Integrating time progression in ecoimmunology studies: beyond immune response intensity

Integrating time progression in ecoimmunology studies: beyond immune response intensity Habitat alterations in agroecosystems may damage amphibian immune capacity. As agroecosystem extension is increasing worldwide, broader-context knowledge on the effects of agroecosystem stressful conditions on amphibian immune capacity is crucial for understanding and management of amphibian global declines. However, most studies on ecoimmunology assume synchronal among- group immune-response peaks, and focus on immune response after standardized time lapses, neglecting its progression. Herein, I compared inflamatory response short-term progression of agro- ecosystem and natural-habitat female and male natterjack toads Epidalea calamita,by measuring swelling response, once per hour, 6 h following an artificial immune challenge with innocuous anti- gen phytohemagglutinin. I also compared maximum magnitude of swelling response, irrespective of the moment when it was reached. Habitat differences arose only 3 h after challenge, when natural- habitat toads showed greater swelling response. Maximum magnitude of swelling response was similar in toads from both habitats. However, agroecosystem toads showed a delayed swelling response as compared with natural-habitat conspecifics, probably as a consequence of agroecosys- tem stressful conditions. Such a delay suggests a weaker immune capacity, and consequently impaired anti-pathogen performance. Regarding sex, swelling response magnitude did not differ between males and females. Female swelling response peaked earlier, but that of males was more sustained in time, which contradicts general across-taxa findings that males show impaired immune response. Interestingly, results suggest that measuring swelling response only after some standar- dized period following stimulation may be a simplistic approach and produce unrealistic results. Consequently, studies on ecoimmunology should implement swelling response progression in order to produce unbiased science. Key words: agroecosystem, amphibian, immunocompetence, natural habitat, immune response progression Variation in prevalence of pathogens (Eizaguirre et al. 2012), as well response (reviewed in Martin et al. 2010). Among human activities, as in natural (Gutie ´ rrez et al. 2013) or anthropogenic environmental habitat fragmentation and pollution are particularly intense in agri- stressors (Pigeon et al. 2013a) that affect host energetic condition, culture lands (Beebee 1983; Watson et al. 2000; Beja and Alca ´ zar may lead to geographic differences in disease dynamics (review in 2003). Also, fertilizers and pesticides used in agriculture contamin- Brearley et al. 2013). Indeed, anthropogenic habitat alterations such ate terrestrial (Teodorescu and Cogalniceanu 2005; Masilionite and _ _ as fragmentation (Berggren 2009) or pollution (Me ´ nard et al. 2010; Maiksteniene 2010) and water ecosystems (Addiscott and Thomas Pilosof et al. 2014) may exert a negative effect on animal immune 2000), with negative effects on fauna health and population V C The Author(s) (2018). Published by Oxford University Press. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy045/5034002 by guest on 13 July 2018 2 Current Zoology, 2018, Vol. 0, No. 0 dynamics (Green et al. 2005). Particularly, pollutants and pesticides 1985) and permeable skins (Bru ¨ hl et al. 2011). Furthermore, in agroecosystems impair immune response of vertebrates (Pigeon amphibians feed mostly on invertebrates (Attademo et al. 2005), et al. 2013b) and invertebrates (Smilanich and Dyer 2012). The fore- so they may ingest pollutants indirectly through diet. Actually, agro- casted expansion of agricultural land in the next years (Veldkamp chemicals have proved negative effects on amphibian health and Lambin 2001), and its important role as fauna reservoir (Dura ´n (Cabagna et al. 2005), and, more specifically, immune capacity et al. 2014), makes the study of agroecosystem dynamics a priority (Albert et al. 2007). Additional understanding of the factors affect- in the context of global change. ing amphibian immune capacity is relevant, particularly in challeng- Indeed, pathogens may severely compromise host’s fitness and ing environments such as agroecosystems, since disease plays a survival, as they consume a part of the host as a resource and jeop- fundamental role in amphibian global declines (De Castro and ardize its health status (Schmid-Hempel 2011). Consequently, im- Bolker 2005). mune systems against pathogens have evolved and are widespread in In this study, I compare immune response magnitude and short- nature (Litman et al. 2005). Vertebrate immune system is a diverse term progression of female and male natterjack toads (Epidalea but highly conservative multi-layer defense of increasing specificity: calamita; Laurenti 1768) from agroecosystems and natural habitats. from general physical barriers to pathogen infiltration, to leukocytes Infections by fungus (May et al. 2011), protozoans (Harris et al. that recognize and combat foreign elements in an organized manner 2013), oomycetes (Ferna ´ ndez-Bena ´ itez et al. 2011), nematodes, and (Wakelin and Apanius 1997). Leukocytes may elicit a generic, innate cestodes (Garcı ´a-Parı ´s et al. 2004) have been reported for this spe- (such as phagocytosis, lysozymes, or oxidant agents), or a highly cies. Presumably, a faster response would prevent pathogen expan- specific, adaptive (such as antibodies) response (Medzhitov 2007). sion more effectively, thus being more successful against disease, Due to its capital role as an anti-pathogen defense, immune capabil- particularly taking into account the high reproduction rates of most ity increases survival (Møller and Saino 2004), and a faster immune pathogens (Pilyugin and Antia 2000). Nevertheless, most studies on response may better preclude pathogen spread in the host and thus immune response neglect its short-term progression, focusing provide a more efficient protection against disease (Rogers et al. instead on the response after a standardized period following im- 2000). mune system stimulation. This approach implicitly assumes synchro- Nonetheless, mounting an immune response entails a number of nal activity of immune response in all individuals (e.g. Alonso- costs (Schmid-Hempel 2011). Firstly, immune response increases Alvarez and Tella 2001; Moreno-Rueda 2010; Carmona-Isunza metabolic rates (Sherman and Stephens 1998) and thus consumes et al. 2013; Zamora-Camacho et al. 2015; Zamora-Camacho et al. energy (Derting and Compton 2003; Demas 2004), as well as 2016). Other studies consider relatively long intervals of time (6, 12, metabolites such as amino acids (Klasing and Calvert 1999). or 24 h) that provide a mid-term view of immune-response time Furthermore, one mechanism of immune defense is oxidative burst trends, often finding an effect only, or neatly more intensely, at early (Lambeth 2007), which, along with collateral reactive oxygen spe- measurements (e.g., Navarro et al. 2003; Scharsack and Kalbe 2014; cies production as a consequence of immune reactions (Sorci and Zhiqiang et al. 2015). Faivre 2009), may cause oxidative stress. Oxidative stress occurs Since the aforementioned agroecosystem conditions may deteri- when the production of pro-oxidant chemical species overwhelms orate amphibian immune system as a consequence of physiological anti-oxidant defenses, with negative effects on physiology homeosta- stress, I predict that immune response will be faster and more sis and