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1. The authors investigated the role of thermoregulation mechanisms and thermal limits on thermal niche differentiation in two hierarchical similar Eucraniini dung beetle assemblages inhabiting arid environments in north-western Argentina. Both dung beetle assemblages showed temporal and thermal niche segregation between all sympatric species, minimising overlap during their daily activity and soil temperature preferences. 2. Thermal tolerance of Eucraniini species was partially modulated by behavioural adaptations and complementary thermoregulatory mechanisms related to eliminating the temperature excess, increasing the metabolic rate promoting an effective regulation of the excess heat that permitted a decrease in their body temperature by evaporative cooling. Based on the physiological syndromes obtained, the authors found a greatest interspecific differentiation that allows us to differentiate each species using ecophysiological traits. Interspecific body size differences in sympatric Eucraniini contributed greatly to the metabolic capacity of individuals to thermoregulate. 3. Eucranium species, the largest species showed the lowest capacity to respond to thermal stress decreasing heat excess temperature, presenting the lowest thermal limit values coinciding with a ‘minithermy’ strategy that explains the preferred lower soil temperatures around 30°C. On the contrary, Anomiopsoides species showed a clear adaptation to being active during periods of the day in which the soil temperature reached over 50°C, adopting a strategy of ‘maxithermy’ by foraging when temperatures are closer to their thermal limits. 4. In conclusion, it can be determined that ecophysiological syndromes related to thermal stress regulation and thermal limits allowed a displacement in thermal niche dimension, minimising interspecific competition and so allowing species coexistence in Eucraniini dung beetles.

Ball-roller dung beetles of the subfamily Scarabaeinae exhibit two nesting patterns ranging from simple nests (nesting pattern IV) to highly complex nests with parental care (nesting pattern V). Most species of the genus Canthon build a simple nest formed by a single brood ball that is abandoned after oviposition. We studied the nesting behaviour and brood ball architecture of two dung beetle species from South America. Canthon unicolor (Blanchard, 1846) and C. histrio (Le Peletier de Saint-Fargeau & Audinet-and Serville, 1828) construct a shallow nest containing a single brood ball. This brood ball is unique in that it is covered with a thick shield layer made of soil. The shield layer provides to the final brood ball an amorphous external aspect, never described in roller dung beetles. It is less dense than the brood ball layer, and provides, an additional post-ovipositional care. Our results suggest that the nesting behaviour observed in C. unicolor and C. histrio can be considered a new variation of the nesting pattern IV in dung beetles, in which post-ovipositional care through the outer shield layer could increase survival chance of the offspring by protecting them from potential predators, desiccation and a variety of other environmental hazards.

We described, for the first time, a case of predation of a non-arthropod species by a dung beetle species. Canthon chalybaeus Blanchard, 1843 kills healthy individuals of the terrestrial snail Bulimulus apodemetes (D’Orbigny, 1835) showing an evident pattern of physical aggressiveness in the attacks using the dentate clypeus and the anterior tibiae. The description of this predatory behaviour was complemented with the analysis of the chemical secretions of the pygidial glands of C. chalybaeus, highlighting those main chemical compounds that, due to their potential toxicity, could contribute to death of the snail. We observed a high frequency of predatory interactions reinforcing the idea that predation in dung beetles is not accidental and although it is opportunistic it involves a series of behavioural sophistications that suggest an evolutionary pattern within Deltochilini that should not only be better studied from a behavioural point of view but also phylogenetically.

A terrestrial test system to investigate the biomagnification potential and tissue-specific distribution of ivermectin, a widely used parasiticide, in the non-target dung beetle Thorectes lusitanicus (Jekel) was developed and validated. Biomagnification kinetics of ivermectin in T. lusitanicus was investigated by following uptake, elimination, and distribution of the compound in dung beetles feeding on contaminated faeces. Results showed that ivermectin was biomagnified in adults of T. lusitanicus when exposed to non-lethal doses via food uptake. Ivermectin was quickly transferred from the gut to the haemolymph, generating a biomagnification factor (BMFk) three times higher in the haemolymph than in the gut after an uptake period of 12 days. The fat body appeared to exert a major role on the biomagnification of ivermectin in the insect body, showing a BMFk 1.6 times higher than in the haemolymph. The results of this study highlight that the biomagnification of ivermectin should be investigated from a global dung-based food web perspective and that the use of these antiparasitic substances should be monitored and controlled on a precautionary basis. Thus, we suggest that an additional effort be made in the development of standardised regulatory recommendations to guide biomagnification studies in terrestrial organisms, but also that it is necessary to adapt existing methods to assess the effects of such veterinary medical products.

