies on the long-lasting sustained immune response (
Nevertheless, several studies did not observe the impact of repeated infections with pathogens on the reproductive abilities of individuals, within the same or the subsequent generations (
Among the numerous species in which immune priming has been observed, the common woodlouse Armadillidium vulgare (Oniscidea, Isopoda, Crustacea) is an appropriate model to investigate this issue. Armadillidium vulgare (Latreille, 1804) mount an immune priming response with two subsequent infections of living Salmonella enterica (Theobald Smith, 1855) injected seven days apart (
In this study, we explored the impact of immune priming with S. enterica on the reproductive ability and the resulting senescence patterns of A. vulgare. Our objectives were: (1) to test whether mounting an immune priming response affects the reproduction of females that successfully survived two consecutive infections with living S. enterica, and (2) to explore to what extent immune priming and reproduction change the senescence patterns of individuals, by using two senescence biomarkers: the β-galactosidase activity and the size of the viable haemocytes (described in
In this experiment, we used the same Armadillidium vulgare line used in the study of
To perform the infections, we used the Salmonella enterica serovar typhimurium J18 strain (
Firstly, we performed the priming procedure on three females’ treatments: either primed (i.e., primo-injected) with the low dose of living S. enterica (SAP, for S. enterica-primed), with sterile LB broth (LPB, for LB-primed) or without priming injection (NP, for non-primed; Fig.
The experimental procedure A the priming procedure was to inject females either with a low dose of living S. enterica (SAP, in red) or sterile LB Broth (LBP, in grey). The non-primed (NP, in green) females did not receive the first injection B SAP, LBP, and NP females received the second, LD50 injection of living S. enterica. Control females (in black) were never injected C all females (SAP, LBP, NP, control) were allowed to reproduce individually in a box with one virgin, non-injected male (brown woodlouse). We checked the survival rates of females, the number of clutches (1 or 2), the time to produce each clutch (number of days), and the number of offspring in each clutch D regularly, we sampled and dissected females that produced the second clutch to analyse haemocytes and β-galactosidase activity. Brackets indicate that not all females produced a second clutch. Approximately eight months later, we waited for the last females to produce their second clutch, and then sampled and dissected the remaining females that produced only one clutch.
Every three days for ca. eight months, we measured the survival rate and the physiological states of all females by observing their ventral faces. The females that were about to lay eggs developed a marsupium following a parturial moult, which is observable under a binocular loupe (
The priming procedure with S. enterica was performed as described in
After their second clutch, females were washed (0.28% NaClO then water). The total haemocyte concentration (number of haemocytes per µL of haemolymph, regardless of the haemocytes’ type), the viability of haemocytes (% of living haemocytes), and the size of viable haemocytes (µM) were measured as described in
After collecting their haemolymph, all females used for haemocyte analysis were dissected in Ringer solution (135 mM sodium chloride, 2 mM potassium chloride, 2 mM calcium chloride, 2 mM sodium bicarbonate) to collect their nerve cords. The β-galactosidase activity was measured as described in
All statistical analysis were performed with RSTUDIO (v.1.4;
Body weight differences of females before reproduction were tested with a linear mixed effects model built with lme4 and car package (
Concerning the first reproductive event, we tested the probability of producing the first clutch with one generalised linear mixed effects model with binary logistic regression (i.e., 1-0;
The total number of offspring (first and second clutches included) was analysed with one linear mixed effects model including the treatment and the weight as fixed effects.
The haemocyte concentrations (number of cells per µL of haemolymph), the size of viable haemocytes and β-galactosidase activity were analysed using linear models with Gaussian distribution, and viability (proportion of viable haemocytes) using one generalised model with Binomial distribution (
For all models (i.e., survival, weight, probability of producing the first and second clutches, time to produce these clutches, the number of offspring in each clutch, haemocyte parameters and β-galactosidase activity), we entered the experimental replicates as random factor. This factor allows to correct the non-independence of samples within the same replicate of treatment (
The R script and the datasets used to perform our analysis are available on the open access repository Mendeley Data https://data.mendeley.com/datasets/gd24nvncvf/2
The treatment had a slight effect on the survival abilities of females after the LD50 injection and before reproduction (X2 = 5.17, df = 2, p = 0.07; Fig.