health (reviewed in Halliwell and Gutteridge 2007). intense in natural-habitat than in agroecosystem toads. Regarding Moreover, strong immune responses may cause health issues, as the impoverishing effect of testosterone on immune response fibrosis, if regenerative activity is excessive (Allen and Wynn 2011), (Muehlenbein and Bribiescas 2005), I expect females to elicit a faster or autoimmune diseases, if the immune system attacks components and more intense immune response than males. of the host itself (Bebo et al. 1998). Consequently, the immune response is under trade-offs with a number of traits (Lochmiller and Deerenberg 2000), such as dispersal (Brown and Shine 2014), escape Materials and Methods performance (Zamora-Camacho et al. 2015), learning capacity Study species (Schleich et al. 2015), social-status maintenance (Thomason et al. Epidalea calamita is a medium-size (50–75 mm of snout–vent length 2013), or reproduction (Nordling et al. 1998), in a number of taxa. in this study system) Bufonid toad that occurs in diverse pristine and Also, costs of immune-system deployment can differ between males anthropized habitats across vast regions of Central and and females, owing to different selective pressures on sexes Southwestern Europe (Go ´ mez-Mestre 2014). They are generalist (Zamora-Camacho et al. 2015). Moreover, a testosterone-mediated predators of a variety of invertebrates (Boomsma and Arntzen compromise of immune function could appear in males, due to 1985), though which they can ingest pesticides (Attademo et al. reduced energy allocation to the immune system to the benefit of 2005). Reproduction takes place during late winter or spring, often reproduction (Muehlenbein and Bribiescas 2005), according to the in small temporary pools, and tadpoles can complete their metamor- immunocompetence handicap hypothesis, proven in numerous taxa phosis in as little as 45 days (Go ´ mez-Mestre 2014). (Folstad and Karter 1992). Among vertebrates, amphibians are particularly susceptible to the negative effects of agroecosystem stressful conditions for several Study sites reasons. Firstly, amphibians undergo complex life cycles, including Field work was conducted in two adjacent habitats in Southwestern an aquatic larva stage followed in most cases by a fairly terrestrial Spain: natural pine grove Pinares de Cartaya, and surrounding agro- adult phase. Therefore, they require a high connectivity between ecosystems (37 20’ N, 7 09’ W). Capture sites were separated by both environments, which habitat fragmentation can damage. 4.5–5 km. This distance is short enough to discard climate differen- Moreover, complex life cycles imply that contamination in water, ces between them, but at once it is greater than maximum dispersal soil, and air may affect amphibians (Liu et al. 2011). Pollutants may of this species (Miaud et al. 2000). On the other hand, this distance also pass through amphibians’ unshelled eggs (Clark and LaZerte is long enough to ensure isolation of habitat characteristics. Indeed, Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy045/5034002 by guest on 13 July 2018 Zamora-Camacho  Time progression of immune response 3 differences in age, body size, indicators of reproductive investment, effect on toad body mass, so I injected the same dose of PHA to all and locomotor performance between E. calamita from these habi- individuals. Afterwards, I measured sole pad thickness as described tats (Zamora-Camacho and Comas, 2017; Zamora-Camacho, above, once per hour, for 6 h following injections. I chose this 2018) suggest different selective pressures in both. The natural pine period because the objectives of this work focus on short-term grove was a Pinus pinea forest, with an undergrowth dominated by immune response. Each time, I calculated sole pad swelling as the Cistus ladanifer, Rosmarinus officinalis, and Pistacia lentiscus. difference between each hourly post-inoculation sole pad thickness Although some controversy exists on the pristine or artificial origin measure and sole pad thickness measure prior to inoculations. of these trees in this region, they are known to be the dominant vege- Therefore, I obtained sole pad swelling on an hourly basis. This tal formation in this area for at least the last 4,000 years (Martı ´nez way, I also obtained maximum sole pad swelling of each individual, and Montero 2004). As for the agroecosystem, it included tradition- irrespective of the moment when it was reached, as a measure of in- al crop lands, which have lately turned into intensive orange, straw- tensity of immune response. Also, in each case, I calculated area berry, and vegetable crops. While I obtained no permission to access under the curve (AUC) of the six measurements of sole pad swelling, private properties for water or soil analyses, land owners reported therefore in h*mm, as an indicator of intensity of swelling response that manure and diverse synthetic NPK fertilizers are regularly incorporating time (Fekedulegn et al. 2007), by applying the trapez- added, as well as gibberellic acid, to enhance plant growth. Also, oidal formula with software GraphPad Prism 7.0. they claimed to use different kinds of insecticides, fungicides, and Once the experiments were finished, I released toads at their cap- herbicides. The substances, the timing of their application, and the ture site. Besides sole pad swelling, toads suffered no detectable amounts spread depend on the type of crop and the discretion of damage in the moment of their release as a consequence of this each farmer, but the substances and the amounts of them that land study. Toad capture and management was conducted according to owners declared to use were very similar. Since E. calamita can Junta de Andalucı ´a research permits (AWG/MGD/MGM/CB) and reproduce in very small rain pools, reproduction sites were wide- the bioethics committee of the University of Granada (permit num- spread in both habitats. Due to warm climatic conditions in these ber 18-CEEA-OH-2013); all applicable guidelines for the care and locations, toads do not hibernate, but aestivate instead. Therefore, handling of animals were followed. the breeding season spans from January to April. Statistics Toad capture and management As data accomplished the criteria of residual homoscedasticity and During the breeding season in 2015, I captured 69 toads (15 females normality, I used parametric statistics (Quinn and Keough 2002). and 18 males from agroecosystem, 18 females and 18 males from I conducted Ordinary Least Squares Linear Models. I performed two- pine grove) by hand. Sexual dimorphism allows undoubted recogni- way ANOVAs to test the effects of habitat, sex, and their interaction tion of sexes: males have purple-to-pinkish vocal sacs in their on 1) hourly sole pad swelling, 2) maximum sole pad swelling, 3) area throats, and blackish nuptial pads in the phalanxes and tubercles of under the curve, and 4) progression of sole pad swelling (by means of a their forelimbs (Go ´ mez-Mestre 2014). During the study, toads were repeated measures ANOVA). Then, I repeated models removing non- kept in individual plastic terraria with wet peat as a substrate and an significant habitat*sex interactions. Statistical analyses were performed opaque plastic plaque as a shelter. Toad body mass was measured with software Statistica 8.0 (StatSoft, Inc., Tulsa, OK, USA). with a balance (model CDS-100, precision 0.01 g). The day after capture, during the evening in order to avoid con- Results founding effects of different moments of circadian rhythms, I meas- ured left forelimb sole