Dung removal increases under higher dung beetle functional diversity regardless of grazing intensification.

Dung removal by macrofauna such as dung beetles is an important process for nutrient cycling in pasturelands. Intensification of farming practices generally reduces species and functional diversity of terrestrial invertebrates, which may negatively affect ecosystem services. Here, we investigate the effects of cattle-grazing intensification on dung removal by dung beetles in field experiments replicated in 38 pastures around the world. Within each study site, we measured dung removal in pastures managed with low- and high-intensity regimes to assess between-regime differences in dung beetle diversity and dung removal, whilst also considering climate and regional variations. The impacts of intensification were heterogeneous, either diminishing or increasing dung beetle species richness, functional diversity, and dung removal rates. The effects of beetle diversity on dung removal were more variable across sites than within sites. Dung removal increased with species richness across sites, while functional diversity consistently enhanced dung removal within sites, independently of cattle grazing intensity or climate. Our findings indicate that, despite intensified cattle stocking rates, ecosystem services related to decomposition and nutrient cycling can be maintained when a functionally diverse dung beetle community inhabits the human-modified landscape.

Season-Long Simplification of Insect Communities in Dung From Cattle Treated With an Extended-Release Formulation of the Parasiticide Eprinomectin.

Cattle treated with LongRange®, an injectable formulation of the parasiticide eprinomectin, fecally excrete insecticidal residues for an extended period post application. We examined the nontarget effect of these residues by comparing insect communities developing in dung of untreated cattle (week 0) with those developing in dung of cattle treated 1, 2, 4, 8, 12, 16, 20, and 24 or 25 weeks previously. Chemical analyses of dung showed that eprinomectin concentrations peaked at 1 week post application and were still detectable at 25 weeks. Results from two separate experiments showed that dung of untreated cattle supported more total insects (beetles, flies, parasitoid wasps) and insect species than did dung of cattle treated for ≤12 weeks (Experiment 1) and ≤25 weeks (Experiment 2) previously. For the two experiments, an effect of residue on individual taxa was either not detected (nine cases) or was determined to suppress insect development in dung of cattle treated for 8-12 weeks (two cases), 12-16 weeks (three cases), 16-20 weeks (two cases), or 24 or 25 weeks (six cases) previously. Flies and their parasitoid wasps were particularly sensitive to residues with suppression often at or near 100%. These results show that cattle treated with LongRange in spring will fecally excrete residues for the entire grazing season with an associated simplification of the dung insect community. The effect of this simplification on the long-term health on dung-breeding populations of insects on pastures and dung degradation was not examined in the present study, but merits future research. Environ Toxicol Chem 2023;42:684-697. © 2023 His Majesty the King in Right of Canada. Environmental Toxicology and Chemistry © 2023 SETAC. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

A Review of Dung Beetle Introductions in the Antipodes and North America: Status, Opportunities, and Challenges.

Following the introduction of cattle, exotic dung beetles (Coleoptera: Aphodiidae, Geotrupidae, Scarabaeidae) were imported into the Antipodes (Australia and New Zealand) and North America (primarily the United States) to accelerate the degradation of cattle dung on pastures. The history of dung beetle introductions between the two regions is similar but has not previously been assessed: this is important as new introductions are continuing in the regions. Here, we review these introduction programs, report on their current status, and discuss methodological advances. In doing so, we examine the accidental introduction of exotic (i.e., adventive) species and the contribution of both deliberately introduced and adventive species to endemic dung beetle faunas. Further, we provide a list of pest and parasite species whose populations can be reduced by dung beetle activity. We also identify a combined total of 37 introduced and 47 adventive dung beetle species that have become established in the Antipodes and North America, with exotic species dominating dung beetle assemblages from pasture habitats. Climatic and edaphic matches, the size of founding populations, abiotic and biotic stressors, and the time of year when releases are made are all critical determinants that affect the success of dung beetle introduction programs. Finally, we discuss opportunities, plus the risks and challenges associated with dung beetle introductions. We hope that this review will aid in the success of future introduction programs, either to enhance ecosystem services in areas that they are needed, or potentially to reestablish native species in regions where they have been extirpated.

Implications of Endectocide Residues on the Survival of Aphodiine Dung Beetles: A Meta-Analysis.

