Abstract
Infectious diseases are considered as a significant factor in the global decline of amphibians. In some vertebrates, the assessment of the individual sexual traits can be useful for assessment of their health status and immunocompetence due to trade-off between them and investment in the immune system. Our aim here was to determine whether the trade-off between the expression of sexual morphological and behavioral traits and investment in the immune system is present in an urodele, the Palmate newt (Lissotriton helveticus). The groups of males were injected by solutions of proinflammatory agent, lipopolysaccharide (LPS) from Escherichia coli serotype O:55:B5, at dosages toxic to vertebrates (2 and 10 mg/kg of body mass) or by saline solution only (control groups). They were subsequently measured for variations in body condition and expression of both morphological (filament length, hind-foot-web, crest) and behavioral (courtship frequency) sexual traits. The injection of either LPS or saline solution did not cause any adverse effect on health in any male of all groups. No significant differences in any of the sexual traits were observed between two groups of males injected by LPS and control groups of males indicating the absence of a trade-off between immune response and expression of sexual traits. Our result suggests that measuring morphological or behavioral sexual traits may not be a useful method for monitoring emergence of infectious diseases in the palmate newt.
Introduction
Infectious diseases caused by Batrachochytrium dendrobatidis, pathogenic bacteria and irridovirus are associated with extinction of some species and decline of different populations worldwide (Daszak, Cunningham and Hyatt, 2010; Chen and Robert, 2011). Their emergence in different populations is associated with host-pathogen regulations dependent on the status of the immune system of the hosts and pathogen virulence (Daszak, Cunningham and Hyatt, 2010; Chen and Robert, 2011). Since the virulence of these pathogens increased in the last two decades (Cunningham et al., 1996; Daszak, Cunningham and Hyatt, 2010; Schadich and Cole, 2010), it has become increasingly important to improve surveillance of animals for assessing their health status and immunocompetence against infectious diseases.
Compared to other vertebrates, the amphibian immune system has been poorly investigated and what we know stems from a limited number of model species only, i.e. two anurans (Xenopus laevis and Rana pipiens) and one urodele (Ambystoma mexicanum) (Chen and Robert, 2011; Rollins-Smith and Woodhams, 2012). It is therefore urgent to extend our knowledge on amphibian immunology by broadening the number and variety of both anuran and urodele model species (Chen and Robert, 2011).
The immune system of vertebrates is phylogenetically conserved, and the amphibian immune system shows high similarity to those of birds and mammals (Froese et al., 2005; Chen and Robert, 2011; Rollins-Smith and Woodhams, 2012). Immune processes have traditionally been divided into two broad, but interconnected, subsystems on the basis of their functions in host defense: the innate and the adaptive immunity (Dunkelberger and Song, 2010). The innate and adaptive immune responses of amphibians were characterized both in vitro and in animal disease models (Schadich et al., 2009; Schadich and Cole, 2010; Chen and Robert, 2011; Rollins-Smith and Woodhams, 2012). The innate immune system includes all of the molecular and cellular defenses that act as the first barrier against infections by providing rapid and non-specific responses against the different invading microbial pathogens. Its molecular defenses are antimicrobial peptides (AMPs) from specialized glands in the skin, stomach and intestines (Moore et al., 1991; Reilly et al., 1994; Rollins-Smith et al., 2009; Schadich, 2009), the complement lytic system of blood and lymph (Green and Cohen, 1977), and lysozymes (Rollins-Smith et al., 2012). Its cellular defenses comprise macrophages and dendritic cells (Manning and Horton, 1982), and T-killer cells (Horton et al., 1996). The adaptive immune response is highly specific but slower, with the main function to control re-infections. It involves both humoral and cell-mediated responses, respectively B lymphocytes which produce antibodies when stimulated by an antigen, and T lymphocytes which regulate antibody production and release pro-inflammatory cytokines (Zimmerman, Vogel and Bowden, 2010; Chen and Robert, 2011; Rollins-Smith and Woodhams, 2012). Cytokines induce sickness behavior (i.e. reduction of activity, food intake, social interactions and behavioral fever) which is thought to enhance immune function by allowing maximum energy allocation (Llewellyn et al., 2011).