pad thickness with a pressure-sensitive Hourly sole pad swelling micrometer (Mitutoyo; precision 0.01 mm), three times, and Within each hourly measurement, only at the third and the fourth I considered sole pad thickness as the average of those three measure- hour after inoculation sole pad swelling differed significantly be- ments. Immediately, I inoculated subcutaneously 0.1 mg of phyto- tween habitats and between sexes, respectively (Table 1), being hemagglutinin (PHA; Sigma Aldrich, L-8754) diluted in 0.01 ml greater in pine grove than in agroecosystem toads 3 h after inocula- isotonic phosphate buffer in each toad’s left forelimb sole pad (Brown tions (Table 1; Figure 1), as predicted, and in males than in females et al. 2011). Phytohemagglutinin is a harmless protein that triggers a four hours after inoculations (Table 1 and Figure 2), contrarily to skin-swelling immune response, in which T-cells as well as other com- expected. Note that habitat differences 2 h after inoculations were ponents of the immune system are involved (Kennedy and Nager marginally non-significant (Table 1). Results remained similar when 2006; Martin et al. 2006). The magnitude of the swelling is directly habitat*sex interactions, non-significant in all cases (Table 1), were proportional to that of the cellular immune response (Parmentier removed (data not shown). et al. 1998; Vinkler et al. 2010). Following Smits et al. (1999), I did not use a phosphate buffer saline (PBS) control in the other forelimb Maximum sole pad swelling sole pad of the same individuals. Smits et al. (1999) proved that PHA Effects of habitat (F ¼ 0.579; P ¼ 0.450), sex (F ¼ 2.726; 1, 65 1, 65 tests can be simplified by omitting PBS controls, with little impact on P ¼ 0.104), and habitat sex interaction (F ¼ 2.760; P ¼ 0.101) on 1, 65 the results, and decreasing handling errors and the coefficient of vari- maximum sole pad swelling, regardless of the moment of the experi- ation owing to measurement inaccuracy. For this reason, PBS controls ment when it was reached, were non-significant. When the interaction are commonly omitted in PHA tests (e.g., Martin 2005; Hale and was removed from the model, both the effect of habitat (F ¼ 0.454; 1, 66 Briskie 2007; Moreno-Rueda 2010; Moreno-Rueda and Redondo P ¼ 0.503) and sex (F ¼ 2.412; P ¼ 0.125) remained similar. 1, 66 2012), also in amphibians (Gervasi and Foufopoulos, 2008; Iglesias- Carrasco et al., 2017). The validity of this method has been assessed in amphibians (Brown et al. 2011; Clulow et al. 2015). Progression of sole pad swelling Habitat (F ¼ 1.10; P ¼ 0.30), sex (F ¼ 0.72; P ¼ 0.40), Repeated measures ANOVAs showed that sole pad swelling peaked 1, 65 1, 65 and their interaction (F ¼ 0.02; P ¼ 0.90) had no significant earlier in pine grove (the repeated measures ANOVA was significant 1, 65 Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy045/5034002 by guest on 13 July 2018 4 Current Zoology, 2018, Vol. 0, No. 0 Table 1. Models testing the effects of habitat, gender, and their Area under the curve interaction on sole pad swelling hourly measurements Habitat (F ¼ 0.213; P ¼ 0.646), sex (F ¼ 0.864; P ¼ 0.356), 1, 65 1, 65 and their interaction (F ¼ 0.907; P ¼ 0.344) showed no signifi- 1, 65 Hours from inoculation Habitat Gender Habitat  Gender cant effect on AUC. Effects of habitat (F ¼ 0.174; P ¼ 0.678) 1, 66 F F F 1, 65 1, 65 1, 65 and sex (F ¼ 0.785; P ¼ 0.379) remained non-significant when 1, 66 ns ns ns 1 0.469 0.759 0.856 habitat*sex interaction was removed from the model. § ns ns 2 3.225 0.028 0.518 ns ns 3 8.436** 0.251 1.568 ns ns 4 1.485 4.915* 0.843 Discussion ns ns ns 5 1.337 0.725 0.185 ns ns ns Pine grove toads deployed a more intense swelling response than 6 0.051 0.367 1.270 agroecosystem toads, but only 3 h after inoculations. At all other Significant results are highlighted in bold. Symbols indicate: ns ¼ non-signifi- times, swelling response was similar in toads from both habitats. cant; ¼ marginally non-significant; *P< 0.05; **P< 0.01. Also, I detected no habitat differences in maximum sole pad swelling nor in AUC, which suggests a swelling response of similar intensity in toads from agroecosystems and natural habitats. Likewise, pris- tine habitat hellbender Cryptobranchus alleganiensis innate immune capacity is similar to that of conspecifics from human-disturbed habitat (Hopkins and DuRant 2011). Those results suggest that, although animals in human-disturbed habitats can show impaired immune capacity (Brearley et al. 2013), compensation may occur under certain circumstances. In fact, urban tree lizards Urosaurus ornatus show reinforced innate immune cap- acity in comparison with individuals from natural sites, which could be triggered by more frequent immune challenges in a circumstance of non-limiting energy intake (French et al. 2008). Both habitats studied here provide toads with similar prey availability, which sug- gests that agroecosystem toads do not suffer reduced energy intake compared to pine grove toads (Zamora-Camacho and Comas 2017). Therefore, no reduction in energy intake could allow agro- ecosystem toads to compensate stressful conditions and elicit a swel- Figure 1. Sole pad swelling peaked earlier in pine grove than in agroecosys- ling response whose magnitude is comparable to that of pine-grove tem toads. Note that habitat differences in maximum sole pad swelling were toads. non-significant. Sample sizes were 33 toads from agroecosystem and 36 However, habitat differences in toad swelling response are more toads from pine grove. Vertical bars represent standard errors. accurately defined in a broader vision of short-term swelling response progression. As a whole, swelling response peaked earlier in pine-grove than in agroecosystem toads. Even though maximum pad swelling magnitude is similar in toads from both habitats, a faster swelling response could reflect a healthier immune system in pine grove toads, and hinder pathogen proliferation more efficiently (Rogers et al. 2000). In fact, agroecosystem conditions are known to reduce amphibian immune capacity (Christin et al. 2003, 2004) and increase infection prevalence as a consequence (Linzey et al. 2003; Rohr et al. 2008). Hence, the finding that agroecosystem stressful conditions impair amphibian immune capacity is not surprising. Nonetheless, results herein are novel to put forward that such impairment does not necessarily represent a reduced magnitude of the response, but may imply a delay of it instead. A delayed response as a consequence of physiological stress triggered by agroecosystem conditions could weaken toad protection against disease, and could thus be at least one of the causes behind agroecosystem toad shorter lifespan found in this system, as compared with pine grove toads Figure 2. Sole pad swelling peaked earlier in female than in male toads. (Zamora-Camacho and Comas 2017). However, gender differences were not significant 3 h after inoculations, while As an alternative explanation, although prey availability does males showed greater immune response one hour later, suggesting more sustained immune response in males. Note that gender differences in max- not differ between both habitats (Zamora-Camacho and Comas imum sole pad swelling were non-significant. Sample sizes were 33 females 2017), energy budgets that toads allocate to different life-history and 36 males. Vertical bars represent standard errors. traits could (Stearns 2000). Actually, agroecosystem toads seem to prioritize energy allocation to reproduction rather than to somatic for habitat: F ¼ 5.464; P< 0.001; Figure 