Barometric pressure decrease induces density-dependent changes in foraging behaviour in a parasitoid fly.

Barometric pressure is an environmental factor involved in the modulation of a variety of activities in insects. Generally, a drop in barometric pressure precedes the arrival of weather conditions that can affect insect activities and life expectancy. We simulated different scenarios of pressure drop in a modified hermetic chamber and studied their influence on the host-seeking behaviour of the larvae of the robber fly Mallophora ruficauda using air stationary olfactometers. In addition, we studied whether larval density modulates orientation to the host under the same scenarios of pressure drop. We found that motivation to search for hosts is affected by the same slope of pressure drop in both low- and high-density larvae. However, larval density modulates the onset of the responses to pressure decrease, as low-density larvae stop searching for hosts more quickly than high-density larvae. This result reflects an avoidance strategy according to which low-density larvae would have a reduced host range and higher risk of mortality and fewer chances to find a suitable host under adverse pressure conditions. Low-density larvae, known to prefer healthy hosts, do not search for parasitized hosts under normal pressure conditions nor under a range of pressure drops, strongly suggesting that host selectivity is not modulated by barometric pressure. This study paves the way to a better understanding of the changes in crucial insect behaviours induced by weather conditions, and provides more knowledge about the risk factors likely to affect insect survival in the context of foraging ecology.

The evolutionary dynamics of venom toxins made by insects and other animals.

Animal venoms are recognised as unique biological systems in which to study molecular evolution. Venom use has evolved numerous times among the insects, and insects today use venom to capture prey, defend themselves from predators, or to subdue and modulate host responses during parasitism. However, little is known about most insect venom toxins or the mode and tempo by which they evolve. Here, I review the evolutionary dynamics of insect venom toxins, and argue that insects offer many opportunities to examine novel aspects of toxin evolution. The key questions addressed are: How do venomous animals evolve from non-venomous animals, and how does this path effect the composition and pharmacology of the venom? What genetic processes (gene duplication, co-option, neofunctionalisation) are most important in toxin evolution? What kinds of selection pressures are acting on toxin-encoding genes and their cognate targets in envenomated animals? The emerging evidence highlights that venom composition and pharmacology adapts quickly in response to changing selection pressures resulting from new ecological interactions, and that such evolution occurs through a stunning variety of genetic mechanisms. Insects offer many opportunities to investigate the evolutionary dynamics of venom toxins due to their evolutionary history rich in venom-related adaptations, and their quick generation time and suitability for culture in the laboratory.

Host gut microorganisms' cues mediate orientation behaviour in the larva of the parasitoid Mallophora ruficauda.

The robber fly Mallophora ruficauda is one of the most important apicultural pests in the Pampas region of Argentina. This species is a parasitoid of scarab beetle larvae. Females lay eggs away from the host, and the larvae perform active search behaviour toward Cyclocephala signaticollis third instar larvae, parasitoid's preferred host. This behaviour is mediated by host-related chemical cues produced in hosts' fermentation chamber. Also, C. signaticollis larvae are attracted to fermentation chamber extracts. As scarab larvae have microbe-rich fermentation chamber, it has been suggested that microorganisms could be involved in the production of these semiochemicals. The aims of this work were first to ascertain the presence of microorganisms in the fermentation chamber of C. signaticollis larvae and second to determine the role of microorganisms in the orientation response of parasitoid and host larvae. We found that microorganisms-free C. signaticollis larvae showed deterioration in their development and did not produce the attractive semiochemicals. Therefore, we isolated fermentation chamber microorganisms of host larvae by means of different cultures media, and then, assayed different microorganisms' stimuli by binary choice tests. We were able to isolate microorganisms and determine that M. ruficauda larvae are attracted to semiochemicals from protein degradation in the fermentation chamber. However, C. signaticollis larvae were not attracted to any semiochemicals associated with microorganisms' activity in the fermentation chamber. Although we were unable to elucidate the exact role of gut microorganisms in host behaviour, we discuss their relevance in parasitoid host-seeking behaviour and host conspecific interaction in M. ruficauda-C. signaticollis system.

Predictive saccades and decision making in the beetle-predating saffron robber fly.