Survival rates A 22 days after the LD50 injection, and B during the reproductive period (ca. eight months). Abbreviations: NP: females non-primed in the priming procedure. LBP: females primed with sterile LB broth. SAP: females primed with 103 living S. enterica. Control: females that have never been injected. NP, LBP and SAP received the LD50 injection. Statistical results of comparisons between treatments are presented in Table S1.
The weight of females after the second infection with S. enterica (before reproduction) was influenced by their treatment (X2 = 8.05, df = 3, p = 0.04; Suppl. material
During the reproductive period, almost all females produced one clutch (SAP: 23/24, LBP: 20/22, NP: 22/22, control: 21/22). The probability to produce the first clutch was neither influenced by the treatment (X2 = 1.09, df = 3, p = 0.77) nor by the weight of females before reproduction (X2 = 0.18, df = 1, p = 0.66) or the interaction between the treatment and the weight (X2 = 2.61, df = 3, p = 0.45). Females were able to produce the first clutch regardless of their treatment or their weight before reproduction.
The time to produce the first clutch was neither influenced by the treatment (X2 = 1.15, df = 3, p = 0.76, Fig.
Interactions effects of body weight and treatment on A the time to produce the first clutch, and B the mean number of offspring in the first clutch per female. Abbreviations: control: never-injected females; NP: non-primed females; LBP: females primed with sterile LB broth, SAP: females primed with 103 living S. enterica. SAP, NP and LBP received the LD50 injection. P-values indicate a significant relationship between x and y axis of the considered treatment (Pearson’s correlation test).
The number of offspring in the first clutch was not influenced by the female’s treatment (X2 = 7.07, df = 3, p = 0.06; Suppl. material
Among the females which produced the first clutch, half produced a second clutch, regardless of the treatment (control: 10/21; NP: 10/19; LBP: 10/20; SAP: 11/23). Hence, the probability to produce the second clutch was not influenced by their treatment (X2 = 0.19, df = 3, p = 0.97). This was neither influenced by the number of offspring in the first clutch (X2 = 0.20, df = 1, p = 0.64), nor by the interaction between the treatment and the weight (X2 = 0.14, df = 3, p = 0.98). However, the weight of the females influenced the probability of producing the second clutch (X2 = 4.60, df = 1, p = 0.03; Fig.
Probability to produce the second clutch according to female’s weight and treatment. Curves were calculated using average marginal effects of the absence/presence of the second clutch (0/1) related to the weight of females. Coloured distributions represent the confident interval for each treatment (95%). Abbreviations: control: never-injected females; NP: non-primed females LBP: females primed with sterile LB broth, SAP: females primed with 103 living S. enterica. SAP, NP and LBP received the LD50 injection.
The time to produce the second clutch (after the first one) and the number of offspring in the second clutch were influenced neither by the treatment (Time: X2 = 3.80, df = 3, p = 0.28; Number of offspring: X2 = 5.38, df = 3, p = 0.14), nor by the weight of females (Time: X2 = 0.97, df = 1, p = 0.32; Number of offspring: X2 = 0.54, df = 1, p = 0.45), the number of offspring in the first clutch (Time: X2 = 1.68, df = 1, p = 0.19; Number of offspring: X2 = 0.27, df = 1, p = 0.60), or the interaction between the treatment and the weight (X2 = 2.53, df = 3, p = 0.46). Regardless of their treatment, body weight, and cost of producing offspring in the first clutch, the females took the same time to produce the second clutch and produced a similar number of offspring in the second clutch.