1. Greenhouse gas (GHG) emissions from livestock contribute significantly to global warming, and a reduction of this source of emissions is crucial in achieving the goal of mitigating global warming. 2. CO2 and CH4 emissions from dung pats were analysed by means of a mesocosm experiment in a Mediterranean ecosystem. The experiment consisted of a total of 30 mesocosms distributed across three treatments: a well‐preserved, undisturbed dung beetle assemblage associated with organic livestock; a dung beetle assemblage that was impoverished as a result of the long‐term use of veterinary medical products; and a control treatment without dung beetles. 3. Corrections related to insect respiration allow researchers to provide more precise measurements of CO2 emissions from dung, especially in the initial and final phases of dung exposure, when the percentage of CO2 emitted by dung beetles can become greater than the emissions from the dung pats themselves. 4. The effects of dung beetles on CO2 and CH4 emissions are much more accentuated in warm‐temperate conditions than in northern temperate areas previously studied. Mediterranean assemblages remove and spread dung faster and more effectively than do northern dung beetle assemblages characterised by a lower functional richness and beetle abundance and biomass. 5. From a livestock management viewpoint, mesocosms representing areas with impoverished dung beetle assemblages, due to the long‐term use of veterinary medical products, such as ivermectin, emitted 1.6‐ and 2.8‐fold higher total CO2 and CH4, respectively, than mesocosms mimicking sites with untreated livestock.

A terrestrial test system to investigate the biomagnification potential and tissue-specific distribution of ivermectin, a widely used parasiticide, in the non-target dung beetle Thorectes lusitanicus (Jekel) was developed and validated. Biomagnification kinetics of ivermectin in T. lusitanicus was investigated by following uptake, elimination, and distribution of the compound in dung beetles feeding on contaminated faeces. Results showed that ivermectin was biomagnified in adults of T. lusitanicus when exposed to non-lethal doses via food uptake. Ivermectin was quickly transferred from the gut to the haemolymph, generating a biomagnification factor (BMF) three times higher in the haemolymph than in the gut after an uptake period of 12 days. The fat body appeared to exert a major role on the biomagnification of ivermectin in the insect body, showing a BMF 1.6 times higher than in the haemolymph. The results of this study highlight that the biomagnification of ivermectin should be investigated from a global dung-based food web perspective and that the use of these antiparasitic substances should be monitored and controlled on a precautionary basis. Thus, we suggest that an additional effort be made in the development of standardised regulatory recommendations to guide biomagnification studies in terrestrial organisms, but also that it is necessary to adapt existing methods to assess the effects of such veterinary medical products.

In heterotherm insects, endothermy implies a high energy cost due to the generation and regulation of body temperature during different activities such as flight, food location, fighting and even walking. We studied the thermoregulation process and the cost of the thermoregulation strategies in two sympatric dung beetles, Sulcophanaeus batesi and S. imperator under heat and cold stress conditions. We used a set of physiological variables to exemplify the capacity of thermolimit respirometry combined with infrared thermography to derive relevant variables capable of describing different thermoregulation syndromes. Habitat use and thermal niche differed notably between S. batesi and S. imperator, reflecting their contrasted thermal requirements. In S. imperator, thermal specialization for high temperatures was observed, being active mainly during the warmer period of the day. On the other hand, thermal adaptation in S. batesi allows its preference for cold exhibiting a morning activity periods, avoiding higher temperatures. The thermophilic strategy used by Sulcophanaeus imperator minimized the energy expenditure produced during the cooling of the body by respiration without thereby endangering higher thermal limits. In this case, S. batesi, the species with a preference for the coldest environments, presented the lowest thermal limits, although the energy cost needed to stay active during cooling was significantly lower than that in S. imperator. Sulcophanaeus imperator and S. batesi showed evident ‘economizing’ strategies associated with hot and cold environmental conditions, respectively. In contrast, if both species experience a deviation from their thermal optimum, a decrement in their performance could be produced.

