Brachyceran Flies Research Articles

Expanding the Mesozoic Record of Early Brachyceran Fly Larvae, including New Larval Forms with Chimera-Type Morphologies.

Diptera are one of the four megadiverse groups of holometabolan insects. Flies perform numerous ecological functions, especially in their larval stages. We can assume that this was already the case in the past; however, fly larvae remain rare in most deposits. Here we report new dipteran larvae preserved in Cretaceous (about 99 Ma) Kachin amber from Myanmar and, even older, Jurassic (about 165 Ma) compression fossils from China. Through light microscopy and micro-CT scanning we explore their peculiar morphology and discuss their possible phylogenetic affinities. Several larvae seem to represent the lineage of Stratiomyomorpha. A few others present characters unique to Xylophagidae (awl-flies), as well as to Athericidae (water sniper-flies), resulting in a chimeric morphology. Understanding the exact relationships of most of these specimens with a particular lineage remains challenging, since they differ considerably from any other known dipteran larvae and present some unique traits. Additionally, we report new specimens of Qiyia jurassica Chen et al., 2014, supposedly parasitic larvae, most likely representatives of Athericidae. These new findings offer valuable insights into the evolution of the early diversification of the brachyceran flies and underscore the importance of immature stages in understanding the evolutionary history and ecology of flies.

New records of immature aquatic Diptera from the Foulden Maar Fossil-Lagerstätte, New Zealand, and their biogeographic implications.

The biogeographical and ecological history of true flies (Diptera) in New Zealand is little known due to a scarcity of fossil specimens. Here, we report a fauna of immature aquatic dipterans from freshwater diatomites of the early Miocene Foulden Maar Fossil-Lagerstätte in southern New Zealand. We document 30 specimens of immature dipterans, mostly pupae, and compare their external morphology to extant aquatic Diptera. Based on the reconstructed paleoenvironment of Foulden Maar, we discuss taxonomic, ecological and taphonomic implications of this early Miocene fauna. Among Chironomidae, one pupal morphotype is attributed to Tanypodinae, one pupal morphotype and one larval morphotype are placed into Chironomus (Chironominae) and a further morphotype into Chironominae incertae sedis. Chaoboridae are represented by a pupal morphotype congeneric or very close to the extant Chaoborus, today globally distributed except for New Zealand. Additional immature specimens are likely larvae and puparia of brachyceran flies but cannot be identified to a narrower range. These finds document an aquatic dipteran fauna in New Zealand in the earliest Miocene and highlight Neogene extinction as a factor in shaping the extant Diptera fauna in New Zealand. Immature aquatic dipterans were a common and likely ecologically important component of the early Miocene Foulden Maar lake. Preservation of larvae and pupae may have been promoted by diatomaceous microbial mats and the light colour of the diatomite likely facilitated spotting of these minute fossils in the field.

A survey of Wolbachia infection in brachyceran flies from Iran.

Wolbachia is a maternally inherited intracellular bacterium that is considered to be the most plentiful endosymbiont found in arthropods. It reproductively manipulates its host to increase the chances of being transmitted to the insect progeny; and it is currently used as a means of suppressing disease vector populations or controlling vector-borne diseases. Studies of the dissemination and prevalence of Wolbachia among its arthropod hosts are important for its possible use as a biological control agent. The molecular identification of Wolbachia relies on different primers sets due to Wolbachia strain variation. Here, we screened for the presence of Wolbachia in a broad range of Brachycera fly species (Diptera), collected from different regions of Iran, using nine genetic markers (wsp, ftsZ, fbpA, gatB, CoxA, gltA, GroEL dnaA, and 16s rRNA), for detecting, assessing the sensitivity of primers for detection, and phylogeny of this bacterium. The overall incidence of Wolbachia among 22 species from six families was 27.3%. The most commonly positive fly species were Pollenia sp. and Hydrotaea armipes. However, the bacterium was not found in the most medically important flies or in potential human disease vectors, including Musca domestica, Sarcophaga spp., Calliphora vicinia, Lucilia sericata, and Chrysomya albiceps. The primer sets of 16s rRNA with 53.0% and gatB with 52.0% were the most sensitive primers for detecting Wolbachia. Blast search, phylogenetic, and MLST analysis of the different locus sequences of Wolbachia show that all the six distantly related fly species likely belonging to supergroup A. Our study showed some primer sets generated false negatives in many of the samples, emphasizing the importance of using different loci in detecting Wolbachia. The study provides the groundwork for future studies of a Wolbachia-based program for control of flies.

