Nalini Puniamoorthy

Unlocking the "Black box": internal female genitalia in Sepsidae (Diptera) evolve fast and are species-specific

BackgroundThe species-specificity of male genitalia has been well documented in many insect groups and sexual selection has been proposed as the evolutionary force driving the often rapid, morphological divergence. The internal female genitalia, in sharp contrast, remain poorly studied. Here, we present the first comparative study of the internal reproductive system of Sepsidae. We test the species-specificity of the female genitalia by comparing recently diverged sister taxa. We also compare the rate of change in female morphological characters with the rate of fast-evolving, molecular and behavioral characters.ResultsWe describe the ectodermal parts of the female reproductive tract for 41 species representing 21 of the 37 described genera and define 19 morphological characters with discontinuous variation found in eight structures that are part of the reproductive tract. Using a well-resolved molecular phylogeny based on 10 genes, we reconstruct the evolution of these characters across the family [120 steps; Consistency Index (CI): 0.41]. Two structures, in particular, evolve faster than the rest. The first is the ventral receptacle, which is a secondary sperm storage organ. It accounts for more than half of all the evolutionary changes observed (7 characters; 61 steps; CI: 0.46). It is morphologically diverse across genera, can be bi-lobed or multi-chambered (up to 80 chambers), and is strongly sclerotized in one clade. The second structure is the dorsal sclerite, which is present in all sepsids except Orygma luctuosum and Ortalischema albitarse. It is associated with the opening of the spermathecal ducts and is often distinct even among sister species (4 characters; 16 steps; CI: 0.56).ConclusionsWe find the internal female genitalia are diverse in Sepsidae and diagnostic for all species. In particular, fast-evolving structures like the ventral receptacle and dorsal sclerite are likely involved in post-copulatory sexual selection. In comparison to behavioral and molecular data, the female structures are evolving 2/3 as fast as the non-constant third positions of the COI barcoding gene. They display less convergent evolution in characters (CI = 0.54) than the third positions or sepsid mating behavior (CICOI= 0.36; CIBEHAV = 0.45).

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.

From kissing to belly stridulation: comparative analysis reveals surprising diversity, rapid evolution, and much homoplasy in the mating behaviour of 27 species of sepsid flies (Diptera: Sepsidae)

Our understanding of how fast mating behaviour evolves in insects is rather poor due to a lack of comparative studies among insect groups for which phylogenetic relationships are known. Here, we present a detailed study of the mating behaviour of 27 species of Sepsidae (Diptera) for which a well‐resolved and supported phylogeny is available. We demonstrate that mating behaviour is extremely diverse in sepsids with each species having its own mating profile. We define 32 behavioural characters and document them with video clips. Based on sister species comparisons, we provide several examples where mating behaviour evolves faster than all sexually dimorphic morphological traits. Mapping the behaviours onto the molecular tree reveals much homoplasy, comparable to that observed for third positions of mitochondrial protein‐encoding genes. A partitioned Bremer support (PBS) analysis reveals conflict between the molecular and behavioural data, but behavioural characters have higher PBS values per parsimony‐informative character than DNA sequence characters.

How sexual selection can drive the evolution of costly sperm ornamentation.

Post-copulatory sexual selection (PSS), fuelled by female promiscuity, is credited with the rapid evolution of sperm quality traits across diverse taxa. Yet, our understanding of the adaptive significance of sperm ornaments and the cryptic female preferences driving their evolution is extremely limited. Here we review the evolutionary allometry of exaggerated sexual traits (for example, antlers, horns, tail feathers, mandibles and dewlaps), show that the giant sperm of some Drosophila species are possibly the most extreme ornaments in all of nature and demonstrate how their existence challenges theories explaining the intensity of sexual selection, mating-system evolution and the fundamental nature of sex differences. We also combine quantitative genetic analyses of interacting sex-specific traits in D. melanogaster with comparative analyses of the condition dependence of male and female reproductive potential across species with varying ornament size to reveal complex dynamics that may underlie sperm-length evolution. Our results suggest that producing few gigantic sperm evolved by (1) Fisherian runaway selection mediated by genetic correlations between sperm length, the female preference for long sperm and female mating frequency, and (2) longer sperm increasing the indirect benefits to females. Our results also suggest that the developmental integration of sperm quality and quantity renders post-copulatory sexual selection on ejaculates unlikely to treat male–male competition and female choice as discrete processes.