The total number of offspring (first and second clutch included) was not influenced by the treatment (X2 = 7.46, df = 3, p = 0.058). Even though control females produced an average of 190 offspring per female, compared to 131 offspring for SAP females (see Suppl. material
For the haemocyte concentrations, no significant effect of any fixed factors was observed (p > 0.05, see Suppl. material
Concerning the senescence biomarkers, the size of viable haemocytes was only influenced by the number of clutches that females produced, with an increase of the cell size in the case of a second clutch production (X2 = 12.99, df = 1, p = 0.003, Fig.
Our study aimed to investigate the impact of immune priming with S. enterica (i.e., two consecutive infections with living pathogens) on the reproductive ability and senescence biomarkers of females of A. vulgare. Fig.
As expected, we showed a protective effect of immune priming on female survival rates: the first encounter with S. enterica improves survival ability of females after the second and lethal infection, confirming previous results described in
Since energetic investment in immunity often reduces available energy to produce offspring, the negative impact of immune responses on reproductive ability is widely observed across invertebrate species (
Most studies that investigated the costs of mounting immune priming showed a negative impact on reproduction (Schwenke et al. 2019). However, from an evolutionary point of view, a biological process inducing higher costs than benefits would be counter-selected. This counter-selection would be particularly strong when the biological process reduces the reproductive ability of individuals because it also reduces the possibility to transmit this process to the next generation. From this statement, it appears that immune priming should be selected during evolution if it does not induce a high cost (
If the energetic resources of individuals are limited and trade-offs are inevitable between reproduction and immune response to infection (
Even though we observed no evident cost on reproduction in females receiving the double infection of S. enterica, the treatment of females indirectly influences their reproductive strategies through body weight. In never-injected (control) females, the lighter ones took a longer time (200 days on average) to produce the first clutch than heavier ones (50 days on average; Fig.
Otherwise, immune priming of double-infected females also induces different effects on the production of the first clutch according to body weight (Fig.
From our point of view, these reproductive patterns in SAP females result from an alteration in energetic resource allocation. During a stressful event occurring in the lifetime of an organism, like an infection, it could opt for the investment of its remaining energy in reproduction, at the expense of growth, in order to maximise fitness before dying (
These two different strategies illustrate a plasticity in resource allocation following two infections with S. enterica that depends on the investment of each female in the different physiological functions, namely somatic maintenance (including response to pathogens and/or growth) and reproduction. However, the total number of offspring per female (first and second clutches included) was influenced neither by the number of infections nor by body weight or the interaction between these parameters. Hence, whatever the allocation strategy of energetic resources in the first clutch in SAP females, the lighter of them mobilise enough energy to finally produce as many offspring as the heavier SAP females. This lack of effect seems explained by the second reproductive event.
Concerning the second reproductive event, the probability of producing the second clutch only depended on the body weight of the females: the heavier the females are, the less likely they are to produce a second clutch (ca. 25%) compared to lighter females (ca. 75%). We suppose that the investment in the first clutch by heavier females is more expensive than for those lighter ones, regardless of treatment. As we have already stated, the costs of producing one marsupium are high, and positively correlated to female size (
Nevertheless, this result raises questions about the intrinsic factors that cause the production of one or two clutch(es) in females of A. vulgare depending on body weight, regardless of treatment. The first assumption about the lower probability to produce two clutches by heavier females is related to the production of better-quality offspring. According to theoretical predictions, females producing a single clutch should provide higher rates of care to their offspring than those producing several clutches (
The second assumption concerning the probability to produce one or two clutches refers to the environmental and physiological conditions of the females before reproduction. By observing the reproductive phenology in four different species of terrestrial isopods,