Internal predictions about the sensory consequences of self-motion, encoded by corollary discharge, are ubiquitous in the animal kingdom, including for fruit flies, dragonflies, and humans. In contrast, predicting the future location of an independently moving external target requires an internal model. With the use of internal models for predictive gaze control, vertebrate predatory species compensate for their sluggish visual systems and long sensorimotor latencies. This ability is crucial for the timely and accurate decisions that underpin a successful attack. Here, we directly demonstrate that the robber fly Laphria saffrana, a specialized beetle predator, also uses predictive gaze control when head tracking potential prey. Laphria uses this predictive ability to perform the difficult categorization and perceptual decision task of differentiating a beetle from other flying insects with a low spatial resolution retina. Specifically, we show that (1) this predictive behavior is part of a saccade-and-fixate strategy, (2) the relative target angular position and velocity, acquired during fixation, inform the subsequent predictive saccade, and (3) the predictive saccade provides Laphria with additional fixation time to sample the frequency of the prey's specular wing reflections. We also demonstrate that Laphria uses such wing reflections as a proxy for the wingbeat frequency of the potential prey and that consecutively flashing LEDs to produce apparent motion elicits attacks when the LED flicker frequency matches that of the beetle's wingbeat cycle.

Pick Your Poison: Molecular Evolution of Venom Proteins in Asilidae (Insecta: Diptera).

Robber flies are an understudied family of venomous, predatory Diptera. With the recent characterization of venom from three asilid species, it is possible, for the first time, to study the molecular evolution of venom genes in this unique lineage. To accomplish this, a novel whole-body transcriptome of Eudioctria media was combined with 10 other publicly available asiloid thoracic or salivary gland transcriptomes to identify putative venom gene families and assess evidence of pervasive positive selection. A total of 348 gene families of sufficient size were analyzed, and 33 of these were predicted to contain venom genes. We recovered 151 families containing homologs to previously described venom proteins, and 40 of these were uniquely gained in Asilidae. Our gene family clustering suggests that many asilidin venom gene families are not natural groupings, as delimited by previous authors, but instead form multiple discrete gene families. Additionally, robber fly venoms have relatively few sites under positive selection, consistent with the hypothesis that the venoms of older lineages are dominated by negative selection acting to maintain toxic function.

Current condition of Bathypogon types (Diptera: Asilidae) described by Frank M. Hull and housed in the South Australian Museum of Natural History with notes on other Bathypogon types.

The current condition of the types of Bathypogon, described by Frank M. Hull 1956-1959, are listed. By his own admission, a portion of the material borrowed from the South Australian Museum was destroyed by "pests". Note is made that the holotype of Bathypogon microdonturus is missing and presumed destroyed. Additions are made to the published data based on data on the Type specimen labels. Photos of the hypandrium of some Bathypogon holotypes that reside in the SA Museum collection are provided.

Entomo-venomics: The evolution, biology and biochemistry of insect venoms.

The insects are a hyperdiverse class containing more species than all other animal groups combined-and many employ venom to capture prey, deter predators and micro-organisms, or facilitate parasitism or extra-oral digestion. However, with the exception of those made by Hymenoptera (wasps, ants and bees), little is known about insect venoms. Here, we review the current literature on insects that use venom for prey capture and predator deterrence, finding evidence for fourteen independent origins of venom usage among insects, mostly among the hyperdiverse holometabolan orders. Many lineages, including the True Bugs (Heteroptera), robber flies (Asilidae), and larvae of many Neuroptera, Coleoptera and Diptera, use mouthpart-associated venoms to paralyse and pre-digest prey during hunting. In contrast, some Hymenoptera and larval Lepidoptera, and one species of beetle, use non-mouthpart structures to inject venom in order to cause pain to deter potential predators. Several recently published insect venom proteomes indicate molecular convergence between insects and other venomous animal groups, with all insect venoms studied so far being potently bioactive cocktails containing both peptides and larger proteins, including novel peptide and protein families. This review summarises the current state of the field of entomo-venomics.

A Dipteran's Novel Sucker Punch: Evolution of Arthropod Atypical Venom with a Neurotoxic Component in Robber Flies (Asilidae, Diptera).