Adaptation to pollination by fungus gnats underlies the evolution of pollination syndrome in the genus Euonymus.

Dipteran insects are known pollinators of many angiosperms, but knowledge on how flies affect floral evolution is relatively scarce. Some plants pollinated by fungus gnats share a unique set of floral characters (dark red display, flat shape and short stamens), which differs from any known pollination syndromes. We tested whether this set of floral characters is a pollination syndrome associated with pollination by fungus gnats, using the genus Euonymus as a model. The pollinator and floral colour, morphology and scent profile were investigated for ten Euonymus species and Tripterygium regelii as an outgroup. The flower colour was evaluated using bee and fly colour vision models. The evolutionary association between fungus gnat pollination and each plant character was tested using a phylogenetically independent contrast. The ancestral state reconstruction was performed on flower colour, which is associated with fungus gnat pollination, to infer the evolution of pollination in the genus Euonymus. The red-flowered Euonymus species were pollinated predominantly by fungus gnats, whereas the white-flowered species were pollinated by bees, beetles and brachyceran flies. The colour vision analysis suggested that red and white flowers are perceived as different colours by both bees and flies. The floral scents of the fungus gnat-pollinated species were characterized by acetoin, which made up >90 % of the total scent in three species. Phylogenetically independent contrast showed that the evolution of fungus gnat pollination is associated with acquisition of red flowers, short stamens and acetoin emission. Our results suggest that the observed combination of floral characters is a pollination syndrome associated with the parallel evolution of pollination by fungus gnats. Although the role of the red floral display and acetoin in pollinator attraction remains to be elucidated, our finding underscores the importance of fungus gnats as potential contributors to floral diversification.

Wide Range of Brachyceran Fly Taxa Attracted to Synthetic and Semi-Synthetic Generic Noctuid Lures and the Description of New Attractants for Sciomyzidae and Heleomyzidae Families.

During field tests implemented in Transcarpathia (West Ukraine) in 2015, 6501 specimens belonging to 26 Brachyceran fly families were collected with traps baited with generic lures (originally developed for noctuid moths) based on fermenting liquid and floral compounds. Isoamyl alcohol-based baits generally attracted more flies than phenylacetaldehyde-based baits and unbaited controls; however, the phenylacetaldehyde-based traps were the most attractive to the Empididae and Milichiidae families. The isoamyl alcohol-based semisynthetic lure showed significant attractivity to the families of Muscidae, Ulidiidae, Sarcophagidae, Calliphoridae, Sciomyzidae, Heleomyzidae, Drosophilidae, Phoridae and Platystomatidae. Additionally, isoamyl alcohol-based semisynthetic lure is the first reported attractant of the Sciomyzidae family. Since our phenylacetaldehyde-based floral lure was also attractive to Heleomyzidae flies, both types of lures can be seen as the first known attractants of this family.

Phylogenetic relationships of Brachycera (Insecta: Diptera) inferred from mitochondrial genome sequences

AbstractBrachyceran flies constitute a large radiation of the order Diptera, but the phylogenetic relationships among them have remained controversial. In this study, we used next-generation sequencing to determine mitochondrial genomes (mitogenomes) for six brachyceran flies. Two of these species represent Milichiidae (Aldrichiomyza flaviventris and Phyllomyza obliqua); two representing Chloropidae (Pachylophus sp.) and Sphaeroceridae (Leptocera erythrocera) are the first sequenced members of these groups; and two species from Lauxaniidae (Homoneura sp.) and Syrphidae (Paragus quadrifasciatus) were newly sequenced. Together with the published mitogenomes, we included a total of 187 species representing 40 dipteran families to investigate the phylogeny of Brachycera. The results strongly supported Brachycera as a monophyletic group. The infra-orders Stratiomyomorpha, Tabanomorpha and Xylophagomorpha were retrieved as the earliest brachyceran lineages, but the clade (Stratiomyomorpha + (Xylophagomorpha + Tabanomorpha)) was not supported. In most analyses, Platypezoidea were recovered as the sister-group to Cyclorrhapha. Syrphoidea were non-monophyletic with respect to Pipunculus. Milichiidae were non-monophyletic because of Pachylophus. Sphaeroceroidea were, in most cases, placed as the sister-group to Ephydroidea. Within Calyptratae, Hippoboscoidea were sister to an assemblage of lineages composed of a muscoid grade and Oestroidea, the latter being monophyletic in the Bayesian analyses using the PhyloBayes site-heterogeneous mixture model.