1)and in female preservation, as both male and female agroecosystem toads in this 5, 325 toads (the repeated measures ANOVA was significant for sex: study system show greater indicators of reproductive investment, F ¼ 2.533; P< 0.029; Figure 2). Repeated measures ANOVA was but shorter lifespans, than pine grove conspecifics (Zamora- 5, 325 not significant for habitat*sex interaction (F ¼ 0.599; P< 0.701). Camacho and Comas 2017). Increased extrinsic mortality by 5, 325 Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy045/5034002 by guest on 13 July 2018 Zamora-Camacho  Time progression of immune response 5 agroecosystem stressful conditions (see the introduction) could be 2000). Generalization of immunocompetence handicap hypothesis reducing agroecosystem toad lifespan. Under those circumstances, is indeed controversial (reviewed in Roberts et al. 2004). However, short-lived agroecosystem toads may divert energy allocation to in this case, the physiological mechanisms underlying male toad reproduction to the detriment of somatic preservation—which is capacity to deploy a more sustained swelling response than females indeed extrinsically constrained—thus compensating lesser repro- remain obscure. ductive events due to reduced lifespan with a higher reproductive Results herein confirm impaired swelling response in agroecosys- investment in each, according to life-history theory (Cox et al. tem toads, which is not surprising, as a number of studies on several 2010). Consequently, reduced energy budget for somatic preserva- taxa have reported similar negative effects on amphibian immune tion, and thus for immune system, could result in a delayed response capacity (Christin et al. 2004; Albert et al. 2007). However, this in agroecosystem toads. Similarly, trade-offs between immune study is novel to find evidence that such impairment does not neces- response and reproduction have indeed been found in a number of sarily involve a reduced magnitude of swelling response: in this case, taxa (Adamo et al. 2001; Ardia 2005; French et al. 2007). it appears as a delay in swelling response. As for sex, swelling In any case, the present results suggest that agroecosystem response magnitude did not differ between males and females. stressful conditions delay toad swelling response, which may impair Female swelling response peaked earlier, but that of males was more protection against pathogens, compared with natural pine grove sustained in time, which does not match previous across-taxa find- toads. In the light of these results, immune response impairment ings that male swelling response is impaired as compared to that of stands among the negative consequences of such conditions on females. amphibian health and populations (Alford et al. 2001; Hegde and In conclusion, most interestingly, these results show that measur- Krishnamurthy 2014), which contributes to define the role of agroe- ing swelling response only after a standardized period following cosystems on amphibian population declines. Actually, amphibian stimulation (Martin et al. 2006) might be simplistically assuming syn- populations are in particular decline in areas that have had pesti- chronal swelling response progression in all individuals. cides applied historically (Davidson 2004). Agriculture land area is Consequently, results might be biased, or important and useful predicted to increase in the near future (Zabel et al. 2014), meaning information could be lost. In fact, results in this work and their that an increasing number of amphibian populations are bound to interpretation would have been completely different if I had measured undergo agroecosystem conditions. Therefore, measures should be swelling response only once, and the moment chosen for the measure taken to milden such conditions, such as limitations in agrochemical would have caused dramatic differences. For instance, swelling re- use (Brodeur et al. 2014) or increased connectivity between amphib- sponse 3 h after inoculations was greater in pine grove than in agro- ian habitats (Naito et al. 2012). ecosystem toads, while it did not differ between sexes at that According to the immunocompetence handicap hypothesis, tes- moment. However, the outlook was completely different 4 h after tosterone exerts suppressive effects on the immune system (Hillgarth inoculations, with no habitat differences in swelling response, but and Wingfield 1997), which results in an across-taxa trend of males males showing greater swelling than females. Finally, if I had made to elicit less intense immune responses than females (Klein 2000). measures only at any other moment, I would have detected no effect Nevertheless, unexpectedly, the only moment when sex differences of habitat or sex. Nevertheless, a broader view including AUC and in swelling response were significant (4 h after inoculations), males maximum magnitude of swelling response, regardless of the moment deployed greater swelling response than females. However, at all when it was deployed, as well as progression of swelling response, other times, sex showed no effect on swelling response. provided an accurate panorama of the actual effects of habitat and Remarkably, maximum sole pad swelling, irrespective of the sex on short-term toad swelling response. Therefore, I urge ecoimmu- moment when it was reached, did not differ significantly between nologists to implement immune response progression in their studies, sexes. Similarly, lack of sex differences in AUC suggests similar or, at least, to assess its synchrony among the groups they are study- intensity of swelling response in males and females. Therefore, hour- ing, in order to obtain realistic, unbiased results in a broader context. ly tests of sex effect on toad swelling response do not seem to sup- port testosterone-induced immunosuppression. Notably, a broader vision of the short-term progression of swel- Acknowledgments ling response showed a significantly earlier swelling response peak The author would like to thank the landowners who provided information on in females than in males. However, sex differences 3 h after inocula- the agrochemicals they use. Daniel Pinto-Navarro gave logistic support. tions, when such female peak took place, were not significant, while Comments by Gregorio Moreno-Rueda and three anonymous reviewers 4 h after inoculations females had a significantly reduced swelling improved the article. This research did not receive any specific grant from response as compared with males. Such progression pattern suggests funding agencies in the public, commercial, or not-for-profit sectors. that male swelling response was not actually slower, but more I assumed the expenses of the work. sustained in time, which should not represent a lower energy invest- ment (Fox et al. 2005), and thus did not reflect impaired male immune response. As a whole, these results did not support male Funding reduced immune capacity (Belliure et al. 2004). Similarly, response This article was partly supported by a Ramo ´ n Areces Foundation postdoctor- to PHA by testosterone-supplemented male European tree frog al fellowship, and a Juan de la Cierva-Formacio ´ n postdoctoral grant from the (Hyla arborea) was not reduced, but enhanced (Desprat et al. 2015). Spanish Ministerio de Economı´a, Industria y Competitividad. 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Response patterns of ces: a high resolution suitability evaluation and its perspectives until 2100 phytohemagglutinin-induced skin swelling at different reproductive phases under climate change conditions. PLOS One 9:e107522. in female striped hamster. Cricetulus barabensis. Acta Theriol Sin 35:74–79. Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy045/5034002 by guest on 13 July 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Zoology Oxford University Press