It is often difficult to compare studies examining the effects of endectocides on dung fauna because of different experimental approaches, for example, active ingredients (eprinomectin, doramectin, ivermectin, moxidectin) and formulations (injectable, pour-on, spiked). To gain a better understanding, we performed a quantitative meta-analysis using 22 studies to assess the overall effect of endectocide residues on the occurrence (presence or absence) and abundance of aphodiine dung beetles. Our results document a positive effect on the occurrence of adult beetles, indicating that adults tend to be attracted to dung with residues. Conversely, larvae are less likely to occur in the presence of residues. Thus, either adults that colonize dung with residues do not lay eggs or, more likely, the larvae that hatch from these eggs die early in development. Abundance of adult and larval stages was shown to be significantly reduced in dung containing residues. When individual endectocides were compared, only ivermectin demonstrated a significantly negative effect on the abundance of both adults and larvae, possibly owing to a small sample size for other agents. In laboratory studies, only dung "spiked" with endectocides reduced the abundance of larvae, whereas during field research, only pour-on applications were shown to reduce the abundance of larvae. The present study further documents the nontarget effects of endectocide residues on dung-dwelling organisms, provides robust evidence on the consequences of different application methods, and emphasizes the need for standardized methodological techniques in future studies. Environ Toxicol Chem 2020;39:863-872. © 2020 SETAC.

Cross-tolerance to Desiccation and Cold in Khapra Beetle (Coleoptera: Dermestidae).

Khapra beetle, Trogoderma granarium Everts, is unusual in two key respects. First, they are among the most cold hardy of stored-product insect pests even though they originate in hot and dry regions of the Indian subcontinent. Second, their larvae can enter into diapause to survive harsh environmental conditions. In the present study, we examined whether these two phenomena are related, i.e., due to cross-tolerance. Cross-tolerance is the tolerance to one ecological stress when induced by a separate stress. To investigate this, khapra beetle larvae were reared at different relative humidities (3, 28, 49, and 79%) in either nondiapausing or diapausing conditions. Then the cold tolerance of larvae was estimated by measuring mortality after different durations at -10°C. For nondiapausing larvae, relative humidity had little effect on cold tolerance with the lethal time to 50% mortality (LT50) occurring between 2 and 4 d. For diapausing larvae, cold tolerance increased with greater desiccation stress with LT50's of 5, 7, 10, and 18 d at 79, 49, 28, and 3% RH, respectively. This suggests that the physiological mechanisms that protect diapausing larvae from desiccation may also increase cold tolerance, even though these insects may rarely be exposed to low temperatures.

Eprinomectin from a sustained release formulation adversely affected dung breeding insects.

The insecticidal activity of parasiticide residues in dung of cattle treated with a sustained release eprinomectin formulation was examined, and an improved eprinomectin dung residue extraction method is presented. Emergent insect abundance and richness were significantly reduced in all post-treatment intervals (7, 14, 28, 56, 84, 112, and 140 d), relative to pre-treatment. Emergent insect diversity was reduced for between 84 and 112 d post-treatment. Collembola were not affected by residues. Chemical analyses subsequently documented residues of eprinomectin in dung of each collection period post-treatment at levels expected based on previously reported excretion profiles for this product. Cattle subcutaneously injected with this product excreted residues that reduced dung-breeding insect emergence for 5 mo post-treatment. The consequences of these long-term non-target effects to pasture ecosystems are not known.

Hyperparasitism of an Avian Ectoparasitic Hippoboscid Fly, Ornithomya anchineuria, by the Mite, Myialges Cf. Borealis, in Alberta, Canada.