Identification and functional characterization of olfactory indolergic receptors in Musca domestica

In mosquitoes, indolic compounds are detected by a group of olfactory indolergic Odorant Receptors (indolORs). The ancient origin of indole and 3-methylindole as chemical signals suggest that they may be detected by insects outside the Culicidae clade. To test this hypothesis, we have identified potential indolOR genes in brachyceran flies based on sequence homology. Because of the crucial roles of indolic compounds in oviposition and foraging, we have focused our attention on the housefly Musca domestica. Using a heterologous expression system, we have identified indolOR transcript expression in the female antennae, and have characterized MdomOR30a and MdomOR49b as 3-methylindole and indole receptors, respectively. We have identified a set of 92 putative indolOR genes encoded in the genomes of Culicoidea, Psychodidae and brachycera, described their phylogenetic relationships, and exon/intron structures. Further characterization of indolORs will impact our understanding of insect chemical ecology and will provide targets for the development of novel odor-based tools that can be integrated into existing vector surveillance and control programs.

Identification and functional characterization of olfactory indolergic receptors in Drosophila melanogaster

Indole-sensitive odorant receptors or indolORs belong to a mosquito-specific expansion as ancient as the Culicidae lineage. Brachyceran flies appeared to lack representative members of this group despite the importance of indolics in this important group of dipterans. To explore whether indolORs occur in other brachyceran species, we searched for candidate indolORs in Drosophila melanogaster. Using phylogenetic tools, we show that D. melanogaster OR30a, OR43a, and OR49b form a distinct monophyletic lineage with mosquito indolORs. To explore a potential functional orthology with indolORs, we expressed these three Drosophila ORs in Xenopus laevis oocytes and measured their responses to a panel of indolic compounds. We provide evidence that OR30a, OR43a, and OR49b exhibit high sensitivity to indoles. Along with the recent discovery of indolORs in the housefly Musca domestica, our findings suggest that indolORs are a widespread feature of the peripheral olfactory systems of Diptera.

Middle Jurassic origin in India: a new look at evolution of Vermileonidae and time-scaled relationships of lower brachyceran flies

AbstractVermileonidae (wormlions) comprises 61 described species in 12 genera and is one of the oddest and most rarely collected dipteran families. Larvae of Vermileonidae are famous for their pitfall prey behaviour, whereas the adults are rarely seen. Here we report, for the first time, the complete mitochondrial genome from members of Vermileonidae. Phylogenetic reconstruction based on a representative sampling of the order reveals new insights into relationships between the Vermileonidae and other members of lower brachyceran flies. A sister-group relationship between Vermileonidae and Xylophagidae is supported, and the higher-level clade relationships are Tabanomorpha + (Muscomorpha + (Xylophagomorpha + Stratiomyomorpha)). Combining mitochondrial genome data with a morphological phylogeny, geographical distribution and geological history, we propose that the Vermileonidae originated in India during the Middle Jurassic, spreading to Africa via land bridges during the Late Jurassic, to the Palaearctic after the collision of India with Laurasia in the Late Oligocene, and to the Nearctic in the Early Miocene, via either North Atlantic or Beringian land bridges. Wet forested regions have proved to be barriers to their dispersal, explaining their absence from Central Africa, South America and eastern North America.

Complete mitochondrial genome of Coenomyia ferruginea (Scopoli). (Diptera, Coenomyiidae)

The dipteran family Coenomyiidae was firstly separated from the Xylophagidae by Akrira Nagatomi in 1975. We sequenced and annotated the mitochondrial genome of Coenomyia ferruginea, the first representative of genera Coenomyia with complete mitochondrial data. This mitogenome is 17,283 bp totally, which consists of 22 transfer RNAs, 13 protein-coding genes, 2 ribosomal RNAs, and 1 non-coding control region. All genes have the conservational arrangement like other published species of brachyceran flies. The nucleotide composition biases toward A and T, the overall A + T% was up to 75.4% of the entire mitogenome. Both Bayesian inference and ML analysis strongly supported the sister relationship between Coenomyiidae and Xylophagidae. Our results also suggested that Xylophagomorpha is the sister group to Stratiomyomorpha.