Integrating time progression in ecoimmunology studies: beyond immune response intensity

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Abstract

Habitat alterations in agroecosystems may damage amphibian immune capacity. As agroecosystem extension is increasing worldwide, broader-context knowledge on the effects of agroecosystem stressful conditions on amphibian immune capacity is crucial for understanding and management of amphibian global declines. However, most studies on ecoimmunology assume synchronal among- group immune-response peaks, and focus on immune response after standardized time lapses, neglecting its progression. Herein, I compared inflamatory response short-term progression of agro- ecosystem and natural-habitat female and male natterjack toads Epidalea calamita,by measuring swelling response, once per hour, 6 h following an artificial immune challenge with innocuous anti- gen phytohemagglutinin. I also compared maximum magnitude of swelling response, irrespective of the moment when it was reached. Habitat differences arose only 3 h after challenge, when natural- habitat toads showed greater swelling response. Maximum magnitude of swelling response was similar in toads from both habitats. However, agroecosystem toads showed a delayed swelling response as compared with natural-habitat conspecifics, probably as a consequence of agroecosys- tem stressful conditions. Such a delay suggests a weaker immune capacity, and consequently impaired anti-pathogen performance. Regarding sex, swelling response magnitude did not differ between males and females. Female swelling response peaked earlier, but that of males was more sustained in time, which contradicts general across-taxa findings that males show impaired immune response. Interestingly, results suggest that measuring swelling response only after some standar- dized period following stimulation may be a simplistic approach and produce unrealistic results. Consequently, studies on ecoimmunology should implement swelling response progression in order to produce unbiased science. Key words: agroecosystem, amphibian, immunocompetence, natural habitat, immune response progression Variation in prevalence of pathogens (Eizaguirre et al. 2012), as well response (reviewed in Martin et al. 2010). Among human activities, as in natural (Gutie ´ rrez et al. 2013) or anthropogenic environmental habitat fragmentation and pollution are particularly intense in agri- stressors (Pigeon et al. 2013a) that affect host energetic condition, culture lands (Beebee 1983; Watson et al. 2000; Beja and Alca ´ zar may lead to geographic differences in disease dynamics (review in 2003). Also, fertilizers and pesticides used in agriculture contamin- Brearley et al. 2013). Indeed, anthropogenic habitat alterations such ate terrestrial (Teodorescu and Cogalniceanu 2005; Masilionite and _ _ as fragmentation (Berggren 2009) or pollution (Me ´ nard et al. 2010; Maiksteniene 2010) and water ecosystems (Addiscott and Thomas Pilosof et al. 2014) may exert a negative effect on animal immune 2000), with negative effects on fauna health and population V C The Author(s) (2018). Published by Oxford University Press. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy045/5034002 by guest on 13 July 2018 2 Current Zoology, 2018, Vol. 0, No. 0 dynamics (Green et al. 2005). Particularly, pollutants and pesticides 1985) and permeable skins (Bru ¨ hl et al. 2011). Furthermore, in agroecosystems impair immune response of vertebrates (Pigeon amphibians feed mostly on invertebrates (Attademo et al. 2005), et al. 2013b) and invertebrates (Smilanich and Dyer 2012). The fore- so they may ingest pollutants indirectly through diet. Actually, agro- casted expansion of agricultural land in the next years (Veldkamp chemicals have proved negative effects on amphibian health and Lambin 2001), and its important role as fauna reservoir (Dura ´n (Cabagna et al. 2005), and, more specifically, immune capacity et al. 2014), makes the study of agroecosystem dynamics a priority (Albert et al. 2007). Additional understanding of the factors affect- in the context of global change. ing amphibian immune capacity is relevant, particularly in challeng- Indeed, pathogens may severely compromise host’s fitness and ing environments such as agroecosystems, since disease plays a survival, as they consume a part of the host as a resource and jeop- fundamental role in amphibian global declines (De Castro and ardize its health status (Schmid-Hempel 2011). Consequently, im- Bolker 2005). mune systems against pathogens have evolved and are widespread in In this study, I compare immune response magnitude and short- nature (Litman et al. 2005). Vertebrate immune system is a diverse term progression of female and male natterjack toads (Epidalea but highly conservative multi-layer defense of increasing specificity: calamita; Laurenti 1768) from agroecosystems and natural habitats. from general physical barriers to pathogen infiltration, to leukocytes Infections by fungus (May et al. 2011), protozoans (Harris et al. that recognize and combat foreign elements in an organized manner 2013), oomycetes (Ferna ´ ndez-Bena ´ itez et al. 2011), nematodes, and (Wakelin and Apanius 1997). Leukocytes may elicit a generic, innate cestodes (Garcı ´a-Parı ´s et al. 2004) have been reported for this spe- (such as phagocytosis, lysozymes, or oxidant agents), or a highly cies. Presumably, a faster response would prevent pathogen expan- specific, adaptive (such as antibodies) response (Medzhitov 2007). sion more effectively, thus being more successful against disease, Due to its capital role as an anti-pathogen defense, immune capabil- particularly taking into account the high reproduction rates of most ity increases survival (Møller and Saino 2004), and a faster immune pathogens (Pilyugin and Antia 2000). Nevertheless, most studies on response may better preclude pathogen spread in the host and thus immune response neglect its short-term progression, focusing provide a more efficient protection against disease (Rogers et al. instead on the response after a standardized period following im- 2000). mune system stimulation. This approach implicitly assumes synchro- Nonetheless, mounting an immune response entails a number of nal activity of immune response in all individuals (e.g. Alonso- costs (Schmid-Hempel 2011). Firstly, immune response increases Alvarez and Tella 2001; Moreno-Rueda 2010; Carmona-Isunza metabolic rates (Sherman and Stephens 1998) and thus consumes et al. 2013; Zamora-Camacho et al. 2015; Zamora-Camacho et al. energy (Derting and Compton 2003; Demas 2004), as well as 2016). Other studies consider relatively long intervals of time (6, 12, metabolites such as amino acids (Klasing and Calvert 1999). or 24 h) that provide a mid-term view of immune-response time Furthermore, one mechanism of immune defense is oxidative burst trends, often finding an effect only, or neatly more intensely, at early (Lambeth 2007), which, along with collateral reactive oxygen spe- measurements (e.g., Navarro et al. 2003; Scharsack and Kalbe 2014; cies production as a consequence of immune reactions (Sorci and Zhiqiang et al. 2015). Faivre 2009), may cause oxidative stress. Oxidative stress occurs Since the aforementioned agroecosystem conditions may deteri- when the production of pro-oxidant chemical