Insularization drives physiological condition of Amazonian dung beetles.

The fragmentation and degradation of otherwise continuous natural landscapes pose serious threats to the health of animal populations, consequently impairing their fitness and survival. While most fragmentation ecology studies focus on habitat remnants embedded withinn terrestrial matrices, the effects of true insularization remains poorly understood. Land-bridge islands created by major dams leads to habitat loss and fragmentation, negatively affecting terrestrial biodiversity. To assess the effects of insularization, we conducted a study on the key aspects of dung beetle physiological condition and body size throughout the Balbina Hydroelectric Reservoir located in the Central Amazon. We assessed these traits at the population and assemblage levels, collecting dung beetles from both forest islands and continuous forest areas while analyzing various landscape variables. We show that landscapes with higher forest cover positively affected dung beetle body size. Interestingly, dung beetle responses to insularization were species-dependent; larger islands tended to host larger individuals of Deltochilum aspericole, while in Canthon triangularis, smaller islands showed larger body sizes. However, individuals from the mainland were larger than those from the islands. Moreover, the proportion of closed-canopy forest in the landscapes also impacted physiological attributes. It negatively affected the body size of Deltochilum aspericole and the lipid mass of Dichotomius boreus, but positively affected the lipid mass of Canthon triangularis. These findings contribute to a better understanding of how habitat fragmentation in aquatic matrices affects the size structure and physiology of insect assemblages. This is essential in formulating effective conservation strategies for preserving biodiversity loss in tropical forest regions and mitigating the consequences of hydropower infrastructure.

Experimental Crosses Between Two Dung Beetle Lineages Show Transgressive Segregation in Physiological Traits.

Physiological traits in insects are intrinsically related to their behavior, fitness, and survival and can reflect adaptations to ecological stressors in different environments, leading to population differentiation that may cause hybrid failure. In this study, we characterized five physiological traits related to body condition (body size, body mass, amount of fat, total hemolymph protein, and phenoloxidase activity) in two geographically separated and recently differentiated lineages of Canthon cyanellus LeConte, 1859 within their natural distribution in Mexico. We also performed experimental hybrid crosses between these lineages to better understand the differentiation process and explore the presence of transgressive segregation over physiological traits in them. We found differences between lineages in all traits except body mass, suggesting selective pressures related to different ecological pressures. These differences were also apparent in the transgressive segregation of all traits in F1 and F2 hybrids, except for phenoloxidase activity. Protein content was sexually dimorphic in both parental lineages but was reversed in hybrids, suggesting a genetic basis for the differences between sexes. The negative sign of transgressive segregation for most traits indicates that hybrids would be smaller, thinner, and generally unfit. Our results suggest that these two lineages may undergo postzygotic reproductive isolation, confirming the cryptic diversity of this species complex.

Contamination effects on sexual selection in wild dung beetles.

Sexual selection influences the expression of secondary sexual traits, which are costly to produce and maintain and are thus considered honest indicators of individual condition. Therefore, sexual selection could select for high-quality individuals able to respond to stressful conditions, with impacts on population-level fitness. We sampled dung beetles from 19 pastures and investigated if contamination by herbicides and veterinary drugs modifies male investment in sexually selected traits and has associated population-level effects. We measured horn size, condition dependence (i.e. size-corrected body mass) and allometry, besides abundance and sexual size dimorphism in three species: Copris incertus, Euoniticellus intermedius and Digitonthophagus gazella. In contrary to our expectations, horn size was independent of contamination and individual condition. However, strong positive allometric relationships were reduced by herbicide contamination for C. incertus and D. gazella and were increased by ivermectin for C. incertus, revealing differential investment in horn production according to body size in contaminated habitats. At the population level, large-horned C. incertus males were more abundant in contaminated pastures, potentially revealing a case of evolutionary rescue by sexual selection or a plastic response to higher population densities. Finally, chemical compounds affected the sexual size dimorphism of all three species, with potential effects on female fecundity or intrasexual selection. Together, our findings indicate that contamination interferes with sexual selection processes in the wild, opening new questions regarding the role of sexual selection in favouring species persistence in contaminated environments.

Insect thermal limits in warm and perturbed habitats: Dragonflies and damselflies as study cases.