Substrate specificity among Diptera in decaying bioenergy wood: can they be conserved by the same measures as are currently applied to beetles?

Although threatened by forestry, our knowledge concerning saproxylic insects is strongly biased towards well-known orders, mainly beetles (Coleoptera). The beetles have, therefore, formed the basis on which conservation measures of other groups have been formulated. Despite being more species-rich, the Diptera have been rather neglected. Moreover, our limited knowledge of the Diptera suggests that their demands on the dead wood substrate differ markedly from that of coleopterans. We tested if this is true by comparing the substrate requirements of dipteran and coleopteran species by analysing the affinities of species assemblages for logging residues differing in age, size, and tree species. Insects were reared out from the same samples of bioenergy wood from clear-cuts in Sweden. 15 species of Brachyceran flies were compared with 56 species of Coleoptera. We found the average level of specialisation to be similar between the two groups, but the dipterans had (contrary to the expectations) a higher proportion of specialists. Affinities for differently aged wood were similar. More dipterans than beetles were associated with the coarsest wood (diameter 9 cm–15 cm). More dipterans than beetles tended to be associated with aspen (Populus tremula), while Coleoptera tended to be more associated than Diptera with oak (Quercus) and spruce (Picea abies). We conclude that most recommendations for conserving the saproxylic beetle fauna also seem to benefit dipterans, but that the dipterans might be even more sensitive to which qualities of the wood that is preserved. The high conservation value of aspen is already recognised and our results for dipterans strengthen this. The high incidence of many dipteran species in logging residues suggests that many dipterans use sun-exposed environments.

Adult Reproductive Tract Morphology and Spermatogenesis in the Black Soldier Fly (Diptera: Stratiomyidae)

Abstract Large-scale production of the black soldier fly [Hermetia illucens (L.) (Diptera: Stratiomyidae)] for use as aquaculture and poultry feed has developed into a global industry. Successful commercialization of the black soldier fly relies on optimizing the production of fecund adults. However, current mass-rearing protocols result in variable production of fertile eggs. To help lay a foundation for a better understanding of factors that may play a role in this variability, the morphology of the black soldier fly male reproductive tract and spermatozoa, associated spermatogenesis process, impact of age on the process, and the female spermatheca morphology were examined with various microscopic techniques (e.g., scanning electron microscope, transition electron microscope, and dissecting scope). The gross morphology of the male reproductive tract and female spermatheca appear to be similar to those found in other brachyceran flies. Male spermatozoa are long (~860 µm overall, ~8 µm head), apparently motile, and possess flagella with a typical 9 + 9 + 2 axoneme triplets. Germ cells go through incomplete mitotic divisions surrounded by somatic cyst cells in the testes. Spermatogenesis appears to be initiated during immature development (cryptocephalic pupa stage). From <24 h to 7 d post-emergence, male aging appeared to impact sperm production.

Comparative Evidence of an Exceptional Impact of Gene Duplication on the Developmental Evolution of Drosophila and the Higher Diptera

The importance of gene duplication in developmental body plan evolution is well established, but for many megadiverse clades such as true flies (Diptera), a comprehensive understanding is still just emerging through comparative genomics. In a survey of 377 developmental gene families, we found that in addition to the pea aphid, which has been previously shown to be genome-wide enriched with gene duplicates and was included as positive control, more than twice as many expanded developmental gene families were observed in Drosophila (49) compared to mosquito (21), flour beetle (20), and honey bee (14). Synonymous sequence divergence estimates and ortholog conservation analyses in additional dipteran genomes revealed that most Drosophila gene duplicates are ancient and accumulated during a time window that reaches back to the origin of brachyceran flies, approximately 180 million years ago. Further, available genetic data suggest that more than half of the Drosophila developmental gene duplicates remained partially or even fully redundant despite their ancient separation. We therefore speculate that the exceptional accumulation of developmental gene duplicates in Drosophila and the higher Diptera was proximally driven by the evolution of fast development, benefiting from increased genetic robustness. At the same time, the concomitant increase of opportunities for gene duplicate diversification appears to have been a source for developmental and phenotypic innovation during the unparalleled diversification of brachyceran Diptera.