Correction to: Scientific Reports https://doi.org/10.1038/s41598-018-33241-0, published online 05 October 2018

1. Greenhouse gas (GHG) emissions from livestock contribute significantly to global warming, and a reduction of this source of emissions is crucial in achieving the goal of mitigating global warming. 2. CO and CH emissions from dung pats were analysed by means of a mesocosm experiment in a Mediterranean ecosystem. The experiment consisted of a total of 30 mesocosms distributed across three treatments: a well-preserved, undisturbed dung beetle assemblage associated with organic livestock; a dung beetle assemblage that was impoverished as a result of the long-term use of veterinary medical products; and a control treatment without dung beetles. 3. Corrections related to insect respiration allow researchers to provide more precise measurements of CO emissions from dung, especially in the initial and final phases of dung exposure, when the percentage of CO emitted by dung beetles can become greater than the emissions from the dung pats themselves. 4. The effects of dung beetles on CO and CH emissions are much more accentuated in warm-temperate conditions than in northern temperate areas previously studied. Mediterranean assemblages remove and spread dung faster and more effectively than do northern dung beetle assemblages characterised by a lower functional richness and beetle abundance and biomass. 5. From a livestock management viewpoint, mesocosms representing areas with impoverished dung beetle assemblages, due to the long-term use of veterinary medical products, such as ivermectin, emitted 1.6- and 2.8-fold higher total CO and CH, respectively, than mesocosms mimicking sites with untreated livestock.

Among macrocyclic lactones (ML), ivermectin (IVM) and moxidectin (MOX) potentially affect all Ecdysozoan species, with dung beetles being particularly sensitive. The comparative effects of IVM and MOX on adult dung beetles were assessed for the first time to determine both the physiological sub-lethal symptoms and pre-lethal consequences. Inhibition of antennal response and ataxia were tested as two intuitive and ecologically relevant parameters by obtaining the lowest observed effect concentration (LOEC) values and interpolating other relevant toxicity thresholds derived from concentration-response curves (IC50, as the concentration of each ML where the antennal response is inhibited by half; and pLC50, as the quantity of ingested ML where partial paralysis was observed by half of treated individuals) from concentration-response curves. Both sub-lethal and pre-lethal symptoms obtained in this study coincided in that IVM was six times more toxic than MOX for adult dung beetles. Values of LOEC, IC50 and pLC50 obtained for IVM and MOX evaluated in an environmental context indicate that MOX, despite needing more time for tis elimination in the faeces, would be twice as harmful to dung beetles as IVM. This approach will be valuable to clarify the real impact of MLs on dung beetle health and to avoid the subsequent environmental consequences.

Ivermectin is the most common endectocide used to control parasites affecting livestock. Short-term physiological and behavioural effects of ivermectin on dung beetles may have long-term consequences for beetle populations and ecosystem functioning. Long-term effects of the use of ivermectin can be estimated by comparing dung assemblages and ecosystem functions in areas with conventional ivermectin-treated livestock and environmentally similar areas in which livestock are not treated with veterinary medical products (organic farming). In this study, we investigated both short-term and long-term effects of the administration of ivermectin on the characteristics of dung beetle assemblages and the services they provided in a protected area (Doñana National Park, SW Spain). We examined short-term dung colonization, dwelling, relocation, and disaggregation rates and the associations between these processes and the key assemblage parameters of species richness, abundance, biomass and functional diversity. Furthermore, we analysed changes in soil physical-chemical properties and processes. Short-term differences were observed in the total amount of dung relocated by dung beetles at different colonization vs. emigration stages, suggesting that dung beetles in this area were affected by the recent treatments of livestock with ivermectin. Moreover, short-term effects could also be responsible for the significant differences in dung spreading rates between sites. Conventional use of ivermectin disrupted ecosystem functioning by affecting species richness, abundance and biomass. The decrease in diversity parameters was related to a reduction in the functional efficiency, which resulted in the long-term accumulation of dung on the ground and considerable changes in soil functionality.