species overwhelms orate amphibian immune system as a consequence of physiological anti-oxidant defenses, with negative effects on physiology homeosta- stress, I predict that immune response will be faster and more sis and health (reviewed in Halliwell and Gutteridge 2007). intense in natural-habitat than in agroecosystem toads. Regarding Moreover, strong immune responses may cause health issues, as the impoverishing effect of testosterone on immune response fibrosis, if regenerative activity is excessive (Allen and Wynn 2011), (Muehlenbein and Bribiescas 2005), I expect females to elicit a faster or autoimmune diseases, if the immune system attacks components and more intense immune response than males. of the host itself (Bebo et al. 1998). Consequently, the immune response is under trade-offs with a number of traits (Lochmiller and Deerenberg 2000), such as dispersal (Brown and Shine 2014), escape Materials and Methods performance (Zamora-Camacho et al. 2015), learning capacity Study species (Schleich et al. 2015), social-status maintenance (Thomason et al. Epidalea calamita is a medium-size (50–75 mm of snout–vent length 2013), or reproduction (Nordling et al. 1998), in a number of taxa. in this study system) Bufonid toad that occurs in diverse pristine and Also, costs of immune-system deployment can differ between males anthropized habitats across vast regions of Central and and females, owing to different selective pressures on sexes Southwestern Europe (Go ´ mez-Mestre 2014). They are generalist (Zamora-Camacho et al. 2015). Moreover, a testosterone-mediated predators of a variety of invertebrates (Boomsma and Arntzen compromise of immune function could appear in males, due to 1985), though which they can ingest pesticides (Attademo et al. reduced energy allocation to the immune system to the benefit of 2005). Reproduction takes place during late winter or spring, often reproduction (Muehlenbein and Bribiescas 2005), according to the in small temporary pools, and tadpoles can complete their metamor- immunocompetence handicap hypothesis, proven in numerous taxa phosis in as little as 45 days (Go ´ mez-Mestre 2014). (Folstad and Karter 1992). Among vertebrates, amphibians are particularly susceptible to the negative effects of agroecosystem stressful conditions for several Study sites reasons. Firstly, amphibians undergo complex life cycles, including Field work was conducted in two adjacent habitats in Southwestern an aquatic larva stage followed in most cases by a fairly terrestrial Spain: natural pine grove Pinares de Cartaya, and surrounding agro- adult phase. Therefore, they require a high connectivity between ecosystems (37 20’ N, 7 09’ W). Capture sites were separated by both environments, which habitat fragmentation can damage. 4.5–5 km. This distance is short enough to discard climate differen- Moreover, complex life cycles imply that contamination in water, ces between them, but at once it is greater than maximum dispersal soil, and air may affect amphibians (Liu et al. 2011). Pollutants may of this species (Miaud et al. 2000). On the other hand, this distance also pass through amphibians’ unshelled eggs (Clark and LaZerte is long enough to ensure isolation of habitat characteristics. Indeed, Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy045/5034002 by guest on 13 July 2018 Zamora-Camacho  Time progression of immune response 3 differences in age, body size, indicators of reproductive investment, effect on toad body mass, so I injected the same dose of PHA to all and locomotor performance between E. calamita from these habi- individuals. Afterwards, I measured sole pad thickness as described tats (Zamora-Camacho and Comas, 2017; Zamora-Camacho, above, once per hour, for 6 h following injections. I chose this 2018) suggest different selective pressures in both. The natural pine period because the objectives of this work focus on short-term grove was a Pinus pinea forest, with an undergrowth dominated by immune response. Each time, I calculated sole pad swelling as the Cistus ladanifer, Rosmarinus officinalis, and Pistacia lentiscus. difference between each hourly post-inoculation sole pad thickness Although some controversy exists on the pristine or artificial origin measure and sole pad thickness measure prior to inoculations. of these trees in this region, they are known to be the dominant vege- Therefore, I obtained sole pad swelling on an hourly basis. This tal formation in this area for at least the last 4,000 years (Martı ´nez way, I also obtained maximum sole pad swelling of each individual, and Montero 2004). As for the agroecosystem, it included tradition- irrespective of the moment when it was reached, as a measure of in- al crop lands, which have lately turned into intensive orange, straw- tensity of immune response. Also, in each case, I calculated area berry, and vegetable crops. While I obtained no permission to access under the curve (AUC) of the six measurements of sole pad swelling, private properties for water or soil analyses, land owners reported therefore in h*mm, as an indicator of intensity of swelling response that manure and diverse synthetic NPK fertilizers are regularly incorporating time (Fekedulegn et al. 2007), by applying the trapez- added, as well as gibberellic acid, to enhance plant growth. Also, oidal formula with software GraphPad Prism 7.0. they claimed to use different kinds of insecticides, fungicides, and Once the experiments were finished, I released toads at their cap- herbicides. The substances, the timing of their application, and the ture site. Besides sole pad swelling, toads suffered no detectable amounts spread depend on the type of crop and the discretion of damage in the moment of their release as a consequence of this each farmer, but the substances and the amounts of them that land study. Toad capture and management was conducted according to owners declared to use were very similar. Since E. calamita can Junta de Andalucı ´a research permits (AWG/MGD/MGM/CB) and reproduce in very small rain pools, reproduction sites were wide- the bioethics committee of the University of Granada (permit num- spread in both habitats. Due to warm climatic conditions in these ber 18-CEEA-OH-2013); all applicable guidelines for the care and locations, toads do not hibernate, but aestivate instead. Therefore, handling of animals were followed. the breeding season spans from January to April. Statistics Toad capture and management As data accomplished the criteria of residual homoscedasticity and During the breeding season in 2015, I captured 69 toads (15 females normality, I used parametric statistics (Quinn and Keough 2002). and 18 males from agroecosystem, 18 females and 18 males from I conducted Ordinary Least Squares Linear Models. I performed two- pine grove) by hand. Sexual dimorphism allows undoubted recogni- way ANOVAs to test the effects of habitat, sex, and their interaction tion of sexes: males have purple-to-pinkish vocal sacs in their on 1) hourly sole pad swelling, 2) maximum sole pad swelling, 3) area throats, and blackish nuptial pads in the phalanxes and tubercles of under the curve, and 4) progression of sole pad swelling (by means of a their forelimbs (Go ´ mez-Mestre 2014). During the study, toads were repeated measures ANOVA). Then, I repeated models removing non- kept in individual plastic terraria with wet peat as a substrate and an significant habitat*sex interactions. Statistical analyses were performed opaque plastic plaque as a shelter. Toad body mass was measured with software Statistica 8.0 (StatSoft, Inc., Tulsa, OK, USA). with