Classification of Springs in the Lake Baikal Region by Macroinvertebrate Communities

The macrozoobenthos communities in 15 cold fresh, 12 mineral (sodium chloride), and 19 thermal springs of the Baikal Rift Zone and adjacent areas have been investigated. The classification of springs on the basis of quantitative indicators of abundance was proposed. According to the dominant group of fauna, three types of communities were identified for the cold springs; six types, for mineral springs; and four types, for thermal springs. It was established that communities with the dominance of Chironomidae, Turbellaria, and Oligochaeta are formed in cold springs and communities with the predominance of Gastropoda (one or two species) prevail in thermal springs. It was noted that mineral springs of medium mineralization are characterized by communities with a prevalence of Amphipoda (Gammarus lacustris); those with high salinity are dominated by Brachyceran flies (Diptera, Brachycera).

Taxon sampling to address an ancient rapid radiation: a supermatrix phylogeny of early brachyceran flies (Diptera)

Early diverging brachyceran fly lineages underwent a rapid radiation approximately 180 Ma, coincident in part with the origin of flowering plants. This region of the fly tree includes 25 000 described extant species with diverse ecological roles such as blood‐feeding (haematophagy), parasitoidism, predation, pollination and wood‐feeding (xylophagy). Early diverging brachyceran lineages were once considered a monophyletic group of families called Orthorrhapha, based on the shared character of a longitudinal break in the pupal skin made during the emergence of the adult. Yet other morphological and molecular evidence generally supports a paraphyletic arrangement of ‘Orthorrhapha’, with strong support for one orthorrhaphan lineage – dance flies and relatives – as the closest relative to all higher flies (Cyclorrhapha), together called Eremoneura. In order to establish a comprehensive estimate of the relationships among orthorrhaphan lineages using a thorough sample of publicly available data, we compiled and analysed a dataset including 1217 taxa representing major lineages and 20 molecular markers. Our analyses suggest that ‘Orthorrhapha’ excluding Eremoneura is not monophyletic; instead, we recover two main lineages of early brachyceran flies: Homeodactyla and Heterodactyla. Homeodactyla includes Nemestrinoidea (uniting two parasitic families Acroceridae + Nemestrinidae) as the closest relatives to the large SXT clade, comprising Stratiomyomorpha, Xylophagidae and Tabanomorpha. Heterodactyla includes Bombyliidae with a monophyletic Asiloidea (exclusive of Bombyliidae) as the closest relatives to Eremoneura. Reducing missing data, modifying the distribution of genes across taxa, and, in particular, removing rogue taxa significantly improved tree resolution and statistical support. Although our analyses rely on dense taxonomic sampling and substantial gene coverage, our results pinpoint the limited resolving power of Sanger sequencing‐era molecular phylogenetic datasets with respect to ancient, hyperdiverse radiations.

Evolution of Lower Brachyceran Flies (Diptera) and Their Adaptive Radiation with Angiosperms.

The Diptera (true flies) is one of the most species-abundant orders of Insecta, and it is also among the most important flower-visiting insects. Dipteran fossils are abundant in the Mesozoic, especially in the Late Jurassic and Early Cretaceous. Here, we review the fossil record and early evolution of some Mesozoic lower brachyceran flies together with new records in Burmese amber, including Tabanidae, Nemestrinidae, Bombyliidae, Eremochaetidae, and Zhangsolvidae. The fossil records reveal that some flower-visiting groups had diversified during the mid-Cretaceous, consistent with the rise of angiosperms to widespread floristic dominance. These brachyceran groups played an important role in the origin of co-evolutionary relationships with basal angiosperms. Moreover, the rise of angiosperms not only improved the diversity of flower-visiting flies, but also advanced the turnover and evolution of other specialized flies.

Phylogenetic Origin and Diversification of RNAi Pathway Genes in Insects.