The responses of immune system of poikilothermic animals, such as amphibians, are slower to develop than those of homoeothermic animals due to their dependence on the environmental temperature. Compared to mammals and birds, amphibians have slower and less diverse adaptive immune responses (Du Pasquier, Schwager and Flajnik, 1989), and their resistance to invading microbial pathogens heavily relies on the different innate immune defenses. Different studies demonstrated the role of the skin AMPs in protection of skin against B. dendrobatidis (Woodhams et al., 2007; Rollins-Smith et al., 2009; Reinert et al., 2010) while the protection against bacterial pathogens is dependent both on skin AMPs and macrophages (Hayes et al., 2006; Schadich et al., 2009). In addition, the analyses of immune responses of the neotenic A. mexicanum, African clawed frog (Xenopus laevis) and a small number of additional salamander species showed slow responses of acquired immune responses and graft rejection rates (Kaufman and Volk, 1994; Raffel et al., 2009; Chen and Robert, 2011) while the immune responses of the other amphibian species have not been studied yet.
To circumvent the high costs of wildlife disease monitoring, the use of reliable indicators of population viability, immunocompetence and health, such as sexual traits was proposed (Ahtiainen et al., 2004; Hegyi et al., 2009). The basic idea is that, assuming that immunity is sufficiently costly, there is an energetic trade-off between reproduction and immunity, so that males fighting against an infection are able to invest less in the production of both morphological and behavioral sexual traits (Ahtiainen et al., 2004; Hegyi et al., 2009). This assumption makes particular sense in amphibians as the vast majority of known amphibian parasites and pathogens rely on aquatic infective stages for transmission and reproduction, and amphibians face the highest risks of infection during their aquatic reproductive phase.
One approach to investigate the trade-off between reproduction and immunity of different animal species is to inject pro-inflammatory agents into live animals. Among these agents, the different lipopolysaccharide (LPS) complexes from the outer membrane of Gram negative bacteria are most frequently used in studies with a number of different vertebrate species including amphibians (Llewellyn et al., 2011). LPS initiates the acute inflammatory responses of macrophages via activation of Tool like receptors 4 (TLR4) and in turn, macrophages release the proinflammatory cytokines (Karima et al., 1999; Bicego et al., 2002; Chilton, Embry and Mitchell, 2012). In vertebrates, these cytokines not only activate the acute response of neutrophils and promote the adaptive immune defenses, but they also might modulate the different sexual traits (Bugbee et al., 1983; Bicego et al., 2002; Llewellyn et al., 2011). However, up to now, the immune responses to LPS were studied in vivo in only four amphibian species, Bufo marinus, Bufo paracnemis, Xenopus laevis and Eupemphix nattereri (Sherman et al., 1991; Sherman and Stephens, 1998; Bicego and Branco, 2002; Bicego et al., 2002; Franco-Belussi and de Oliveira, 2011; Llewellyn et al., 2011). These studies showed that the response in these species occurs between one to thirty days after treatment. Similar responses could be also present in other amphibian groups as it was found that LPS from Escherichia coli 026:B5 activated the production of B lymphocytes of the urodele axolotl A. mexicanus in vitro after three day treatments (Salvadori and Tournefier, 1996) indicating its in vivo potential. However, up to now, in vivo effects of LPS have not been studied in any urodele species.
