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Thermal performance curves (TPCs) have been estimated in multiple ectotherm species to understand their thermal plasticity and adaptation and to predict the effect of global warming. However, TPCs are typically assessed under constant temperature regimes, so their reliability for predicting thermal responses in the wild where temperature fluctuates diurnally and seasonally remains poorly documented. Here, we use distant latitudinal populations of five species of sepsid flies (Diptera: Sepsidae) from the temperate region (Europe, North Africa, North America) to compare estimates derived from constant TPCs with observed development rate under fluctuating temperatures in laboratory and field conditions. TPCs changed across gradients in that flies originating from higher latitudes showed accelerated development at higher temperatures, an adaptive response. TPCs were then used to predict development rates observed under fluctuating temperatures; these predictions were relatively accurate in the laboratory but not the field. Interestingly, the precision of TPC predictions depended not only on the resolution of temperature data, with daily and overall temperature summing performing better than hourly temperature summing, but also on the frequency of temperatures falling below the estimated critical minimum temperature. Hourly temperature resolution most strongly underestimated actual development rates, because flies apparently either did not stop growing when temperatures dropped below this threshold, or they sped up their growth when the temperature rose again, thus most severely reflecting this error. We conclude that when flies do not encounter cold temperatures, TPC predictions based on constant temperatures can accurately reflect performance under fluctuating temperatures if adequately adjusted for nonlinearities, but when encountering cold temperatures, this method is more error‐prone. Our study emphasizes the importance of the resolution of temperature data and cold temperatures in shaping thermal reaction norms. Thermal performance curve (TPC) can be used to predict the response of ectotherms to thermal changes. This study, carried out on coprophagous flies, highlights the usefulness and limitations of TPC in predicting the effect of temperature fluctuations on development rate under laboratory and field conditions.

The influence of pre-copulatory courtship on mate acquisition has been well explored but the effects of copulatory courtship (or behaviours occurring after intromission) on reproductive fitness largely remains underexplored. In insects, behaviour performed during copulation often involves rapid, successive movements with several limbs and wing beats, which are often difficult to observe and quantify. There is also a lack of understanding of how behavioural plasticity can compensate for physical impairments (e.g. loss of limbs) during copulation. Here, we studied the reproductive consequences of limb loss using two North American (NA) and two European (EU) populations of Sepsis punctum flies that are known to employ various limbs to interact with females during copulation. We observed plasticity in copulatory behaviour as a consequence of limb loss, giving rise to novel behavioural characters. The loss of different sets of limbs (forelegs vs. midlegs vs. hindlegs) significantly decreased mating success (i.e., successful copulations), but individuals that did copulate, did not vary in fertilisation success (i.e., number of offspring), suggesting that plasticity in copulatory behaviour has consequences for mate acquisition and less for fertilization success. Notably, we observed population level differences in baseline copulatory behaviour, and higher levels of plasticity for EU populations when compensating for limb loss impairments. Our results suggest that limb loss in S. punctum males influences the behavioural repertoire performed during copulation, but population-specific differences in plasticity may be able to compensate for reproductive success. Significance statement Courtship influences non-random mate choice via mate acquisition and fertilisation success. Across the courtship phases (pre-copulatory, copulatory, and post-copulatory), copulatory behaviours remain a mystery. The widespread dung fly, Sepsis punctum , displays a variety of contact behaviours using all sets of limbs. We observed continental differences between North American and European populations, with North American populations displaying a wider repertoire of copulatory behaviours. We also observed that the loss of different sets of limbs (e.g. forelegs, midlegs, hindlegs) impacted mate acquisition to different extents but it did not significantly influence fertilization success. Notably, novel copulatory behaviours and behavioural plasticity could rescue copulatory courtship particularly with the loss of midlegs that are important for behavioural interactions in sepsid flies.

Sexual selection can displace traits acting as ornaments or armaments from their viability optimum in one sex, ultimately giving rise to sexual dimorphism. The degree of dimorphism should not only mirror the strength of sexual selection but also the net viability costs of trait maintenance at equilibrium. As the ability of organisms to bear exaggerated traits will depend on their condition, more sexually dimorphic traits should also exhibit greater sex differences in condition dependence. While this has been demonstrated among traits within species, similar patterns are expected across the phylogeny. We investigated this prediction within and across 11 (sub)species of sepsid flies with varying mating systems. When estimating condition dependence for seven sexual and nonsexual traits that vary in their sexual dimorphism, we not only found a positive relationship between the sex difference in allometric slopes (our measure of condition dependence) and relative trait exaggeration within species but also across species for those traits expected to be under sexual selection. Species with more pronounced male aggression further had relatively larger and more condition-dependent male fore- and midlegs. Our comparative study suggests a common genetic/developmental basis of sexual dimorphism and sex-specific plasticity that evolves across the phylogeny—and that the evolution of size consistently alters scaling relationships and thus contributes to the allometric variation of sexual armaments or ornaments in animals.