Hippoboscid flies (Diptera: Hippoboscidae) include species that are ectoparasites of birds in the Northern Hemisphere, but little is known regarding their taxonomy, parasites, avian host associations, or geographical distribution in North America. In late August of 2013 and 2014, we collected hippoboscid flies from live birds trapped in mist nets as part of a banding study in Cypress Hills Interprovincial Park in southeastern Alberta, Canada. A total of 113 birds comprising 9 species was examined in 2013. Of these, 18 individuals were infested with 1-3 Ornithomya anchineuria Speiser (n = 22 flies; prevalence = 15.9%). Eight of these flies carried 1-8 adult female epidermoptid mites anchored to their ventral, posterior abdomens. Each female was associated with clusters of up to 30 stalked eggs. The first pair of tarsi on adult female mites was highly modified as anchors, indicating permanent attachment through the host cuticle. Morphological traits identified these mites as Myialges cf. borealis Mironov, Skirnisson, Thorarinsdottier and Nielsen. Cytochrome c oxidase subunit 1 ( COX1) gene sequences obtained for 2 mites were distinct from those previously reported for species of Myialges, being most similar to Myialges trinotoni Cooreman. The paucity of available gene sequences for Myialges and related genera of epidermoptid mites prevents any further conclusions regarding taxonomy. These findings extend previous reports of O. anchineuria from Pacific and Atlantic coasts of Canada inland to the central migratory flyway of the Northern Great Plains and expand the limited information available for Myialges spp.

Analysis and dissipation of the antiparasitic agent ivermectin in cattle dung under different field conditions.

Cattle treated with the veterinary parasiticide ivermectin fecally excrete residues. The authors report the exposition and dissipation characteristics of these residues in dung of ivermectin-treated cattle and in soil beneath this dung on pastures in Canada, France, Switzerland, and The Netherlands. Residues were quantified for dung collected from cattle after 3 d, 7 d, 14 d, and 28 d posttreatment and subsequently exposed in the field for up to 13 mo. The authors optimized a high-performance liquid chromatography-fluorescence detection method to detect ivermectin residues in dung and soil matrices. They showed that a solid phase extraction and purification step generally can be eliminated to reduce the time and cost of these analyses. They also found that the addition of water to relatively dry samples improves the extraction efficiency of residues. They then analyzed the field samples to document differences in ivermectin dissipation in cattle dung among sites, with 50% dissipation times of up to 32 d and 90% dissipation times >396 d. They further showed that the dissipation characteristics of residues are comparable between dung of ivermectin-treated cattle and dung to which ivermectin has been added directly. Lastly, they report the first use of a desorption electrospray ionization-high-resolution-mass spectrometric method to detect residues of metabolites in a dung matrix. Environ Toxicol Chem 2016;35:1924-1933. © 2016 SETAC.

Nontarget effects of ivermectin residues on earthworms and springtails dwelling beneath dung of treated cattle in four countries.

The authorization of veterinary medicinal products requires that they be assessed for nontarget effects in the environment. Numerous field studies have assessed these effects on dung organisms. However, few studies have examined effects on soil-dwelling organisms, which might be exposed to veterinary medicinal product residues released during dung degradation. The authors compared the abundance of earthworms and springtails in soil beneath dung from untreated cattle and from cattle treated 0 d, 3 d, 7 d, 14 d, and 28 d previously with ivermectin. Study sites were located in different ecoregions in Switzerland (Continental), The Netherlands (Atlantic), France (Mediterranean), and Canada (Northern Mixed Grassland). Samples were collected using standard methods from 1 mo to 12 mo after pat deposition. Ivermectin concentrations in soil beneath dung pats ranged from 0.02 mg/kg dry weight (3 mo) to typically <0.006 mg/kg dry weight (5-7 mo). Earthworms were abundant and species-rich at the Swiss and Dutch sites, less common with fewer species at the French site, and essentially absent at the Canadian site. Diverse but highly variable communities of springtails were present at all sites. Overall, results showed little effect of residues on either earthworms or springtails. The authors recommend that inclusion of soil organisms in field studies to assess the nontarget effects of veterinary medicinal products be required only if earthworms or springtails exhibit sensitivity to the product in laboratory tests. Environ Toxicol Chem 2016;35:1959-1969. © 2015 SETAC.

Effects of ivermectin application on the diversity and function of dung and soil fauna: Regulatory and scientific background information.