Disturbance (e.g. loss of plant cover) increases ambient temperature which can be lethal for ectotherm insects especially in hot places. We compared the thorax temperatures of 26 odonate species as a function of body size, habitat quality ("conserved" and cooler vs "perturbed" and warmer) and suborder (Anisoptera vs Zygoptera), as well as critical thermal maximum (CTmax) and as a function of habitat quality in Argia pulla (Zygoptera) and Orthemis ferruginea (Anisoptera). We expected thorax temperatures to differ between suborders based on their differences in body size and habitat quality status, and that populations in perturbed sites would have higher critical thermal maxima compared to those in conserved sites. This study was done in a tropical region with high ambient temperatures. Anisopterans had a higher body temperature than zygopterans, with no difference between habitats. Thoracic and air temperature were positively related, yet body temperatures were higher than the ambient temperature. A. pulla had higher CTmax in the perturbed sites, while O. ferruginea showed the opposite trend. Microenvironmental changes increase the ambient temperature, perhaps filtering insect species. The apparent resilience of odonates to disturbance should be examined more closely (using more species), especially in small species like the zygopterans which appear to be more strongly affected by ambient temperature.

Heritable responses to combined effects of heat stress and ivermectin in the yellow dung fly.

In current times of global change, several sources of stress such as contaminants and high temperatures may act synergistically. The extent to which organisms persist in stressful conditions will depend on the fitness consequences of multiple simultaneously acting stressors and the genetic basis of compensatory genetic responses. Ivermectin is an antiparasitic drug used in livestock that is excreted in dung of treated cattle, causing severe negative consequences on non-target fauna. We evaluated the effect of a combination of heat stress and exposure to ivermectin in the yellow dung fly, Scathophaga stercoraria (Diptera: Scathophagidae). In a first experiment we investigated the effects of high rearing temperature on susceptibility to ivermectin, and in a second experiment we assayed flies from a latitudinal gradient to assess potential effects of local thermal adaptation on ivermectin sensitivity. The combination of heat and ivermectin synergistically reduced offspring survival, revealing severe effects of the two stressors when combined. However, latitudinal populations did not systematically vary in how ivermectin affected offspring survival, body size, development time, cold and heat tolerance. We also found very low narrow-sense heritability of ivermectin sensitivity, suggesting evolutionary constraints for responses to the combination of these stressors beyond immediate maternal or plastic effects. If the revealed patterns hold also for other invertebrates, the combination of increasing climate warming and ivermectin stress may thus have severe consequences for biodiversity. More generally, our study underlines the need for quantitative genetic analyses in understanding wildlife responses to interacting stressors that act synergistically and threat biodiversity.

Heat shock proteins and antioxidants as mechanisms of response to ivermectin in the dung beetle Euoniticellus intermedius.

Ivermectin is the most common antiparasitic drug used in livestock in many regions of the world. Its residues are excreted in dung, threatening non-target fauna such as dung beetles, fundamental for cleaning dung in pastures. However, it is unclear which are the physiological mechanisms used by dung beetles to cope with ivermectin. Here we evaluated experimentally the physiological responses of the dung beetle Euoniticellus intermedius to ivermectin-induced stress. We measured metabolic rates, heat shock protein 70 (Hsp70) expression, antioxidant capacity, and oxidative damage in lipids in both males and females exposed to a sublethal dose. Compared to control beetles, ivermectin-treated males and females had increased metabolic rates. Moreover, ivermectin-treated females increased their expression of Hsp70 whereas males increased their antioxidant capacity. No changes in the levels of oxidative damage to lipids were detected for either sex, suggesting a process of hormesis, such that exposure to a moderate concentration of ivermectin could stimulate the action of a protective mechanism against oxidative stress, that differs between sexes. However, it does not exclude the possibility that damage to other biomolecules might have occurred. Sexual differences in physiological responses can be interpreted as the result of hormonal differences or life-history trade-offs that favor different mechanisms in females and males. Hsps and antioxidants are involved in the physiological response of beetles to ivermectin and may be key in providing resistance to this contaminant in target and non-target species, including dung beetles.

Effect of Chemical Pollution and Parasitism on Heat Tolerance in Dung Beetles (Coleoptera: Scarabaeinae).