Here, we investigated therefore whether the expression of the sexual traits reliably signals immunocompetence and health status in male Palmate newts Lissotriton helveticus and whether it could be used as a reliable indicator for disease surveillance. In the Palmate newt Lissotriton helveticus, an urodele species, males express striking morphological and behavioral sexual traits, a caudal filament at the end of the tail, hind-foot webs, and a courtship display behavior, as the most important determinants for female mate choice (Cornuau et al., 2012). Both types of sexual traits could be modulated by bacterial LPS induced trade-offs between reproduction and immunity. Therefore, these sexual traits could be good indicators of immunocompetence and health status of animals in different populations. The aim of our study was to assess whether the bacterial LPS can affect the sexual traits in L. helveticus males.
Material and methods
Animals
Palmate newts were all caught with a net at the early beginning of the reproductive season in two natural ponds and were all released back in the breeding pond (or pond of origin) at the end of the experiment. For the monitoring of morphological traits, 123 males were caught at Etang Bouteve (Mourtis, N42.90295 E0.77374, France, altitude: 1682 m). For the monitoring of courtship display, 60 males and 45 females were caught in Caumont (N43.01182 E1.09142, France, altitude: 438 m). These individuals were maintained at our laboratory. Each of them was individually marked with a subcutaneous visible elastomer implant (VIE, Northwest Marine Technology, Washington, Shaw Island, WA, USA) at the basis of one of the four legs.
Treatments with LPS and monitoring of morphological traits
Animals were injected intraperitoneally with 0.02 ml of a solution composed either of LPS (E. coli serotype 055:B5, Sigma, Lyon) in saline solution (0.9%), or of saline solution only. This LPS contains the lipid A, a component required for interaction with newt macrophages, and highly conserved among different bacterial species (Chilton, Embry and Mitchell, 2012; Schadich, Mason and Cole, 2013). The 33 newts were challenged with 0.14 mg of LPS/ml (low dose, which approximately corresponds to a 2 mg of LPS/kg for an average sized-individual of 1.4 g as used by Llewellyn et al., 2011 in B. marinus and Bicego et al., 2002 in B. paracnemis), 30 newts with 0.7 mg of LPS/ml (high dose, which approximately corresponds to a 10 mg of LPS/kg for an average sized-individual), and 60 newts with a saline solution (control). These dosages are known to be toxic in experimental mice and other vertebrates (Piccioni et al., 2013). Individuals were randomly assigned to an experimental treatment (control, low and high doses of LPS), and all morphological traits were homogenously distributed among these treatments at the start of the experiment (MANOVA: , ).
After injection, individuals were placed in an opaque tank together with 11 other individuals. To avoid a potential confounding tank effect, these tank groups were composed of 6 males injected with LPS (either males injected with low or high doses) and 6 males injected with the saline solution (except for three tanks in which 7 males were injected with a low dose of LPS). Tanks contained 10 liters of water, an air-pump to oxygenate the water, one shelter perforated with 17 holes, and plants collected in the native pond. We used a natural light/dark cycle (12 h:12 h, Reptisun 2.0, ZooMed) and maintained the animal facility at 18°C (± 1). Individuals were fed ad libitum with larvae of chironomids, daphnia and tubifex.
On the day of injection and 32 days later, individuals were weighted with a digital scale (accuracy: 0.01 g) and photographed on a millimeter paper background (grid 1 mm2). The photo images were analyzed using the ImageJ software (http://rsbweb.nih.gov/ij/) to measure snout-vent-length (SVL), filament length, hind-foot-web size and crest size. The male body condition (BCI) was calculated as the residual of the linear regression of the cube root of body mass on SVL, as recommended for amphibian species (Băncilă et al., 2010). The development of each trait ( trait) was calculated as the value of the trait measured at the start of the experiment minus the value of the trait measured at the end of the experiment. The values of traits were homogenously distributed between tanks (Kruskal-Wallis rank sum test, SVL: , , ; body mass: , , ; BCI: , , ; filament length: , , ; hind-foot-web size: , , ; crest size: , , ).
Treatments with LPS and monitoring of courtship display
Individuals were measured for SVL, weight and filament length as described above. They were housed in three outdoor tanks by groups of 20 males and 15 females per tank. Each tank had a volume of 1000 liters, was filled with water and covered by a net to avoid escape and predation. It contained shelters and aquatic plants from the pond of origin.