Among macrocyclic lactones (ML), ivermectin (IVM) and moxidectin (MOX) potentially affect all Ecdysozoan species, with dung beetles being particularly sensitive. The comparative effects of IVM and MOX on adult dung beetles were assessed for the first time to determine both the physiological sub-lethal symptoms and pre-lethal consequences. Inhibition of antennal response and ataxia were tested as two intuitive and ecologically relevant parameters by obtaining the lowest observed effect concentration (LOEC) values and interpolating other relevant toxicity thresholds derived from concentration-response curves (IC50, as the concentration of each ML where the antennal response is inhibited by half; and pLC50, as the quantity of ingested ML where partial paralysis was observed by half of treated individuals) from concentration-response curves. Both sub-lethal and pre-lethal symptoms obtained in this study coincided in that IVM was six times more toxic than MOX for adult dung beetles. Values of LOEC, IC50 and pLC50 obtained for IVM and MOX evaluated in an environmental context indicate that MOX, despite needing more time for its elimination in the faeces, would be half as harmful to dung beetles as IVM. This approach will be valuable to clarify the real impact of MLs on dung beetle health and to avoid the subsequent environmental consequences.

Ivermectin is the most common endectocide used to control parasites affecting livestock. Short-term physiological and behavioural effects of ivermectin on dung beetles may have long-term consequences for beetle populations and ecosystem functioning. Long-term effects of the use of ivermectin can be estimated by comparing dung assemblages and ecosystem functions in areas with conventional ivermectin-treated livestock and environmentally similar areas in which livestock are not treated with veterinary medical products (organic farming). In this study, we investigated both short-term and long-term effects of the administration of ivermectin on the characteristics of dung beetle assemblages and the services they provided in a protected area (Doñana National Park, SW Spain). We examined short-term dung colonization, dwelling, relocation, and disaggregation rates and the associations between these processes and the key assemblage parameters of species richness, abundance, biomass and functional diversity. Furthermore, we analysed changes in soil physical-chemical properties and processes. Short-term differences were observed in the total amount of dung relocated by dung beetles at different colonization vs. emigration stages, suggesting that dung beetles in this area were affected by the recent treatments of livestock with ivermectin. Moreover, short-term effects could also be responsible for the significant differences in dung spreading rates between sites. Conventional use of ivermectin disrupted ecosystem functioning by affecting species richness, abundance and biomass. The decrease in diversity parameters was related to a reduction in the functional efficiency, which resulted in the long-term accumulation of dung on the ground and considerable changes in soil functionality.

The beetle Omorgus suberosus (F.) is a facultative predator of eggs of the olive ridley turtle Lepidochelys olivacea (Eschscholtz). Laboratory and field investigations were conducted in order to characterize volatile attractants of O. suberosus and to explore the potential for application of these volatiles in a selective mass trapping method. Headspace sorptive extraction (HSSE) coupled to thermo-desorption gas chromatography–mass spectrometry (TD-GC-MS) analysis of the volatile constituents from beetles or turtle nests revealed 24 potential compounds. However, electroantennographic (EAG) measurements revealed antennal sensitivity only to indole, linoleic acid, trimethylamine, dimethyl sulphide, dimethyl disulphide and ammonia. Behavioural tests showed that these compounds are highly attractive to O. suberosus. Field trapping experiments revealed that indole and ammonia were more attractive than the other volatile compounds and showed similar attractiveness to that produced by conventional baits (chicken feathers). The use of a combined bait of indole and NH3 would therefore be the most effective trap design. The data presented are the first to demonstrate effective massive capture of O. suberosus using an attractant-based trapping method. These findings have potential for the development of an efficient mass trapping method for control of this beetle as part of efforts towards conservation of L. olivacea at La Escobilla in Oaxaca, Mexico.