Ecosystem services provided by insects are threatened by recent increasing global temperatures, particularly in the tropics, where insects live close to their thermal limits. Given that tolerance to high temperatures depends on individual metabolism and physiological stress response, it may also be sensitive to other stressors that are common in natural and human-modified environments, such as pollution and parasite pressure. The effects of multiple stressors could be synergistic and can be particularly relevant in insects that provide highly valuable ecosystem services, such as dung beetles in cattle pastures. Here we measured heat tolerance (critical thermal maximum, CTmax) in dung beetles exposed to ivermectin, a toxic parasiticide excreted in cattle dung, with known negative effects on coprophagous fauna, and in beetles exposed to an immune challenge. We also exposed a group of beetles to a combination of both ivermectin and immune challenge to test for potential synergistic effects of both stressors. Contrary to our predictions, CTmax did not change with ivermectin exposure, but increased in immune-challenged beetles. As found in other insects, CTmax was higher in larger beetles, highlighting the importance of body size on thermal tolerance in ectotherms. We discuss potential mechanisms responsible of increased heat tolerance in immune-challenged beetles and highlight the importance of natural and human-induced environmental pressures that now interact with global warming and threaten ecosystem services provided by wild animals.

Insect responses to heat: physiological mechanisms, evolution and ecological implications in a warming world.

Surviving changing climate conditions is particularly difficult for organisms such as insects that depend on environmental temperature to regulate their physiological functions. Insects are extremely threatened by global warming, since many do not have enough physiological tolerance even to survive continuous exposure to the current maximum temperatures experienced in their habitats. Here, we review literature on the physiological mechanisms that regulate responses to heat and provide heat tolerance in insects: (i) neuronal mechanisms to detect and respond to heat; (ii) metabolic responses to heat; (iii) thermoregulation; (iv) stress responses to tolerate heat; and (v) hormones that coordinate developmental and behavioural responses at warm temperatures. Our review shows that, apart from the stress response mediated by heat shock proteins, the physiological mechanisms of heat tolerance in insects remain poorly studied. Based on life-history theory, we discuss the costs of heat tolerance and the potential evolutionary mechanisms driving insect adaptations to high temperatures. Some insects may deal with ongoing global warming by the joint action of phenotypic plasticity and genetic adaptation. Plastic responses are limited and may not be by themselves enough to withstand ongoing warming trends. Although the evidence is still scarce and deserves further research in different insect taxa, genetic adaptation to high temperatures may result from rapid evolution. Finally, we emphasize the importance of incorporating physiological information for modelling species distributions and ecological interactions under global warming scenarios. This review identifies several open questions to improve our understanding of how insects respond physiologically to heat and the evolutionary and ecological consequences of those responses. Further lines of research are suggested at the species, order and class levels, with experimental and analytical approaches such as artificial selection, quantitative genetics and comparative analyses.

Dung Beetle Body Condition: A Tool for Disturbance Evaluation in Contaminated Pastures.

The use of veterinary medical products and herbicides is a common practice in intensified livestock systems. These compounds affect nontarget organisms that perform important ecosystem functions, such as dung beetles. The assessment of body condition allows us to determine how individuals respond to changes in the environment. However, assessments of how contamination associated with cattle farming affects coprophagous insects such as dung beetles have not been conducted in natural systems. In the present study, we evaluated the effect of ivermectin (an antiparasitic drug) and herbicides on the body condition of 3 species of dung beetles collected in the field: Copris incertus, Euoniticellus intermedius, and Digitonthophagus gazella. We recorded 3 condition indicators (body size, lipid mass, and muscle mass) of beetles collected from 19 livestock ranches in northeastern Mexico. In general, the use of ivermectin had adverse effects on C. incertus and E. intermedius whereas the effects were positive for D. gazella. Conversely, the use of herbicides had adverse effects on D. gazella and positive effects on C. incertus. The different effects of ivermectin and herbicides found in males and females show that sex can be important in determining individual responses to environmental contamination. Importantly, we provide the first evidence under natural conditions that native and exotic species of dung beetles are highly sensitive to different types of livestock management, with veterinary medications and herbicides having the ability to alter body condition. Changes in dung beetle condition can reduce the ecosystem services that dung beetles provide in livestock systems. Environ Toxicol Chem 2019;38:2392-2404. © 2019 SETAC.