Courtship frequency was measured for each male by counting the number of times this male was observed in courtship. Three observation sessions (S0, S1 and S2) were performed. One session corresponded to three days of observation and sessions were separated by three days. In the first session (S0), observations were conducted continuously from 6:00 am to 7:00 pm. It allowed to determining the time of day when most males interacted with females: 6:00-12:00 am and 5:00-7:00 pm. In the following observation sessions S1 and S2, the observer performed subsequent observations during these activity peaks. Observations were performed by one observer (RP) walking around the three tanks until seeing a courting male. Daily observations corresponded to a succession of 15 min of focal observations during which one tank was observed at a time and all individuals in the tank observed altogether. Every 15 min a different tank was observed, and the tank order was changed every day. All tanks were similarly observed and all tanks had similar total courtship frequencies (, ). As the VIE marking did not allow the remote identification of the males, we caught them with a net and identified each of them. Then, they were immediately released into the tank. We counted the number of times each male was seen courting during all sessions to obtain an index of courtship frequency.
Male newts were assigned to the experimental treatment the night before observation S1 and S2. In each tank, at around 11:00 pm, 10 males were injected with a saline solution (control) and 10 males with LPS (10 mg/kg). Individuals of each group were immediately released back in their initial outdoor tank. Courtship frequency was recorded the day following injection, and for a total of three days (observation session S1). Then, females were removed from the tank for a three-day period without observations, and placed back before the second injection of the males. Male traits (filament length, SVL, weight and courtship frequency) were homogenously distributed among experimental treatments (control and LPS) (MANOVA: , ). Males were injected a second time and a second observation period (S2) was performed under the conditions as described above. The post-injection courtship frequency was defined as the total number of courtship observed during the two observational periods S1 and S2.
Statistical analysis
Statistical analyses were performed using R (R Development Core Team, 2009, version 2.9.2). We built generalized linear mixed models (GLMMs) to investigate the impact of the experimental treatments on filament length, hind-foot-web size, crest size and courtship frequency. The experimental treatment (low dose of LPS versus saline, high dose of LPS versus saline, or high dose versus low dose) was set as an explanatory fixed variable and all models included tank identity as a random factor. We investigated the impact of the experimental treatment on courtship frequency in a similar way, including the number of courtship observed during S1 and S2 as a dependent variable, the experimental treatment (high dose of LPS versus saline) as an independent fixed variable and tank identity as a random factor. For all models, the link function was identity because all models had a Gaussian distribution of error terms. We evaluated the effect of tank identity by testing whether its removal from the model caused a significant decrease in the model fit and found that this was never significant (all ).
Results
Effect of LPS on L. helveticus survival
All of the animals survived the experiment and no adverse effects on their health was observed neither in the two groups of males injected by LPS nor in the control groups of males injected by saline. Notably, over the experiment, SVL, body mass, BCI, filament length and hind-foot-web size increased for all individuals of all treatments, while crest size decreased (fig. 1).
Development of morphological traits over the experiment according to experimental treatment (mean ± SE). a) Evolution of BCI (body condition index). b) Development of filament length. c) Development of HFW (hind-foot-web). d) Development of crest size. Black bars correspond to individuals injected with LPS, white bars correspond to individuals injected with saline solution (control), HD corresponds to high dose treatment (10 mg of LPS/kg) and LD corresponds to low dose treatment (2 mg of LPS/kg). NS means that . The development of each trait ( trait) was calculated as the value of the trait measured at the start of the experiment minus the value of the trait measured at the end of the experiment.