SEXUAL SELECTION ACCOUNTS FOR THE GEOGRAPHIC REVERSAL OF SEXUAL SIZE DIMORPHISM IN THE DUNG FLY, SEPSIS PUNCTUM (DIPTERA: SEPSIDAE)

Sexual size dimorphism (SSD) varies widely across and within species. The differential equilibrium model of SSD explains dimorphism as the evolutionary outcome of consistent differences in natural and sexual selection between the sexes. Here, we comprehensively examine a unique cross‐continental reversal in SSD in the dung fly, Sepsis punctum. Using common garden laboratory experiments, we establish that SSD is male‐biased in Europe and female‐biased in North America. When estimating sexual (pairing success) and fecundity selection (clutch size of female partner) on males under three operational sex ratios (OSRs), we find that the intensity of sexual selection is significantly stronger in European versus North American populations, increasing with male body size and OSR in the former only. Fecundity selection on female body size also increases strongly with egg number and weakly with egg volume, however, equally on both continents. Finally, viability selection on body size in terms of intrinsic (physiological) adult life span in the laboratory is overall nil and does not vary significantly across all seven populations. Although it is impossible to prove causality, our results confirm the differential equilibrium model of SSD in that differences in sexual selection intensity account for the reversal in SSD in European versus North American populations, presumably mediating the ongoing speciation process in S. punctum.

A plea for digital reference collections and other science-based digitization initiatives in taxonomy: Sepsidnet as exemplar

1Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, 2Oxford University Museum of Natural History, Oxford, England, 3Natural History Museum, London, England, 4Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Prague, Czech Republic, 5Institute of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland, 6Escuela de Biología, Universidad de Costa Rica, Ciudad Universitaria, San Pedro, Costa Rica, 7Smithsonian Tropical Research Institute, Balboa, Panama, 8Departamento de Morfologia e Fisiologia Animal, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, São Paulo, Brazil, 9Natural History Museum, Entomology Section, Venice, Italy and 10University Scholars Programme, National University of Singapore, Singapore

Ivermectin sensitivity is an ancient trait affecting all ecdysozoa but shows phylogenetic clustering among sepsid flies

Avermectins are potent and popular veterinary pharmaceuticals used globally to fight parasites of livestock and humans. By disturbing ion channel transport through the membrane, avermectins are effective against endo‐ and ectoparasitic round and horsehair worms (Nematoida), insects, or ticks (Arthropoda), but not against Plathelminthes, including flatworms (Trematoda) and tapeworms (Cestoda), or segmented worms (Annelida). Unfortunately, excreted avermectins have strong nontarget effects on beneficial arthropods such as the insect community decomposing livestock dung, ultimately impeding this important ecosystem function to the extent that regulators mandate standardized eco‐toxicological tests of dung organisms worldwide. We show that the ancient phylogenetic pattern and qualitative mechanism of avermectin sensitivity is conserved and compatible with most recent phylogenomic hypotheses grouping the Nematoida with the Arthropoda as Ecdysozoa (molting animals). At the species level, we demonstrate phylogenetic clustering in ivermectin sensitivities of 23 species of sepsid dung flies (Diptera: Sepsidae). This clustered 500‐fold quantitative variation in sensitivity may indicate recent lineage‐specific responses to selection, but more likely reflects pre‐existing genetic variation with pleiotropic effects on eco‐toxicological responses to pollutants. Regardless, our results question the common practice in eco‐toxicology of choosing single test species to infer detrimental effects on entire species communities, which should ideally assess a representative taxonomic sample.