The application of veterinary medical products to livestock can impact soil organisms in manure-amended fields or adversely affect organisms that colonize dung pats of treated animals and potentially retard the degradation of dung on pastures. For this reason, the authorization process for veterinary medicinal products in the European Union includes a requirement for higher-tier tests when adverse effects on dung organisms are observed in single-species toxicity tests. However, no guidance documents for the performance of higher-tier tests are available. Hence, an international research project was undertaken to develop and validate a proposed test method under varying field conditions of climate, soil, and endemic coprophilous fauna at Lethbridge (Canada), Montpellier (France), Zurich (Switzerland), and Wageningen (The Netherlands). The specific objectives were to determine if fecal residues of an anthelmintic with known insecticidal activity (ivermectin) showed similar effects across sites on 1) insects breeding in dung of treated animals, 2) coprophilous organisms in the soil beneath the dung, and 3) rates of dung degradation. By evaluating the effects of parasiticides on communities of dung-breeding insects and soil fauna under field conditions, the test method meets the requirements of a higher-tier test as mandated by the European Union. The present study provides contextual information on authorization requirements for veterinary medicinal products and on the structure and function of dung and soil organism communities. It also provides a summary of the main findings. Subsequent studies on this issue provide detailed information on different aspects of this overall project. Environ Toxicol Chem 2016;35:1914-1923. © 2015 SETAC.

Plant-herbivore interactions in a trispecific hybrid swarm of Populus: assessing support for hypotheses of hybrid bridges, evolutionary novelty and genetic similarity.

UNLABELLED: Natural systems of hybridizing plants are powerful tools with which to assess evolutionary processes between parental species and their associated arthropods. Here we report on these processes in a trispecific hybrid swarm of Populus trees. Using field observations, common garden experiments and genetic markers, we tested the hypothesis that genetic similarities among hosts underlie the distributions of 10 species of gall-forming arthropods and their ability to adapt to new host genotypes. KEY FINDINGS: the degree of genetic relatedness among parental species determines whether hybridization is primarily bidirectional or unidirectional; host genotype and genetic similarity strongly affect the distributions of gall-forming species, individually and as a community. These effects were detected observationally in the wild and experimentally in common gardens; correlations between the diversity of host genotypes and their associated arthropods identify hybrid zones as centres of biodiversity and potential species interactions with important ecological and evolutionary consequences. These findings support both hybrid bridge and evolutionary novelty hypotheses. However, the lack of parallel genetic studies on gall-forming arthropods limits our ability to define the host of origin with their subsequent shift to other host species or their evolution on hybrids as their final destination.

Validation of a standard field test method in four countries to assess the toxicity of residues in dung of cattle treated with veterinary medical products.

Registration of veterinary medical products includes the provision that field tests may be required to assess potential nontarget effects associated with the excretion of product residues in dung of treated livestock (phase II, tier B testing). However, regulatory agencies provide no guidance on the format of these tests. In the present study, the authors report on the development of a standardized field test method designed to serve as a tier B test. Dung was collected from cattle before and up to 2 mo after treatment with a topical application of a test compound (ivermectin). Pats formed of dung from the different treatments were placed concurrently in the field to be colonized by insects. The abundance, richness, and diversity of insects developing from egg to adult in these pats were compared across treatments using analysis of variance tests. Regression analyses were used to regress abundance, richness, and diversity against residue concentrations in each treatment. Results of the regression were used to estimate mean lethal concentration (LC50) values. The robustness of the method and the repeatability of its findings were assessed concurrently in 4 countries (Canada, France, Switzerland, and The Netherlands) in climatically diverse ecoregions. Results were generally consistent across countries, and support the method's formal adoption by the European Union to assess the effects of veterinary medical product residues on the composition and diversity of insects in dung of treated livestock. Environ Toxicol Chem 2016;35:1934-1946. © 2015 Crown in the right of Canada. Published by Wiley Periodicals Inc., on behalf of SETAC.

Effects of pitfall trap lid transparency and habitat structure on the catches of carabid beetles (Coleoptera: Carabidae) in tame pasture.

Captures of insects in pitfall traps are affected by features of trap design that may confound the interpretation of data. One such feature is a lid suspended over the opening of the trap to exclude debris and rainwater. In this study, we tested whether use of these lids affected captures of carabid beetles by altering the light conditions at the opening to the trap. In one experiment, we examined the effects of lid transparency (opaque, semitransparent, or transparent) on catch rates. In a second experiment, we manipulated the heights (high, medium, or low) of vegetation adjacent to the traps to test for lid transparency and vegetation height interactions. We found that significantly more carabids were captured with use of transparent lids compared with other lid transparencies. Fewest Agonum cupreum Dejean, 1831, were captured with use of opaque lids. No other effects were detected. Given these results, we advocate the use of transparent lids, which provide the benefits of traditional opaque lids while minimizing the effects of lid use on light conditions at the opening to the trap.

