Nontoxic effects of thymol, carvacrol, cinnamaldehyde, and garlic oil on dung beetles: A potential alternative to ecotoxic anthelmintics.

The sustainability of the traditional extensive livestock sector will only be possible if healthy dung-decomposing insect communities are preserved. However, many current pharmaceutical anthelmintics are harmful to dung beetles, their presence can have a negative impact on biological systems. Phytochemical anthelmintics are an alternative to ecotoxic synthetic pharmaceutical anthelmintics, although ecotoxicological tests of their possible indirect effects on dung beetles are required to demonstrate their viability. In this study, the potential ecotoxicity of thymol, carvacrol, cinnamaldehyde and garlic oil (diallyl disulfide and diallyl trisulfide) were tested for the first time. Inhibition of antennal response was measured as a relevant parameter by obtaining relevant toxicity thresholds derived from concentration‒response curves, such as the IC50. All phytochemical compounds tested were demonstrated to be suitable alternative candidates to the highly ecotoxic compound ivermectin, considering their non-toxicity to nontarget organisms. Residues of the phytochemical antiparasitics found in cattle droppings were extremely low, even undetectable in the case of diallyl disulfide and diallyl trisulfide. Furthermore, our results showed that none of the phytochemical compounds have ecotoxic effects, even at extremely high concentrations, including those almost 1000 times higher than what is most likely to be found in dung susceptible to ingestion by dung beetles in the field. We can conclude that the four selected phytochemical compounds meet the requirements to be considered reliable alternatives to ecotoxic veterinary medicinal products, such as ivermectin.

Links Between Feeding Preferences and Electroantennogram Response Profiles in Dung Beetles: The Importance of Dung Odor Bouquets.

The detection of dung odors is a crucial step in the food-searching behavior of dung beetles (Coleoptera: Scarabaeoidea). Yet, whether certain compounds characteristic of a given dung type contribute to a 'choosy generalism' behavior proposed for this taxonomic group is unknown. To address this, we analyzed the chemical composition of three types of dung (cow, horse, and rabbit) and conducted behavioral and electroantennogram (EAG) bioassays on 15 species of dung beetles using 19 volatile organic compounds representing the three dung samples. Chemical analyses revealed substantial qualitative and quantitative differences among dung types. When offered these food options in an olfactometer, 14 species exhibited a feeding preference. Surprisingly, all 19 compounds used in the EAG assays elicited antennal responses, with species displaying different olfactory profiles. The relationship between behavioral preferences and electrophysiological profiles highlighted that species with different food preferences had differences in antennal responses. Moreover, a specific set of EAG-active compounds (nonanal, sabinene, acetophenone, ρ-cresol, 2-heptanone, 1H-indole, and 6-methyl-5-hepten-2-one) were the strongest drivers in the distinct sensory profiles of the trophic preference groups. Our results point to the importance of the whole bouquet of dung-emanating compounds in driving food-searching behavior, but specific volatiles could aid in determining highly marked trophic preferences in certain species.

First observation on the predation of a non-arthropod species by a dung beetle species: The case of Canthon chalybaeus and the snail Bulimulus apodemetes.

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.

Biomagnification and body distribution of ivermectin in dung beetles.

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.

Thermoregulatory syndromes of two sympatric dung beetles with low energy costs.

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.

Author Correction: First assessment of the comparative toxicity of ivermectin and moxidectin in adult dung beetles: Sub-lethal symptoms and pre-lethal consequences.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

First assessment of the comparative toxicity of ivermectin and moxidectin in adult dung beetles: Sub-lethal symptoms and pre-lethal consequences.

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 residues disrupt dung beetle diversity, soil properties and ecosystem functioning: An interdisciplinary field study.

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.

Isolation and determination of ivermectin in post-mortem and in vivo tissues of dung beetles using a continuous solid phase extraction method followed by LC-ESI+-MS/MS.

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.

Identification and evaluation of semiochemicals for the biological control of the beetle Omorgus suberosus (F.) (Coleoptera: Trogidae), a facultative predator of eggs of the sea turtle Lepidochelys olivacea (Eschscholtz).

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.

Low doses of ivermectin cause sensory and locomotor disorders in dung beetles.

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.

Acorn consumption improves the immune response of the dung beetle Thorectes lusitanicus.

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.