Standardized laboratory tests with 21 species of temperate and tropical sepsid flies confirm their suitability as bioassays of pharmaceutical residues (ivermectin) in cattle dung

Veterinary pharmaceuticals excreted in the dung of treated livestock can have strong non-target effects on the dung organism community. We report results of ecotoxicological tests with ivermectin for 21 species of temperate (Europe, North America) and tropical (Asia, Central America) black scavenger flies (Diptera: Sepsidae), using standardized methods developed previously for the yellow dung fly and the face fly. Our study documents great variation in ivermectin sensitivity of more than two orders of magnitude among species and even populations within species: estimated lethal effect concentrations LC(50) (at which 50% of the flies died) ranged from 0.05 to 18.55 μg/kg dung fresh weight (equivalent to 0.33-132.22 μg/kg dung dry weight). We also show that controlled laboratory tests can--within reasonable limits-be extended to the field or to laboratory settings without climate control, as obtained LC(50) were roughly similar. In addition to lethal effects, our study revealed relevant sub-lethal effects at lower ivermectin concentrations in terms of prolonged development, smaller body size and reduced juvenile growth rate. Finally, oviposition choice experiments showed that females generally do not discriminate against dung containing ivermectin residues. We conclude that sepsid flies are well suited test organisms for pharmaceutical residues in the dung of livestock due to their ease and speed of rearing and handling, particularly in the tropics, where high-tech laboratory equipment is often not available.

Secondarily reduced foreleg armature in Perochaeta dikowi sp.n. (Diptera: Cyclorrhapha: Sepsidae) due to a novel mounting technique

Abstract The males of almost all sepsid species have strongly modified forelegs that are used to clamp the female’s wingbase during mounting. Here, we describe a new species in the genus Perochaeta whose males have unmodified forelegs. We use DNA sequence data for ten genes to reconstruct the position of Perochaeta on the phylogenetic tree for Sepsidae, and reveal that the lack of foreleg armature in Perochaeta dikowi sp.n. is secondary. Through the study of the mating behaviour of the new species, we demonstrate that the loss of armature is correlated with a new mounting technique during which the males of P. dikowi do not use the foreleg to clamp the female’s wingbase. Instead, the male approaches the female from behind and bends his abdomen forwards in order to establish genital contact. Our study shows how data from morphology, phylogenetics, and behavioural biology can complement each other to yield a deeper understanding of how changes in morphology and behaviour are correlated.

Differential investment in pre‐ vs. post‐copulatory sexual selection reinforces a cross‐continental reversal of sexual size dimorphism in Sepsis punctum (Diptera: Sepsidae)

Theory predicts that males have a limited amount of resources to invest in reproduction, suggesting a trade‐off between traits that enhance mate acquisition and those that enhance fertilization success. Here, we investigate the relationship between pre‐ and post‐copulatory investment by comparing the mating behaviour and reproductive morphology of four European and five North American populations of the dung fly Sepsis punctum (Diptera) that display a reversal of sexual size dimorphism (SSD). We show that the geographic reversal in SSD between the continents (male biased in Europe, female biased in North America) is accompanied by differential investment in pre‐ vs. post‐copulatory traits. We find higher remating rates in European populations, where larger males acquire more matings and consequently have evolved relatively larger testes and steeper hyper‐allometry with body size. American populations, in sharp contrast, display much reduced, if any, effect of body size on those traits. Instead, North American males demonstrate an increased investment in mate acquisition prior to copulation, with more mounting attempts and a distinctive abdominal courtship display that is completely absent in Europe. When controlling for body size, relative female spermathecal size is similar on both continents, so we find no direct evidence for the co‐evolution of male and female internal reproductive morphology. By comparing allopatric populations of the same species that apparently have evolved different mating systems and consequently SSD, we thus indirectly demonstrate differential investment in pre‐ vs. post‐copulatory mechanisms increasing reproductive success.