Energy storage, body size and immune response of herbivore beetles at two different elevations in Costa Rica

The physiological condition and immune responses of organisms living at different elevations are expected to display local adaptations to the different climatic and biotic conditions. Small ectotherms with specialized diets are highly susceptible to environmental change, as their life cycle is largely affected by temperature and by the presence of specific resources. This study focuses on two species of rolled-leaf beetles (Chrysomelidae), both present at two different elevations in Costa Rica: Cephaloleia belti Baly, a diet-generalist, and Chelobasis bicolor Gray, a diet-specialist. Body size, energetic condition (lipid storage), muscle mass and immune condition (melanization response) were compared in beetle populations from a tropical rainforest (50 m elevation) and a tropical montane forest (2 000 m elevation). These measurements reflect current and past nutritional condition and hence are good estimators of individual fitness. We analyzed a total of 172 individuals from both species, sexes and elevations. We predicted that beetles at higher elevation would display larger body size, higher energetic condition and lower immune response associated with metabolic requirements and parasitic pressure in colder temperatures. In general, beetles at high altitude were larger, had more lipids and muscle and showed stronger melanization (i.e., immune response) than beetles at low altitudes. Such differences among populations at different elevations may reflect local adaptation to altitude or phenotypic plasticity. However, the effect of elevation was not equivalent amongst species or sex. Measuring physiological traits in organisms with broad elevational distributions could be useful in understanding possible species responses to climate change.

La condición fisiológica y la respuesta inmune de poblaciones que habitan a distintas altitudes pueden mostrar adaptación local a las condiciones abióticas y bióticas locales. Entre los organismos más susceptibles al cambio ambiental están los ectotermos pequeños con dietas especializadas, debido a que su ciclo de vida es, en gran medida, afectado tanto por la temperatura como por la presencia de recursos específicos. Este estudio se enfoca en dos especies de escarabajos (Chrysomelidae) presentes a lo largo de un gradiente altitudinal en Costa Rica: Cephaloleia belti Baly, una especie de dieta generalista, y Chelobasis bicolor Gray, de dieta especialista. Se comparó el tamaño corporal, la condición energética (almacenamiento de lípidos), la masa muscular y la condición inmunológica (respuesta de melanización) entre poblaciones de escarabajos de un bosque tropical (a 50 m de altitud) y un bosque tropical montano (a 2 000 m de altitud). Las variables seleccionadas reflejan las condiciones nutricionales actuales y pasadas y son buenos estimadores de la adecuación individual. Se analizaron un total de 172 escarabajos de ambas especies, sexos y elevaciones. Predecimos que los escarabajos a mayor elevación tendrán mayor tamaño corporal, mayor condición energética y menor respuesta inmune asociadas con los requerimientos metabólicos y la presión de los parásitos asociados con ambientes fríos. En general, los escarabajos a mayor altitud fueron más grandes, tuvieron más lípidos y masa muscular, y presentaron una mayor respuesta inmune de melanización que los escarabajos a menor altitud. Es posible que las diferencias entre las poblaciones a diferentes altitudes reflejen adaptaciones locales a las condiciones ambientales a distintas altitudes o plasticidad fenotípica. Sin embargo, el efecto de la altitud no fue de la misma magnitud entre las dos especies ni entre los dos sexos. La medición de caracteres fisiológicos en organismos ectotérmicos de amplia distribución altitudinal puede ser útil para entender las respuestas de ciertas especies al cambio climático.

Trans-generational effects of ivermectin exposure in dung beetles.

Ivermectin is a powerful antiparasitic drug commonly used in cattle. Ivermectin residues are excreted in dung, threatening non-target coprophagous fauna such as dung beetles. This can have severe ecological and economic consequences for dung degradation and soil fertility. Even though the negative effects of direct ivermectin exposure on dung-degrading organisms are well known, effects could extend across generations. Here, we tested the effects of paternal or maternal exposure to ivermectin on offspring in the dung beetle Euoniticellus intermedius. This species is a classic study subject in ecotoxicology and sexual selection because males have a cephalic horn that is under intense selection via male-male competition. After confirming a negative effect of ivermectin on the number of emerged beetles, we found trans-generational effects of ivermectin exposure on the horn size of male offspring. Surprisingly however, this trans-generational effect only occurred when only the father was exposed. We detected no trans-generational effects of ivermectin exposure on offspring number, sex ratio or body size. Our results confirm that ivermectin not only has a strong effect on exposed individuals but also in their progeny. Our study opens new questions about the mechanisms responsible for parental effects and their long-term fitness consequences in contaminated habitats.