A new analytical method based on solvent extraction, followed by continuous solid-phase extraction (SPE) clean-up using a polymeric sorbent, was demonstrated to be applicable for the detection of ivermectin in complex biological matrices of dung beetles (hemolymph, excreta or dry tissues) using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI+–MS/MS). Using a signal-to-noise ratio of 3:1, the limit of detection (LOD) in the insect matrices at trace levels was 0.01 ng g–1 and the limit of quantification (LOQ) was 0.1 ng g–1. The proposed method was successfully used to quantitatively determine the levels of ivermectin in the analysis of small samples in in vivo and post mortem samples, demonstrating the usefulness for quantitative analyses that are focused on future pharmacokinetic and bioavailability studies in insects and the establishment of a new protocol to study the impact of ivermectin on non-target arthropods such as dung beetles and other insects that are related with the “dung community”. Because satisfactory precision and accuracy values were obtained in both in vivo matrices, we suggest that the method can be consistently used for quantitative determinations that are focused on future pharmacokinetic and bioavailability studies in insects. Furthermore, this new analytical method was successfully applied to biological samples of dead dung beetles from the field suggesting that the method can be used to establish a new routine analysis of ivermectin residues in insect carcasses that is applied to complement typical mortality tests.

Dung roller beetles of the genus Canthon (Coleoptera: Scarabaeinae) emit an odorous secretion from a pair of pygidial glands. To investigate the chemical composition of these secretions, we used stir bar sorptive extraction (SBSE), coupled with gas chromatography–mass spectrometry (GC–MS) for analysis of extracts of pygidial gland secretions secreted by the dung roller beetles Canthon femoralis femoralis and Canthon cyanellus cyanellus. Chemical analyses of volatiles collected from pygidial gland secretions comprise a great diversity of the functional groups. Chemical profile comparisons showed high intra- and interspecific variability. The pygidial gland secretion of Canthon f. femoralis was dominated by sesquiterpene hydrocarbons, whereas the profile of Canthon c. cyanellus was dominated by carboxylic acids. The different pygidial secretions have a high diversity of chemical compounds suggesting a multifunctional nature involving some key functions in the biology. We discuss the biological potential of these compounds found in the pygidial glands of each species with respect to their ecological and behavioral relevance.

Ivermectin is a veterinary pharmaceutical generally used to control the ecto- and endoparasites of livestock, but its use has resulted in adverse effects on coprophilous insects, causing population decline and biodiversity loss. There is currently no information regarding the direct effects of ivermectin on dung beetle physiology and behaviour. Here, based on electroantennography and spontaneous muscle force tests, we show sub-lethal disorders caused by ivermectin in sensory and locomotor systems of Scarabaeus cicatricosus, a key dung beetle species in Mediterranean ecosystems. Our findings show that ivermectin decreases the olfactory and locomotor capacity of dung beetles, preventing them from performing basic biological activities. These effects are observed at concentrations lower than those usually measured in the dung of treated livestock. Taking into account that ivermectin acts on both glutamate-gated and GABA-gated chloride ion channels of nerve and muscle cells, we predict that ivermectin’s effects at the physiological level could influence many members of the dung pat community. The results indicate that the decline of dung beetle populations could be related to the harmful effects of chemical contamination in the dung.

Ivermectin is a veterinary pharmaceutical generally used to control the ecto- and endoparasites of livestock, but its use has resulted in adverse effects on coprophilous insects, causing population decline and biodiversity loss. There is currently no information regarding the direct effects of ivermectin on dung beetle physiology and behaviour. Here, based on electroantennography and spontaneous muscle force tests, we show sub-lethal disorders caused by ivermectin in sensory and locomotor systems of Scarabaeus cicatricosus, a key dung beetle species in Mediterranean ecosystems. Our findings show that ivermectin decreases the olfactory and locomotor capacity of dung beetles, preventing them from performing basic biological activities. These effects are observed at concentrations lower than those usually measured in the dung of treated livestock. Taking into account that ivermectin acts on both glutamate-gated and GABA-gated chloride ion channels of nerve and muscle cells, we predict that ivermectin € s effects at the physiological level could influence many members of the dung pat community. The results indicate that the decline of dung beetle populations could be related to the harmful effects of chemical contamination in the dung.