Sexual size dimorphism (SSD) arises when the net effects of natural and sexual selection on body size differ between the sexes. Quantitative SSD variation between taxa is common, but directional intraspecific SSD reversals are rare. We combined micro- and macroevolutionary approaches to study geographic SSD variation in closely related black scavenger flies. Common garden experiments revealed stark intra- and interspecific variation: Sepsis biflexuosa is monomorphic across the Holarctic, while S. cynipsea (only in Europe) consistently exhibits female-biased SSD. Interestingly, S. neocynipsea displays contrasting SSD in Europe (females larger) and North America (males larger), a pattern opposite to the geographic reversal in SSD of S. punctum documented in a previous study. In accordance with the differential equilibrium model for the evolution of SSD, the intensity of sexual selection on male size varied between continents (weaker in Europe), whereas fecundity selection on female body size did not. Subsequent comparative analyses of 49 taxa documented at least six independent origins of male-biased SSD in Sepsidae, which is likely caused by sexual selection on male size and mediated by bimaturism. Therefore, reversals in SSD and the associated changes in larval development might be much more common and rapid and less constrained than currently assumed.

Coprophagous insects frequently encounter veterinary medication residues that are excreted unmetabolized in livestock dung. These residues often negatively affect insect survival, reproduction, and ecosystem services and may contribute to the rapid decline in insect populations. Ivermectin is an antiparasitic drug widely used to treat parasites in livestock. While it has long been recognized that ivermectin residues affect insect survival, the potential interactive effects between ivermectin exposure and other ecologically relevant abiotic stressors remain poorly understood. Here, we study these effects in the black scavenger fly Sepsis neocynipsea, which depends on cow dung for reproduction. Using a fully factorial experimental design, we test whether the effects of ivermectin exposure on adult survival interact with heat and desiccation stress and whether the severity of these effects depends on an individual's size and sex. We found that ivermectin exposure had strong negative impacts on adult survival overall, but that mortality was approximately three times higher in females compared to males. The combination of ivermectin exposure, heat, and desiccation stress drastically reduced survival. Interestingly, individuals simultaneously exposed to heat and ivermectin stress survived better—on average— than individuals only exposed to ivermectin, suggesting potential hormetic effects. Taken together, our findings highlight how the complex interactions between veterinary pharmaceuticals and abiotic stressors could drive changes in coprophagous insect populations and their ecological functions. Veterinary medication residues in livestock dung, such as the antiparasitic drug ivermectin, can negatively affect insects that rely on dung for reproduction, impacting survival, reproduction, and ecosystem functions. This study on the dung fly Sepsis neocynipsea reveals that ivermectin exposure significantly reduces adult survival, particularly in females and larger individuals, and interacts with heat stress to produce non‐additive effects, suggesting cross‐resistance. These findings underscore the complex interplay between insecticides and abiotic stressors in driving changes in insect populations and ecosystem dynamics.

The dipteran fauna from the Brazilian Amazon remains poorly known, including that of forensic interest, such as the Sepsidae. The sepsid fauna of two secondary forests in the eastern Amazon was surveyed utilizing rotting bovine lung and feces (humans, bovines, and monkeys).We obtained 17 sespid species in six genera, most of them on bovine dung and rotting bovine lung. Two new species are described herein: Archisepsis bosque sp. nov. and Archisepsis verae sp. nov. The genus Palaeosepsis Duda, 1926 and the species Archisepsis polychaeta (Ozerov, 1993), Archisepsis diversiformis (Ozerov, 1993), Microsepsis mitis (Curran, 1927), Meropliosepsis sexsetosa Duda, 1926, and Palaeosepsis cf. golovastik Ozerov, 2004 are newly recorded from the Brazilian Amazon.