a balance (model CDS-100, precision 0.01 g). The day after capture, during the evening in order to avoid con- Results founding effects of different moments of circadian rhythms, I meas- ured left forelimb sole pad thickness with a pressure-sensitive Hourly sole pad swelling micrometer (Mitutoyo; precision 0.01 mm), three times, and Within each hourly measurement, only at the third and the fourth I considered sole pad thickness as the average of those three measure- hour after inoculation sole pad swelling differed significantly be- ments. Immediately, I inoculated subcutaneously 0.1 mg of phyto- tween habitats and between sexes, respectively (Table 1), being hemagglutinin (PHA; Sigma Aldrich, L-8754) diluted in 0.01 ml greater in pine grove than in agroecosystem toads 3 h after inocula- isotonic phosphate buffer in each toad’s left forelimb sole pad (Brown tions (Table 1; Figure 1), as predicted, and in males than in females et al. 2011). Phytohemagglutinin is a harmless protein that triggers a four hours after inoculations (Table 1 and Figure 2), contrarily to skin-swelling immune response, in which T-cells as well as other com- expected. Note that habitat differences 2 h after inoculations were ponents of the immune system are involved (Kennedy and Nager marginally non-significant (Table 1). Results remained similar when 2006; Martin et al. 2006). The magnitude of the swelling is directly habitat*sex interactions, non-significant in all cases (Table 1), were proportional to that of the cellular immune response (Parmentier removed (data not shown). et al. 1998; Vinkler et al. 2010). Following Smits et al. (1999), I did not use a phosphate buffer saline (PBS) control in the other forelimb Maximum sole pad swelling sole pad of the same individuals. Smits et al. (1999) proved that PHA Effects of habitat (F ¼ 0.579; P ¼ 0.450), sex (F ¼ 2.726; 1, 65 1, 65 tests can be simplified by omitting PBS controls, with little impact on P ¼ 0.104), and habitat sex interaction (F ¼ 2.760; P ¼ 0.101) on 1, 65 the results, and decreasing handling errors and the coefficient of vari- maximum sole pad swelling, regardless of the moment of the experi- ation owing to measurement inaccuracy. For this reason, PBS controls ment when it was reached, were non-significant. When the interaction are commonly omitted in PHA tests (e.g., Martin 2005; Hale and was removed from the model, both the effect of habitat (F ¼ 0.454; 1, 66 Briskie 2007; Moreno-Rueda 2010; Moreno-Rueda and Redondo P ¼ 0.503) and sex (F ¼ 2.412; P ¼ 0.125) remained similar. 1, 66 2012), also in amphibians (Gervasi and Foufopoulos, 2008; Iglesias- Carrasco et al., 2017). The validity of this method has been assessed in amphibians (Brown et al. 2011; Clulow et al. 2015). Progression of sole pad swelling Habitat (F ¼ 1.10; P ¼ 0.30), sex (F ¼ 0.72; P ¼ 0.40), Repeated measures ANOVAs showed that sole pad swelling peaked 1, 65 1, 65 and their interaction (F ¼ 0.02; P ¼ 0.90) had no significant earlier in pine grove (the repeated measures ANOVA was significant 1, 65 Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy045/5034002 by guest on 13 July 2018 4 Current Zoology, 2018, Vol. 0, No. 0 Table 1. Models testing the effects of habitat, gender, and their Area under the curve interaction on sole pad swelling hourly measurements Habitat (F ¼ 0.213; P ¼ 0.646), sex (F ¼ 0.864; P ¼ 0.356), 1, 65 1, 65 and their interaction (F ¼ 0.907; P ¼ 0.344) showed no signifi- 1, 65 Hours from inoculation Habitat Gender Habitat  Gender cant effect on AUC. Effects of habitat (F ¼ 0.174; P ¼ 0.678) 1, 66 F F F 1, 65 1, 65 1, 65 and sex (F ¼ 0.785; P ¼ 0.379) remained non-significant when 1, 66 ns ns ns 1 0.469 0.759 0.856 habitat*sex interaction was removed from the model. § ns ns 2 3.225 0.028 0.518 ns ns 3 8.436** 0.251 1.568 ns ns 4 1.485 4.915* 0.843 Discussion ns ns ns 5 1.337 0.725 0.185 ns ns ns Pine grove toads deployed a more intense swelling response than 6 0.051 0.367 1.270 agroecosystem toads, but only 3 h after inoculations. At all other Significant results are highlighted in bold. Symbols indicate: ns ¼ non-signifi- times, swelling response was similar in toads from both habitats. cant; ¼ marginally non-significant; *P< 0.05; **P< 0.01. Also, I detected no habitat differences in maximum sole pad swelling nor in AUC, which suggests a swelling response of similar intensity in toads from agroecosystems and natural habitats. Likewise, pris- tine habitat hellbender Cryptobranchus alleganiensis innate immune capacity is similar to that of conspecifics from human-disturbed habitat (Hopkins and DuRant 2011). Those results suggest that, although animals in human-disturbed habitats can show impaired immune capacity (Brearley et al. 2013), compensation may occur under certain circumstances. In fact, urban tree lizards Urosaurus ornatus show reinforced innate immune cap- acity in comparison with individuals from natural sites, which could be triggered by more frequent immune challenges in a circumstance of non-limiting energy intake (French et al. 2008). Both habitats studied here provide toads with similar prey availability, which sug- gests that agroecosystem toads do not suffer reduced energy intake compared to pine grove toads (Zamora-Camacho and Comas 2017). Therefore, no reduction in energy intake could allow agro- ecosystem toads to compensate stressful conditions and elicit a swel- Figure 1. Sole pad swelling peaked earlier in pine grove than in agroecosys- ling response whose magnitude is comparable to that of pine-grove tem toads. Note that habitat differences in maximum sole pad swelling were toads. non-significant. Sample sizes were 33 toads from agroecosystem and 36 However, habitat differences in toad swelling response are more toads from pine grove. Vertical bars represent standard errors. accurately defined in a broader vision of short-term swelling response progression. As a whole, swelling response peaked earlier in pine-grove than in agroecosystem toads. Even though maximum pad swelling magnitude is similar in toads from both habitats, a faster swelling response could reflect a healthier immune system in pine grove toads, and hinder pathogen proliferation more efficiently (Rogers et al. 2000). In fact, agroecosystem conditions are known to reduce amphibian immune capacity (Christin et al. 2003, 2004) and increase infection prevalence as a consequence (Linzey et al. 2003; Rohr et al. 2008). Hence, the finding that agroecosystem stressful conditions impair amphibian immune capacity is not surprising. Nonetheless, results herein are novel to put forward that such impairment does not necessarily represent a reduced magnitude of the response, but may imply a delay of it instead. A delayed response as a consequence of physiological stress triggered by agroecosystem conditions could weaken toad protection against disease, and could thus be at least one of the causes behind agroecosystem toad shorter lifespan found in this system, as compared with pine grove toads Figure 2. Sole pad swelling peaked earlier in female than in male toads. (Zamora-Camacho and Comas 2017). However, gender differences were not significant 3 h after inoculations, while As an alternative explanation, although prey availability does males showed greater immune response one hour later, suggesting more sustained immune response in males. Note that gender differences in max- not differ between both habitats (Zamora-Camacho and Comas imum sole pad swelling were non-significant. Sample sizes were 33 females 2017), energy budgets that toads allocate to different life-history and 36 