In some cases external morphology is not sufficient to discern between populations of a species, as occurs in the dung beetle Canthon humectus hidalgoensis Bates; and much less to determine phenotypic distances between them. FTIR-ATR spectroscopy show several advantages over other identification techniques (e.g. morphological, genetic, and cuticular hydrocarbons analysis) due to the non-invasive manner of the sample preparation, the relative speed of sample analysis and the low-cost of this technology. The infrared spectrum obtained is recognized to give a unique ‘fingerprint’ because vibrational spectra are specific and unique to the molecular nature of the sample. In our study, results showed that proteins, amino acids and aromatic ethers of insect exocuticle have promising discriminative power to discern between different populations of C. h. hidalgoensis. Furthermore, the correlation between geographic distances between populations and the chemical distances obtained by proteins + amino acids + aromatic ethers was statistically significant, showing that the spectral and spatial information available of the taxa together with appropriated chemometric methods may help to a better understanding of the identity, structure, dynamics and diversity of insect populations.

Thorectes lusitanicus, a typically coprophagous species is also actively attracted to oak acorns, consuming, burying them, and conferring ecophysiological and reproductive advantages to both the beetle and the tree. In this study, we explored the possible relation between diet shift and the health status of T. lusitanicus using a generalist entomopathogenic fungus (Metarhizium anisopliae) as a natural pathogen. To measure the health condition and immune response of beetles, we analysed the protein content in the haemolymph, prophenoloxidase (proPO) content, phenoloxidase (PO) activity and mortality of beetles with diets based on either acorns or cow dung. Protein content, proPO levels and PO levels in the haemolymph of T. lusitanicus were found to be dependent on the type of diet. Furthermore, the beetles fed with acorns developed a more effective proPO-PO system than the beetles fed with cow dung. Furthermore, a significant decrease in mortality was observed when infected individuals were submitted to an acorn-based diet. In addition to enhancing an understanding of the relevance of dietary change to the evolutionary biology of dung beetles, these results provide a more general understanding of the ecophysiological implications of differential dietary selection in the context of fitness.

Chemical ecology comprises the study of interactions of organisms with their environment that are mediated by the chemicals they produced. In general, chemicals that mediate interactions between organisms are called semiochemicals. Semiochemicals are divided into two major groups depending on whether the interactions are intraspecific (pheromones) or interspecific (allelochemics). The development of chemical ecology has been stimulated by the interest in knowing the details of the chemical interactions between organisms and the benefit of exploring new semiochemicals. In recent years, studies of chemical ecology have contributed to the conservation of biodiversity, demonstrating how valuable this can be in many ways. In the present context, chemical ecology studies open up a field of knowledge for the conservation of the olive ridley sea turtle (Lepidochelys olivacea), specie in danger of extinction. This study provides a powerful basis for the development of a new control method for reducing the impact of the beetle Omorgus soberosus Fabricius (Coleoptera: Trogidae), which significantly affects the survival of eggs of L. olivacea on Santuario La Escobilla Beach in Oaxaca, Mexico.

Thorectes lusitanicus, a typically coprophagous species is also actively attracted to oak acorns, consuming, burying them, and conferring ecophysiological and reproductive advantages to both the beetle and the tree. In this study, we explored the possible relation between diet shift and the health status of T. lusitanicus using a generalist entomopathogenic fungus (Metarhizium anisopliae) as a natural pathogen. To measure the health condition and immune response of beetles, we analysed the protein content in the haemolymph, prophenoloxidase (proPO) content, phenoloxidase (PO) activity and mortality of beetles with diets based on either acorns or cow dung. Protein content, proPO levels and PO levels in the haemolymph of T. lusitanicus were found to be dependent on the type of diet. Furthermore, the beetles fed with acorns developed a more effective proPO-PO system than the beetles fed with cow dung. Furthermore, a significant decrease in mortality was observed when infected individuals were submitted to an acorn-based diet. In addition to enhancing an understanding of the relevance of dietary change to the evolutionary biology of dung beetles, these results provide a more general understanding of the ecophysiological implications of differential dietary selection in the context of fitness. © 2013 Verdu et al.