Sepsid flies (Diptera: Sepsidae) are important model insects for sexual selection research. In order to develop mitochondrial (mt) genome data for this significant group, we sequenced the first complete mt genome of the sepsid fly Nemopoda mamaevi Ozerov, 1997. The circular 15,878 bp mt genome is typical of Diptera, containing all 37 genes usually present in bilaterian animals. We discovered inaccurate annotations of fly mt genomes previously deposited on GenBank and thus re-annotated all published mt genomes of Cyclorrhapha. These re-annotations were based on comparative analysis of homologous genes, and provide a statistical analysis of start and stop codon positions. We further detected two 18 bp of conserved intergenic sequences from tRNAGlu-tRNAPhe and ND1-tRNASer(UCN) across Cyclorrhapha, which are the mtTERM binding site motifs. Additionally, we compared automated annotation software MITOS with hand annotation method. Phylogenetic trees based on the mt genome data from Cyclorrhapha were inferred by Maximum-likelihood and Bayesian methods, strongly supported a close relationship between Sepsidae and the Tephritoidea.

The Sepsidae family, commonly known as black scavenger flies or ensign flies, is a diverse group of flies found worldwide. In India, this family is represented by 45 species across 11 genera under two subfamilies: Sepsinae and Toxopodinae. Out of which 8 species are found in Assam. Despite their ecological importance as decomposers and scavengers, the distribution of Indian Sepsidae remains poorly documented. This study aims to create a preliminary distribution map of Sepsidae species in India using historical records and the open-source geographic information system software QGIS. Occurrence data for Indian Sepsidae species were compiled from published literature, museum specimens, and online biodiversity databases. The data were cleaned, georeferenced, and mapped using QGIS. Preliminary analysis reveals that the majority of records are concentrated in the northeastern states of India, particularly Assam and West Bengal. Other regions, such as the Western Ghats and central India, show fewer records, likely due to under sampling rather than true absence. Several species appear to have wide distributions spanning multiple states, while others seem to be more localized. This study provides the first comprehensive attempt to map the historical distribution of Sepsidae in India. The resulting maps highlight regions that require further sampling to better understand the true diversity and range of this family. Future work will focus on incorporating additional data sources, analyzing patterns of endemism, and assessing the potential impact of environmental factors on Sepsidae distribution. The generated maps can serve as a baseline for monitoring changes in Sepsidae populations and guide targeted surveys to fill knowledge gaps.

Clinal variation in body size and related life history traits is common and has stimulated the postulation of several ecogeographical rules. Whereas some clinal patterns are clearly adaptive, the causes of others remain obscure. We investigated intra-specific body size, development time and female fecundity (egg size and number) clines across 13 European populations of the dung fly Sepsis fulgens spanning 20° latitude from southern Italy to Estonia in a genetic common garden approach. Despite very short generation times (ca. 2 weeks at 24 °C), we found a converse Bergmann cline (smaller size at higher latitudes). As development time did not change with latitude (flat cline), integral growth rate thus likely declines towards the pole. At the same time, early fecundity, but not egg size, increased with latitude. Rather than being mediated by seasonal time constraints, the body size reduction in the northernmost flies from Estonia could suggest that these are marginal, edge populations, as when omitting them the body size cline became flat as well. Most of the other sepsid species investigated to date also show flat body size clines, a pattern that strikingly differs from Drosophila. We conclude that S. fulgens life history traits appear to be shaped by similar environmental pressures and selective mechanisms across Europe, be they adaptive or not. This reiterates the suggestion that body size clines can result as a secondary consequence of selection pressures shaping an entire life history syndrome, rendering them inconsistent and unpredictable in general.

The gene expression pattern specified by an animal regulatory sequence is generally viewed as arising from the particular arrangement of transcription factor binding sites it contains. However, we demonstrate here that regulatory sequences whose binding sites have been almost completely rearranged can still produce identical outputs. We sequenced the even-skipped locus from six species of scavenger flies (Sepsidae) that are highly diverged from the model species Drosophila melanogaster, but share its basic patterns of developmental gene expression. Although there is little sequence similarity between the sepsid eve enhancers and their well-characterized D. melanogaster counterparts, the sepsid and Drosophila enhancers drive nearly identical expression patterns in transgenic D. melanogaster embryos. We conclude that the molecular machinery that connects regulatory sequences to the transcription apparatus is more flexible than previously appreciated. In exploring this diverse collection of sequences to identify the shared features that account for their similar functions, we found a small number of short (20-30 bp) sequences nearly perfectly conserved among the species. These highly conserved sequences are strongly enriched for pairs of overlapping or adjacent binding sites. Together, these observations suggest that the local arrangement of binding sites relative to each other is more important than their overall arrangement into larger units of cis-regulatory function.