Predatory robber flies (Diptera, Asilidae) have been suspected to be venomous due to their ability to overpower well-defended prey. However, details of their venom composition and toxin arsenal remained unknown. Here, we provide a detailed characterization of the venom system of robber flies through the application of comparative transcriptomics, proteomics and functional morphology. Our results reveal asilid venoms to be dominated by peptides and non-enzymatic proteins, and that the majority of components in the crude venom is represented by just ten toxin families, which we have named Asilidin1-10. Contrary to what might be expected for a liquid-feeding predator, the venoms of robber flies appear to be rich in novel peptides, rather than enzymes with a putative pre-digestive role. The novelty of these peptides suggests that the robber fly venom system evolved independently from hematophagous dipterans and other pancrustaceans. Indeed, six Asilidins match no other venom proteins, while three represent known examples of peptide scaffolds convergently recruited to a toxic function. Of these, members of Asilidin1 closely resemble cysteine inhibitor knot peptides (ICK), of which neurotoxic variants occur in cone snails, assassin bugs, scorpions and spiders. Synthesis of one of these putative ICKs, U-Asilidin1-Mar1a, followed by toxicity assays against an ecologically relevant prey model revealed that one of these likely plays a role as a neurotoxin involved in the immobilization of prey. Our results are fundamental to address these insights further and to understand processes that drive venom evolution in dipterans as well as other arthropods.

Buzz Kill: Function and Proteomic Composition of Venom from the Giant Assassin Fly Dolopus genitalis (Diptera: Asilidae).

Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly Dolopus genitalis using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies (Lucilia cuprina) produced rapid contractile paralysis (PD50 at 1 min = 3.1 µg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/µL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/ß defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom.

Genomic and transcriptomic resources for assassin flies including the complete genome sequence of Proctacanthus coquilletti (Insecta: Diptera: Asilidae) and 16 representative transcriptomes.

A high-quality draft genome for Proctacanthus coquilletti (Insecta: Diptera: Asilidae) is presented along with transcriptomes for 16 Diptera species from five families: Asilidae, Apioceridae, Bombyliidae, Mydidae, and Tabanidae. Genome sequencing reveals that P. coquilletti has a genome size of approximately 210 Mbp and remarkably low heterozygosity (0.47%) and few repeats (15%). These characteristics helped produce a highly contiguous (N50 = 862 kbp) assembly, particularly given that only a single 2 × 250 bp PCR-free Illumina library was sequenced. A phylogenomic hypothesis is presented based on thousands of putative orthologs across the 16 transcriptomes. Phylogenetic relationships support the sister group relationship of Apioceridae + Mydidae to Asilidae. A time-calibrated phylogeny is also presented, with seven fossil calibration points, which suggests an older age of the split among Apioceridae, Asilidae, and Mydidae (158 mya) and Apioceridae and Mydidae (135 mya) than proposed in the AToL FlyTree project. Future studies will be able to take advantage of the resources presented here in order to produce large scale phylogenomic and evolutionary studies of assassin fly phylogeny, life histories, or venom. The bioinformatics tools and workflow presented here will be useful to others wishing to generate de novo genomic resources in species-rich taxa without a closely-related reference genome.

Australian species of Ommatius Wiedemann (Diptera: Asilidae) with an anepimeral bristle.

The presence of an anepimeral bristle in Australian Ommatius Wiedemann species is newly recorded and descriptions of seven new species possessing this character are presented: O. aquilonaris sp. nov., O. burwelli sp. nov., O. imaginis sp. nov., O. limbatus sp. nov., O. melasmus sp. nov., O. musselbrookensis sp. nov. and O. radamnis sp. nov. All species occur in Queensland but O. melasmus sp. nov. also occurs in New South Wales and O. musselbrookensis sp. nov. also occurs in the Northern Territory. A key to separate the species is presented.

Longivena, a new robber-fly genus from Brazil (Diptera, Asilidae, Asilinae).

Longivena gen. n. and five new species are described and illustrated from caatinga and cerrado habitats from Brazil: Longivenadigitata sp. n., type-species (Maranhão, Bahia, Minas Gerais and Mato Grosso do Sul states), Longivenabilobata sp. n. (Maranhão state), Longivenaflava sp. n. (Mato Grosso do Sul state), Longivenalimeiraoliverai sp. n. (Maranhão state), Longivenaspatulata sp. n. (Maranhão state). An illustrated key is also provided.

Review of the species of Michotamia from China with a description of a new species (Diptera, Asilidae).

Three species of Michotamia are recorded from China. Of these Michotamia aurata (Fabricius, 1794) was previously reported from Hainan and Taiwan. Michotamia assamensis Joseph & Parui, 1995 is recorded from China and Laos for the first time, and Michotamia yunnanensissp. n., is described and figured. A key to the known species from China is provided. A new name, Michotamia subnigra, is given to Michotamia nigra Scarbrough & Hill, 2000, which is preoccupied by Michotamia nigra (Meijere, 1911).