Ivermectin alters reproductive success, body condition and sexual trait expression in dung beetles.

Ivermectin is a very common parasiticide used in livestock. It is excreted in the dung and has negative effects on survival and reproduction of dung-degrading organisms, including dung beetles. Here we exposed the dung beetle Euoniticellus intermedius to different concentrations of ivermectin in the food and evaluated reproductive success and the expression of traits associated with survival and reproduction under laboratory conditions. It is the first time the effects of ivermectin were evaluated on offspring physiological condition and the expression of a secondary sexual trait. We also registered the number of emerged beetles, sex ratio and body size of emerged adult beetles. Besides reducing the number of emerged beetles and body size, as found in the same and other insects, ivermectin at high doses reduced muscle mass while at intermediate doses it increased lipid mass. Ivermectin changed offspring sex ratio and at high doses increased the size of male horn, which is an important trait defining the male mating success. Our results highlight the importance of regulating parasiticide usage in livestock in order to maintain ecosystem services provided by dung beetles and confirm that contaminants impose new environmental conditions that not only impact on wild animal survival, but also on evolutionary processes such as sexual selection.

The combination of the functionalities of feedback circuits is determinant for the attractors' number and size in pathway-like Boolean networks.

Molecular regulation was initially assumed to follow both a unidirectional and a hierarchical organization forming pathways. Regulatory processes, however, form highly interlinked networks with non-hierarchical and non-unidirectional structures that contain statistically overrepresented circuits or motifs. Here, we analyze the behavior of pathways containing non-unidirectional (i.e. bidirectional) and non-hierarchical interactions that create motifs. In comparison with unidirectional and hierarchical pathways, our pathways have a high diversity of behaviors, characterized by the size and number of attractors. Motifs have been studied individually showing that feedback circuit motifs regulate the number and size of attractors. It is less clear what happens in molecular networks that usually contain multiple feedbacks. Here, we find that the way feedback circuits couple to each other (i.e., the combination of the functionalities of feedback circuits) regulate both the number and size of the attractors. We show that the different expected results of epistasis analysis (a method to infer regulatory interactions) are produced by many non-hierarchical and non-unidirectional structures. Thus, these structures cannot be correctly inferred by epistasis analysis. Finally, we show that the combinations of functionalities, combined with other network properties, allow for a better characterization of regulatory structures.

Effects of an herbicide on physiology, morphology, and fitness of the dung beetle Euoniticellus intermedius (Coleoptera: Scarabaeidae).

Some agrochemical compounds threaten nontarget organisms and their functions in the ecosystem. The authors experimentally evaluated the effects of one of the most common herbicide mixtures used worldwide, containing 2,4-dichlorophenoxyacetic acid and picloram, on dung beetles, which play fundamental roles in the function of natural and managed ecosystems. The present study employed techniques of physiology and geometric morphometrics, besides including fitness measurements, to assess the effects of the herbicide in the introduced beetle Euoniticellus intermedius. Because herbicide components promote oxidative stress and affect survival in certain insects, the authors predicted negative effects on the beetles. Unexpectedly, no effect of herbicide concentration was found on clutch size, sex ratio, and fluctuating asymmetry, and it even increased physiological condition and body size in exposed beetles. Because the studied species presents 2 male morphs, the authors, for the first time, evaluated the effect of a pollutant on the ratio of these morphs. Contrary to the prediction, the herbicide mixture increased the proportion of major males. Thus, the herbicide does not threaten populations of the studied beetles. The present study discusses how both negative and positive effects of pollutants on wild animals modify natural and sexual selection processes occurring in nature, which ultimately impact population dynamics. The authors recommend the use of physiological and geometric morphometrics techniques to assess the impact of pollutants on nontarget animals. Environ Toxicol Chem 2017;36:96-102. © 2016 SETAC.

Effect of Ecological Restoration on Body Condition of a Predator.