Testing the 'hybrid susceptibility' and 'phenological sink' hypotheses using the P. balsamifera - P. deltoides hybrid zone and septoria leaf spot [Septoria musiva].

Hybrid genotypes that arise between plant species frequently have increased susceptibility to arthropod pests and fungal pathogens. This pattern has been attributed to the breakdown of plant defenses ('Hybrid susceptibility' hypothesis) and (or) to extended periods of susceptibility attributed to plant phenologies in zones of species overlap and (or) hybridization ('phenological sink' hypothesis). We examined these hypotheses by assessing the susceptibility of parental and hybrid Populus host genotypes to a leaf spot disease caused by the fungal pathogen Septoria musiva. For this purpose, 214 genotypes were obtained from morphologically pure zones of P. balsamifera and P. deltoides, and from an intervening zone of overlap and hybridization on the drainage of the Red Deer River, Alberta, Canada. Genotypes were identified as P. balsamifera, P. deltoides, or hybrid using a suite of 27 species-specific SNP markers. Initially the genetic structure of the hybrid zone was characterized with 27.7% of trees classified as admixed individuals. To test the hybrid susceptibility hypothesis, a subset of 52 genotypes was inoculated with four isolates of S. musiva. Levels of susceptibility were P. balsamifera > F1 hybrid > P. deltoides. A further 53 genotypes were grown in a common garden to assess the effect of genotype on variation in leaf phenology. Leaf phenology was more variable within the category of hybrid genotypes than within categories of either parental species. Leaf phenology was also more variable for the category of trees originating in the hybrid (P. balsamifera - P. deltoides [hybrid and parental genotypes combined]) zone than in adjacent pure zones of the parental species. The results from the inoculation experiment support the hybrid intermediacy hypothesis. The results from the common garden experiment support the 'phenological sink' hypothesis. These findings have greatly increased our understanding of the epidemiology and ecology of fungal pathogens in plant hybrid zones.

How to test nontarget effects of veterinary pharmaceutical residues in livestock dung in the field.

To register veterinary medicinal products (VMPs) as parasiticides on pastured animals, legislation in the European Union requires an environmental risk assessment to test the potential nontarget effects of fecal residues on dung-dwelling organisms. Products with adverse effects in single-species laboratory tests require further, higher-tier testing to assess the extent of these effects on entire communities of dung-dwelling organisms under more realistic field or semifield conditions. Currently, there are no documents specifically written to assist researchers in conducting higher-tier tests or to assist regulators in interpreting the results of such tests in an appropriate context. Here we provide such a document, written by members of the SETAC Advisory Group DOTTS (Dung Organism Toxicity Testing Standardization) with research experience on dung fauna in central and southern Europe, Canada, Australia, and South Africa. This document briefly reviews the organisms that make up the dung community and their role in dung degradation, identifies key considerations in the design and interpretation of experimental studies, and makes recommendations on how to proceed.

The host range of the male-killing symbiont Arsenophonus nasoniae in filth fly parasitioids.

The Son-killer bacterium, Arsenophonus nasoniae, infects Nasonia vitripennis (Hymenoptera: Pteromalidae), a parasitic wasp that attacks filth flies. This gammaproteobacterium kills a substantial amount of male embryos produced by an infected female. Aside from male death, the bacterium does not measurably affect the host, and how it is maintained in the host population is unknown. Interestingly, this bacterial symbiont can be transmitted both vertically (from mother to offspring) and horizontally (to unrelated Nasonia wasps developing in the same fly host). This latter mode may allow the bacterium to spread throughout the ecological community of filth flies and their parasitoids, and to colonize novel species, as well as permit its long-term persistence. We tested 11 species of filth flies and 25 species of their associated parasitoids (representing 28 populations from 16 countries) using diagnostic PCR to assess the bacterium's actual host range. In addition to 16S rRNA, two loci were targeted: the housekeeping gene infB, and a sequence with high homology to a DNA polymerase gene from a lysogenic phage previously identified from other insect symbionts. We identified infections of A. nasoniae in four species of parasitoids, representing three taxonomic families. Highly similar phage sequences were also identified in three of the four species. These results identify the symbiont as a generalist, rather than a specialist restricted solely to species of Nasonia, and also that horizontal transmission may play an important role in its maintenance.