RNA interference (RNAi) refers to the set of molecular processes found in eukaryotic organisms in which small RNA molecules mediate the silencing or down-regulation of target genes. In insects, RNAi serves a number of functions, including regulation of endogenous genes, anti-viral defense, and defense against transposable elements. Despite being well studied in model organisms, such as Drosophila, the distribution of core RNAi pathway genes and their evolution in insects is not well understood. Here we present the most comprehensive overview of the distribution and diversity of core RNAi pathway genes across 100 insect species, encompassing all currently recognized insect orders. We inferred the phylogenetic origin of insect-specific RNAi pathway genes and also identified several hitherto unrecorded gene expansions using whole-body transcriptome data from the international 1KITE (1000 Insect Transcriptome Evolution) project as well as other resources such as i5K (5000 Insect Genome Project). Specifically, we traced the origin of the double stranded RNA binding protein R2D2 to the last common ancestor of winged insects (Pterygota), the loss of Sid-1/Tag-130 orthologs in Antliophora (fleas, flies and relatives, and scorpionflies in a broad sense), and confirm previous evidence for the splitting of the Argonaute proteins Aubergine and Piwi in Brachyceran flies (Diptera, Brachycera). Our study offers new reference points for future experimental research on RNAi-related pathway genes in insects.

Broadband Photoreceptors Are Involved in Violet Light Preference in the Parasitoid Fly Exorista Japonica.

Phototaxis has been described in many insects, which are often attracted to specific wavelengths of light. However, little is known about phototaxis in parasitoid insect species that are potentially useful for integrated pest management. In this study, we investigated the wavelength dependency of the phototactic behavior of the parasitoid fly Exorista japonica and its possible mechanism. Multiple-choice tests with six monochromatic stimuli revealed that the flies were specifically attracted to violet light peaking at 405 nm, which was unexpected because insects are generally attracted to ultraviolet or green light. We measured the spectral sensitivity of the compound eye, and found that the sensitivity peaked at 340 nm, as in other brachyceran flies. We used statistical modeling and optimization of the process parameters to predict the type of photoreceptor contributing to the violet preference. The analysis revealed that the wavelength preference could be explained by linear models of the quanta received by photoreceptors, including the R1-6 broadband receptors. The broadband receptors appear to contribute positively, whereas the R7-8 narrowband receptors contribute negatively to achieve the violet preference; i.e., spectral opponency might be involved.

A remarkable brachyceran fly (Diptera: Tabanomorpha) from Late Cretaceous Burmese amber

A new brachyceran fly (Diptera: Tabanomorpha), Pseudorhagio zhangi gen. et sp. nov., is described from Late Cretaceous Burmese amber. It is tentatively placed as Family incertae sedis in Tabanomorpha and distinguished from other Tabanomorpha by the following unusual combination of characters: head wider than thorax; body densely covered with fine and short setae, devoid of macrosetae; scutum strongly convex, nearly spherical; scutellum rather small, convex; antennal flagellum elongated, tapering, unsegmented; vein R4 perpendicular to R5, and strongly curved; crossvein m-m very long, strongly sinuated; tibial spur formula 0, 2, 0. This discovery further confirms the high diversity of Tabanomorpha in Late Cretaceous Burmese amber. An updated list of brachyceran species in Burmese amber is given.

A field test of the effect of spiked ivermectin concentrations on the biodiversity of coprophagous dung insects in Switzerland.

Veterinary medical product residues can cause severe damage in the dung ecosystem. Depending on the manner of application and the time after treatment, the excreted concentration of a given pharmaceutical varies. The popular anthelmintic drug ivermectin can be applied to livestock in several different ways and is fecally excreted over a period of days to months after application. In a field experiment replicated in summer and autumn, the authors mixed 6 ivermectin concentrations plus a null control into fresh cow dung to assess the reaction of the dung insect community. Taxon richness of the insect dung fauna emerging from the dung, but not Hill diversity ((1) D) or the total number of individuals (abundance), decreased as ivermectin concentration increased. Corresponding declines in the number of emerging insects were found for most larger brachyceran flies and hymenopteran parasitoids, but not for most smaller nematoceran flies or beetles (except Hydrophilidae). Parallel pitfall traps recovered all major dung organism groups that emerged from the experimental dung, although at times in vastly different numbers. Ivermectin generally did not change the attractiveness of dung: differences in emergence therefore reflect differences in survival of coprophagous offspring of colonizing insects. Because sample size was limited to 6 replicates, the authors generally recommend more than 10 (seasonal) replicates and also testing higher concentrations than used in the present study as positive controls in future studies. Results accord with parallel experiments in which the substance was applied and passed through the cow's digestive system. In principle, therefore, the authors' experimental design is suitable for such higher-tier field tests of the response of the entire dung community to pharmaceutical residues, at least for ivermectin. Environ Toxicol Chem 2016;35:1947-1952. © 2015 SETAC.