males. Vertical bars represent standard errors. traits could (Stearns 2000). Actually, agroecosystem toads seem to prioritize energy allocation to reproduction rather than to somatic for habitat: F ¼ 5.464; P< 0.001; Figure 1)and in female preservation, as both male and female agroecosystem toads in this 5, 325 toads (the repeated measures ANOVA was significant for sex: study system show greater indicators of reproductive investment, F ¼ 2.533; P< 0.029; Figure 2). Repeated measures ANOVA was but shorter lifespans, than pine grove conspecifics (Zamora- 5, 325 not significant for habitat*sex interaction (F ¼ 0.599; P< 0.701). Camacho and Comas 2017). Increased extrinsic mortality by 5, 325 Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy045/5034002 by guest on 13 July 2018 Zamora-Camacho  Time progression of immune response 5 agroecosystem stressful conditions (see the introduction) could be 2000). Generalization of immunocompetence handicap hypothesis reducing agroecosystem toad lifespan. Under those circumstances, is indeed controversial (reviewed in Roberts et al. 2004). However, short-lived agroecosystem toads may divert energy allocation to in this case, the physiological mechanisms underlying male toad reproduction to the detriment of somatic preservation—which is capacity to deploy a more sustained swelling response than females indeed extrinsically constrained—thus compensating lesser repro- remain obscure. ductive events due to reduced lifespan with a higher reproductive Results herein confirm impaired swelling response in agroecosys- investment in each, according to life-history theory (Cox et al. tem toads, which is not surprising, as a number of studies on several 2010). Consequently, reduced energy budget for somatic preserva- taxa have reported similar negative effects on amphibian immune tion, and thus for immune system, could result in a delayed response capacity (Christin et al. 2004; Albert et al. 2007). However, this in agroecosystem toads. Similarly, trade-offs between immune study is novel to find evidence that such impairment does not neces- response and reproduction have indeed been found in a number of sarily involve a reduced magnitude of swelling response: in this case, taxa (Adamo et al. 2001; Ardia 2005; French et al. 2007). it appears as a delay in swelling response. As for sex, swelling In any case, the present results suggest that agroecosystem response magnitude did not differ between males and females. stressful conditions delay toad swelling response, which may impair Female swelling response peaked earlier, but that of males was more protection against pathogens, compared with natural pine grove sustained in time, which does not match previous across-taxa find- toads. In the light of these results, immune response impairment ings that male swelling response is impaired as compared to that of stands among the negative consequences of such conditions on females. amphibian health and populations (Alford et al. 2001; Hegde and In conclusion, most interestingly, these results show that measur- Krishnamurthy 2014), which contributes to define the role of agroe- ing swelling response only after a standardized period following cosystems on amphibian population declines. Actually, amphibian stimulation (Martin et al. 2006) might be simplistically assuming syn- populations are in particular decline in areas that have had pesti- chronal swelling response progression in all individuals. cides applied historically (Davidson 2004). Agriculture land area is Consequently, results might be biased, or important and useful predicted to increase in the near future (Zabel et al. 2014), meaning information could be lost. In fact, results in this work and their that an increasing number of amphibian populations are bound to interpretation would have been completely different if I had measured undergo agroecosystem conditions. Therefore, measures should be swelling response only once, and the moment chosen for the measure taken to milden such conditions, such as limitations in agrochemical would have caused dramatic differences. For instance, swelling re- use (Brodeur et al. 2014) or increased connectivity between amphib- sponse 3 h after inoculations was greater in pine grove than in agro- ian habitats (Naito et al. 2012). ecosystem toads, while it did not differ between sexes at that According to the immunocompetence handicap hypothesis, tes- moment. However, the outlook was completely different 4 h after tosterone exerts suppressive effects on the immune system (Hillgarth inoculations, with no habitat differences in swelling response, but and Wingfield 1997), which results in an across-taxa trend of males males showing greater swelling than females. Finally, if I had made to elicit less intense immune responses than females (Klein 2000). measures only at any other moment, I would have detected no effect Nevertheless, unexpectedly, the only moment when sex differences of habitat or sex. Nevertheless, a broader view including AUC and in swelling response were significant (4 h after inoculations), males maximum magnitude of swelling response, regardless of the moment deployed greater swelling response than females. However, at all when it was deployed, as well as progression of swelling response, other times, sex showed no effect on swelling response. provided an accurate panorama of the actual effects of habitat and Remarkably, maximum sole pad swelling, irrespective of the sex on short-term toad swelling response. Therefore, I urge ecoimmu- moment when it was reached, did not differ significantly between nologists to implement immune response progression in their studies, sexes. Similarly, lack of sex differences in AUC suggests similar or, at least, to assess its synchrony among the groups they are study- intensity of swelling response in males and females. Therefore, hour- ing, in order to obtain realistic, unbiased results in a broader context. ly tests of sex effect on toad swelling response do not seem to sup- port testosterone-induced immunosuppression. Notably, a broader vision of the short-term progression of swel- Acknowledgments ling response showed a significantly earlier swelling response peak The author would like to thank the landowners who provided information on in females than in males. However, sex differences 3 h after inocula- the agrochemicals they use. Daniel Pinto-Navarro gave logistic support. tions, when such female peak took place, were not significant, while Comments by Gregorio Moreno-Rueda and three anonymous reviewers 4 h after inoculations females had a significantly reduced swelling improved the article. This research did not receive any specific grant from response as compared with males. Such progression pattern suggests funding agencies in the public, commercial, or not-for-profit sectors. that male swelling response was not actually slower, but more I assumed the expenses of the work. sustained in time, which should not represent a lower energy invest- ment (Fox et al. 2005), and thus did not reflect impaired male immune response. As a whole, these results did not support male Funding reduced immune capacity (Belliure et al. 2004). Similarly, response This article was partly supported by a Ramo ´ n Areces Foundation postdoctor- to PHA by testosterone-supplemented male European tree frog al fellowship, and a Juan de la Cierva-Formacio ´ n postdoctoral grant from the (Hyla arborea) was not reduced, but enhanced (Desprat et al. 2015). Spanish Ministerio de Economı´a, Industria y Competitividad. 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