A test using Wolbachia bacteria to identify Eurasian source populations of cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham), in North America.

Previous research using mitochondrial haplotypes indicates that North American populations of cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham), originated from at least two separate introductions from source populations in Eurasia. We tested this hypothesis by comparing the genetic variation of symbiotic Wolbachia bacteria in C. obstrictus among seven North American and four European populations. Because Wolbachia are maternally inherited, infections acquired by a host species at one geographic location theoretically may be present in derivative populations that have established in new regions. Use of the conserved MLST Wolbachia genes gatB, coxA, hcpA, fbpA, and ftsZ identified one strain present in all beetles. Use of the variable wsp gene identified three distinct isolates of this strain that appear to co-occur in all populations and potentially in all individuals. Use of the variable wspB gene provided independent support for the presence of these isolates and evidence of a wspB pseudogene. The lack of genetic variation for these Wolbachia genes among host populations prevents their use to clarify the origins of C. obstrictus in North America. However, the results are an interesting example illustrating disjunction in genetic variation between mitochondria and a maternally-inherited symbiont.

Further evidence for the absence of bacteria in horsehair worms (Nematomorpha: Gordiidae).

We used molecular techniques to characterize bacteria associated with the nematomorph Gordius robustus (Leidy). This worm is a parasite of the fall field cricket, Gryllus pennsylvanicus (Burmeister), which is infected with the symbiotic bacteria, Wolbachia. Because of this close association, our a priori expectation was that G. robustus may be similarly infected. However, results of denaturing gradient gel electrophoresis and sequencing of amplified 16S rDNA failed to detect any bacteria (symbiotic or non-symbiotic) in G. robustus. These unexpected findings suggest that G. robustus has no internal bacterial community and indicate that close association with a Wolbachia-infected host is insufficient for the transmission of bacteria from insect to nematomorph.

Lethal and sublethal toxic effects of a test chemical (ivermectin) on the yellow dung fly (Scathophaga stercoraria) based on a standardized international ring test.

A standardized bioassay using the yellow dung fly, Scathophaga stercoraria L. (Diptera: Scathophagidae), was developed to test the lethal and sublethal toxicity of parasiticide residues in livestock dung. The repeatability of the bioassay was assessed for the parasiticide ivermectin in 13 tests performed by seven laboratories in Germany, the United Kingdom, Switzerland, and Canada. Test results had an acceptable range of heterogeneity. The calculated median effective concentration for 50% (EC50) egg-to-adult mortality was 20.9 +/- 19.1 microg ivermectin/kg dung fresh weight (FW) (mean +/- standard deviation; range, 6.33-67.5 microg/kg). Mortality was not observed below a calculated no-observable-effect concentration (NOEC) of 8.1 +/- 7.7 microg/kg FW. However, prolonged development time (and, in a subset of tests, reduced body size) was observed above a calculated NOEC of 0.8 +/- 0.8 microg/kg FW. An oviposition site choice test revealed that yellow dung fly females do not discriminate among dung of different ivermectin concentrations. Thus, the yellow dung fly is suitably sensitive, and the methods are sufficiently repeatable, to support use of this standardized bioassay by the international community in the registration of new veterinary pharmaceuticals.

Wolbachia infection in Australasian and North American populations of Haematobia irritans (Diptera: Muscidae).

Buffalo fly (Haematobia irritans exigua) is a major pest of beef and dairy cattle in northern Australia. Global warming is expected to increase the southern range of buffalo fly. Chemical control is compromised by resistance and may not be feasible in extensive production systems and there is rapidly growing market preference for beef and dairy products produced in low-chemical systems. Wolbachia are vertically transmitted intracellular bacteria that can profoundly influence host reproduction and fitness and are of increasing interest for use in biocontrol programs. To determine whether Australian flies are infected with Wolbachia, buffalo flies were collected from 12 cattle herds around Australia and assayed by standard PCR for the Wolbachia wsp gene. H. i. exigua from Indonesia and horn fly (H. i. irritans) from Canada were also tested. All H. i. exigua samples tested were negative for Wolbachia infection whereas a very strong signal for Wolbachia was obtained from H. i. irritans.