Citation: Amphibia-Reptilia 35, 1 (2014) ; 10.1163/15685381-00002928
Effect of LPS on morphological traits
The SVL, body mass, BCI, filament length, hind-foot-web size and crest size did not differ between the group of males injected by a low dose of LPS and its associated control group (SVL: , , body mass: , , BCI: , , filament length: , , hind-foot-web size: , , crest size: , , fig. 1). These traits also did not differ between the group of males injected by a high dose of LPS and its associated control group ( SVL: , , body mass: , , BCI: , , filament length: , , hind-foot-web size: , , crest size: , , fig. 1). There was also no difference between the groups of males injected by low and high dose of LPS in the SVL (, ), body mass (, ), BCI (, ), filament length (, ), hind-foot-web size (, ) and crest size (, ). The two control groups did not differ statistically in any of the traits ( SVL: , ; body mass: , ; BCI: , ; filament length: , ; hind-foot-web size: , ; crest size: , ; fig. 1).
Effect of LPS on courtship display frequency
Post-injection courtship frequency (S1 + S2) was not affected by the experimental treatment as no difference was noted between the LPS treatment group and the control group (, , fig. 2).
Post-injection courtship frequency according to experimental injection (LPS or saline solution, mean ± SE). NS means that . Courtship frequency was measured for each male by counting the number of times this male was observed in courtship after experimental injection.
Citation: Amphibia-Reptilia 35, 1 (2014) ; 10.1163/15685381-00002928
Discussion
The palmate newts might be well protected from effects of proinflammatory agents like bacterial LPS that have profound effects in humans and other vertebrates. Our analyses showed that injection of the LPS did not affect either survival or expression of morphological and behavioral sexual traits in L. helveticus.
Our findings showed that the reproductive traits of L. helveticus may not be included in the immune trade-off during bacterial infections as the injection of males by LPS did not cause any significant effect on animal survival, health and sexual traits. These results are consistent with the broader resistance of amphibians to the effects of the LPS as the doses that were used in this study as well as studies with frogs and toads did not cause the extensive dysregulation of immune defenses which they caused in different mammalian species during experimental treatments (Sherman et al., 1991; Sherman and Stephens, 1998; Zou et al., 2000; Bicego and Branco, 2002; Bicego et al., 2002; Franco-Belussi and de Oliveira, 2011; Llewellyn et al., 2011). Such resistance of amphibians to LPS might be due to the weak interactions of the lipid components of the LPS with TLR4 of macrophages (Berczi, Bertók and Bereznai, 1966; Bleicker et al., 1983; Bugbee et al., 1983) and/or neutralization of the LPS by the AMPs and serum LPS binding protein (Schadich, Mason and Cole, 2013). Such obvious resistance of L. helveticus is definitely ecologically relevant as it shows that the pivotal reproductive traits of the infected males could not be affected by LPS toxemia associated with bacterial disease.
At this stage it is not possible to completely rule out the possible interactions of the LPS with the host immune cells. Furthermore, due to the observed effects of the LPS on stimulation of B lymphocytes of the axolotl (Salvadori and Tournefier, 1996), we could still expect that the LPS injection yield the similar immune activation in other newt species as in frogs, despite the absence of its obvious effects on sexual traits of males of L. helveticus. Generally, such a non-activation would be surprising given that (i) LPS is a firmly established immune elicitor which has been shown to efficiently activate the immune system in a large range of taxa including insects, birds, mammals, reptiles and anuran amphibians, and (ii) LPS stimulates both innate and acquired immunity, which both are phylogenetically highly conservative in vertebrates, despite a few particularities (Chen and Robert, 2011; Rollins-Smith and Woodhams, 2012). More specifically, LPS has an impact in vivo on the metabolism and activity of frogs (Sherman et al., 1991; Sherman and Stephens, 1998; Zou et al., 2000; Bicego and Branco, 2002; Bicego et al., 2002; Llewellyn et al., 2011; Franco-Belussi and de Oliveira, 2011) and the newt immune system is not dramatically different from that of frogs (Todd, 2007; Raffel et al., 2009). Nevertheless, the possibility that the immune system of male L. helveticus did not respond at all to the LPS cannot be discarded, and it deserves to be investigated further.