Ecological restoration attempts to recover the structure and function of ecosystems that have been degraded by human activities. A crucial test of ecosystem recovery would be to determine whether individuals in restored environments are as healthy as those in conserved environments. However, the impact of restoration on physiology of terrestrial animals has never been tested. Here, we evaluated the effect of two restoration methods on body condition measured as body size, body mass, lipid and muscle content of the spider Nephila clavipes in a tropical dry forest that has suffered chronic disturbance due to cattle grazing. We used experimental plots that had been excluded from disturbance by cattle grazing during eight years. Plots were either planted with native trees (i. e. maximal intervention), or only excluded from disturbance (i. e. minimal intervention), and were compared with control conserved (remnants of original forest) and disturbed plots (where cattle is allowed to graze). We predicted (1) better body condition in spiders of conserved and restored sites, compared to disturbed sites, and (2) better body condition in plots with maximal intervention than in plots with minimal intervention. The first prediction was not supported in males or females, and the second prediction was only supported in females: body dry mass was higher in planted than in conserved plots for spiders of both sexes and also higher that in disturbed plots for males, suggesting that plantings are providing more resources. We discuss how different life histories and environmental pressures, such as food availability, parasitism, and competition for resources can explain our contrasting findings in male and female spiders. By studying animal physiology in restoration experiments it is possible to understand the mechanistic basis of ecological and evolutionary processes that determine success of ecological restoration.

Cuticular antifungals in spiders: density- and condition dependence.

Animals living in groups face a high risk of disease contagion. In many arthropod species, cuticular antimicrobials constitute the first protective barrier that prevents infections. Here we report that group-living spiders produce cuticular chemicals which inhibit fungal growth. Given that cuticular antifungals may be costly to produce, we explored whether they can be modulated according to the risk of contagion (i.e. under high densities). For this purpose, we quantified cuticular antifungal activity in the subsocial crab spider Diaea ergandros in both natural nests and experimentally manipulated nests of varying density. We quantified the body-condition of spiders to test whether antifungal activity is condition dependent, as well as the effect of spider density on body-condition. We predicted cuticular antifungal activity to increase and body-condition to decrease with high spider densities, and that antifungal activity would be inversely related to body-condition. Contrary to our predictions, antifungal activity was neither density- nor condition-dependent. However, body-condition decreased with density in natural nests, but increased in experimental nests. We suggest that pathogen pressure is so important in nature that it maintains high levels of cuticular antifungal activity in spiders, impacting negatively on individual energetic condition. Future studies should identify the chemical structure of the isolated antifungal compounds in order to understand the physiological basis of a trade-off between disease prevention and energetic condition caused by group living, and its consequences in the evolution of sociality in spiders.

A mismatch between the perceived fighting signal and fighting ability reveals survival and physiological costs for bearers.

Signals of fighting indicate an animal's intention to attack and so they serve to prevent costly aggressive encounters. However, according to theory, a signal that is different in design (i.e. a novel signal) but that fails to inform fighting intentions will result in negative fitness consequences for the bearer. In the present study we used males of the territorial damselfly Hetaerina americana, which have a red wing spot during territory defense that has evolved as a signal of fighting ability. By producing a novel signal (covering the red spot with blue ink) in territory owners, we investigated: a) the behavioral responses by conspecific males; b) survival cost and c) three physiological mediators of impaired survival: muscular fat reserves, muscle mass and immune ability. We predicted that males with the novel signal would be attacked more often by conspecifics as the former would fail to convey fighting ability and intentions adequately. This will result in lower survival and physiological condition for the novel signal bearers. We found that, compared to control males (males whose red spot was not changed), experimental males had reduced survival, were less able to hold a territory, and had a reduced muscle mass. It seems that spot modified males were not able to effectively communicate their territory tenancy, which may explain why they lost their defended sites. Our results provide support for theoretical models that a novel signal that fails to informing fighting ability may lead to a fitness cost for bearers.

Support for the immunocompetence handicap hypothesis in the wild: hormonal manipulation decreases survival in sick damselflies.

The immunocompetence handicap hypothesis (ICHH) states that hormones enhance sexual trait expression but impair immunity. Previous tests of the ICHH have been hampered by experimental design problems. Here, we report on an experimental test of the ICHH that includes manipulations of both hormones and infections in males of the territorial damselfly, Hetaerina americana, with accurate survival measurements. We conducted a fully factorial experiment subjecting each individual to one of three topical treatments: methoprene (a juvenile hormone analog), acetone, or control, and one of three injection treatments: bacteria, PBS, or control. We measured survival of manipulated males in both the wild and in captivity. As predicted, survival was most heavily impaired in methoprene-bacteria males than in the other groups in the wild, and no survival differences emerged in captive animals. This result confirms that survival is one cost an animal pays for increased hormonal levels. This corroborates theoretical predictions of the ICHH.