For the senescence biomarkers, we only observed an effect of the investment in reproduction on the size of the viable haemocytes: regardless of treatment, females that have produced a second clutch had larger viable haemocytes than those of females that produced only one clutch. The size of haemocytes in A. vulgare increases with the age of individuals, making this morphometric trait a biomarker of senescence (
Finally, we observed no effect of the treatment or the reproductive event(s) on the haemocyte parameters of females (concentration and viability). We conclude that several months after S. enterica infection(s), immune cell production is no longer impacted by infection(s) (
Our study aimed to investigate the impact of immune priming with S. enterica on life-history traits and senescence biomarkers in A. vulgare. While current studies in various species show negative effects of immune priming, we only found an indirect effect of immune priming by body weight of females that could indirectly impact reproduction. However, we observed no strong effects of consecutive infections with S. enterica in the reproductive ability of female. Even though the absence of evidence for cost(s) does not mean that there is no cost at all, the fact that only a few studies reporting the absence of costs of immune priming or transgenerational immune priming could be explained by the difficulty to publish non-significant results. The publishing bias towards significant results of immune priming costs can change our view of evolutionary implications (
We would like to thank the reviewer and editor for their very constructive comments. We would also like to thank Richard Cordaux (Team Manager) for supporting our publication proposal, Benjamin Macchi (Teaching assistant) for his technical support and Sebastien Thobie for reviewing the English language. We sincerely thank Julie Tee for her careful proofreading of the manuscript and her corrections to the English language.
This work was supported by the Vivipary and Immune Priming grant (PEPS EXOMOD CNRS), State-Region Planning Contracts, the European Regional Development Fund, the French National Centre for Scientific Research, the French Ministry of Higher Education and Research, and the University of Poitiers.
Tables S1–S4, Figures S1–S3
Data type: Pdf file.
Explanation note: Table S1. Pairwise comparisons (Tukey adjustment) of survival rates according to females’ treatments (NP: non-primed females; LBP: females primed with sterile LB broth; SAP: females primed with 103 S. enterica in the priming procedure; Control: never-injected females). Figure S1. Body weight of females before reproduction according to their priming treatment. NI: never-injected, control females; NP: non-primed females; LBP: females primed with sterile LB broth, SAP: females primed with 103 living S. enterica. NP, LBP and SAP females received the LD50 injection. Mean ± SE: Control = 0.15g ± 0.008, NP = 0.12g ± 0.01, LBP = 0.14g ± 0.007, SAP = 0.12g ± 0.007). Table S2. Pairwise comparisons (Tukey adjustment) of body weight of females before reproduction, according to the treatments (NP: non-primed females; LBP: females primed with sterile LB Broth; SAP: females primed with 103 S. enterica during the priming procedure; Control: never-injected females). Figure S2. Number of offspring in the first clutch according to the females’ treatment. NI: never-injected, control females; NP: non-primed females; LBP: females primed with sterile LB broth, SAP: females primed with 103 living S. enterica. NP, LBP and SAP females received the LD50 injection. Table S3. Pairwise comparisons (Tukey adjustment) of the number of offspring in the first clutch according to females’ treatments (NP: non-primed females; LBP: females primed with sterile LB Broth; SAP: females primed with 103 S. enterica during the priming procedure; Control: never-injected females). Figure S3. Total number of offspring (first and second clutches included) according to the females’ treatment. NI: never-injected, control females; NP: non-primed females; LBP: females primed with sterile LB broth, SAP: females primed with 103 living S. enterica. NP, LBP and SAP females received the LD50 injection. Table S4. Average marginal effects of the interaction between the probability to produce a second clutch and the body weight of females by treatment (NP: non-primed, Control females; LBP: females primed with sterile LB Broth; SAP: females primed with 103 S. enterica in the priming procedure; Control: never-injected females). Table S5. Pairwise comparisons (Tukey adjustment) of the total number of offspring according to females’ treatments (NP: non-primed, Control females; LBP: females primed with sterile LB Broth; SAP: females primed with 103 S. enterica in the priming procedure; Control: never-injected females). Table S6. Statistical results of haemocyte parameters and senescence biomarkers analysis (generalized linear mixed effect models).