Another, more plausible, explanation for the absence of an observed impact of LPS on morphological and behavioral sexual traits is that LPS did activate the newt immune system but this activation was not measured in our study for many and non-exclusive reasons. First, the trade-off between immune response and expressing sexual traits might exist but we did not observe it because the immune reaction of newts may have been slow and still below the threshold of detectability during our second experiment. In anurans, an activation of the immune system by LPS may sometimes take up to 30 days (Llewellyn et al., 2011). However, our first experiment lasted 32 days and yet we did not observe any impact of the LPS injection on morphological traits.
Further, given that the LPS is inert, non-replicating and non-pathogenic, it may activate the immune system but, contrary to a pathogen or parasite, does not incur deleterious pathogenic costs, as it does not replicate or generate its own metabolic products in the host. Male palmate newts may only pay a relatively small energetic cost and may be able to sustain the challenge imposed by LPS to their immune system. Thus, there may not be a trade-off between immunity and reproduction in L. helveticus. Similar results were found in the zebra finch Taeniopygia guttata in which sexual traits appear condition-dependent but not linked to immune capacity (Birkhead, Fletcher and Pellatt, 1998).
A trade-off may exist in some environmental conditions and be hidden in other ones due to ceiling or floor effects. Indeed, the evolutionary and ecological history of a population, such as earlier intense exposure to bacteria, and particularly to E. coli, could play an important role in the apparent lack of response to an LPS challenge, as previously found in the House sparrow Passer domesticus (Lee, Martin and Wikelski, 2005). Moreover, good environmental conditions, such as high food availability, may improve general body condition of the individuals and hide a trade-off between immunity and sexual traits that is sometimes revealed in harsh environmental conditions (e.g. Jacot et al., 2005; Leman et al., 2009). Stress of captivity or high initial parasitic load in the newt population, on the other side, may prevent energy mobilization to mount an immune response. For example, acute stress can lead to short-term decreases in circulating lymphocytes in amphibians (Maule and VanderKooi, 1999). In line with this idea one could argue that the act of injection (without LPS) could initiate a response from the innate immune system, because inserting a needle into the skin damages dermal tissue (Brown, Shilton and Shine, 2011), or even introduces bacteria from the skin in the peritonea, and that this could mask an impact of LPS. We find this possibility improbable, as we did not observe any decrease of BCI or sexual traits over the experiment, suggesting that the injection itself was not costly to the individuals.
Finally, the social context of our study may also contribute to hide effects of LPS. Males were housed in groups of males in the first experiment and with females in the second experiment. In song sparrows, the effect of an LPS injection is much less noticeable during the breeding season than during other parts of the year and in zebra finch experimental evidence shows that reproductive adults may overcome or mask sickness when the social context present opportunities to access to reproduction (Owen-Ashley and Wingfield, 2006; Lopes et al., 2012).
Here our aim was to provide a simple, low cost method to assess immunocompetence in an urodele, the Palmate newt. We did not find any indication of a potential trade-off between the response to an injection of LPS and the expression of sexual traits in male palmate newts. This result suggests that measuring morphological or behavioral sexual traits may not be a useful method to monitor immunocompetence in this species. However, our study was only a first step and further studies are needed to fully rule out the potential of sexual traits to signal individual and population health status in the wild, as contrary to LPS, natural pathogenic load is likely to incur strong costs to the hosts.
Acknowledgements
Adelaïde Sibeaux is thanked for technical assistance. Catching permits n°2009-13 (Ariège) and n°2009-12 (Haute Garonne). This work was supported by the Ministère de la Recherche (PhD fellowship to JHC), two CNRS grants to AL and DS, the BioDiversa-project RACE and the Observatoire Homme-Milieu Pyrénées Haut-Vicdessos.
Ethical standards
The experiments comply with the current laws of the country in which they were performed.
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Footnotes
Associated Editor: Caitlin Gabor
