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Abstract BackgroundThe blowflyLucilia cuprinais a destructive parasite of sheep that causes flystrike or myiasis. Larvae consume the animal’s living flesh, producing large wounds that can lead to death. The main aim of this study was to identify genes that may play important roles in the behavior and physiology ofL. cuprinalarvae. MethodsAn RNA-Seq analysis of RNA from whole larvae at different developmental stages and third-instar head and gut tissues was used to identify sensory receptors and other genes relevant to the physiology ofL. cuprinalarvae. In addition, CRISPR/Cas9 gene editing was used to obtain a loss-of-function mutation for theL. cuprinaodorant coreceptor gene (LcupOrco). The response of mutant larvae and adult females to fresh and rotten meat at different temperatures was evaluated. ResultsThe RNA-Seq analysis suggested that odorant (OR), gustatory, ionotropic, andPickpocketreceptors may not play a central role in theL. cuprinalarval sensory signaling and digestive systems. Rather, ATP-binding cassettes (ABCs) were highly enriched in head and gut RNA, and odorant-binding proteins (OBPs) only in the head. To confirm that ORs are not essential for larval detection of rotten beef, diet-choice assays were performed including larvae and adults homozygous for a null mutation inLcupOrco. While the attraction of adult females to rotten beef was disrupted,LcupOrcomutant larvae showed no change in diet preference. ConclusionsThe expression pattern of the ABC and OBP gene families suggests a central role in the sensory system of theL. cuprinalarva for these receptors. Behavioral assays showed that ORs are essential for the adult female response to rotten beef, but not for larval behavior. These findings are consistent with high levels of expression ofLcupOrcoin the adult female antenna but very low expression in larvae. Graphical abstract 

What circumstances might justify deliberate, full extinction of a species? 

Abstract The New World Screwworm, Cochliomyia hominivorax (Calliphoridae), is the most important myiasis-causing species in America. Screwworm myiasis is a zoonosis that can cause severe lesions in livestock, domesticated and wild animals, and occasionally in people. Beyond the sanitary problems associated with this species, these infestations negatively impact economic sectors, such as the cattle industry. Here, we present a chromosome-scale assembly of C. hominivorax’s genome, organized in 6 chromosome-length and 515 unplaced scaffolds spanning 534 Mb. There was a clear correspondence between the D. melanogaster linkage groups A–E and the chromosomal-scale scaffolds. Chromosome quotient (CQ) analysis identified a single scaffold from the X chromosome that contains most of the orthologs of genes that are on the D. melanogaster fourth chromosome (linkage group F or dot chromosome). CQ analysis also identified potential X and Y unplaced scaffolds and genes. Y-linkage for selected regions was confirmed by PCR with male and female DNA. Some of the long chromosome-scale scaffolds include Y-linked sequences, suggesting misassembly of these regions. These resources will provide a basis for future studies aiming at understanding the biology and evolution of this devastating obligate parasite. 

Drosophila suzukii (D. suzukii) (Matsumura, 1931; Diptera: Drosophilidae), also known as spotted wing Drosophila , is a worldwide pest of fruits with soft skins such as blueberries and cherries. Originally from Asia, D. suzukii is now present in the Americas and Europe and has become a significant economic pest. Growers largely rely on insecticides for the control of D. suzukii . Genetic strategies offer a species-specific environmentally friendly way for suppression of D. suzukii populations. We previously developed a transgenic strain of D. suzukii that produced only males on a diet that did not contain tetracycline. The strain carried a single copy of the FL19 construct on chromosome 3. Repeated releases of an excess of FL19 males led to suppression of D. suzukii populations in laboratory cage trials. Females died as a consequence of overexpression of the tetracycline transactivator (tTA) and tTA-activated expression of the head involution defective proapoptotic gene. The aim of this study was to generate additional male-only strains that carried two copies of the FL19 transgene through crossing the original line with a piggyBac jumpstarter strain. Males that carried either two chromosome 3 or a singleX-linked transgene were identified through stronger expression of the red fluorescent protein marker gene. The brighter fluorescence of the X-linked lines was likely due to dosage compensation of the red fluorescent protein gene. In total, four X-linked lines and eleven lines with two copies on chromosome 3 were obtained, of which five were further examined. All but one of the strains produced only males on a diet without tetracycline. When crossed with wild type virgin females, all of the five two copy autosomal strains examined produced only males. However, the single copy X-linked lines did not show dominant female lethality. Five of the autosomal lines were further evaluated for productivity (egg to adult) and male competition. Based on these results, the most promising lines have been selected for future population suppression experiments with strains from different geographical locations. 

Abstract Blow flies (Diptera: Calliphoridae) occur worldwide and exhibit a wide range of larval feeding habits, including saprophagy, coprophagy, parasitism and predation. Understanding their biology is critical for medical and veterinary science and ecology. Calliphorids thrive across a range of habitats and exhibit complex life histories, with larvae developing immersed in their food substrate, while adults are free‐living and have diverse feeding strategies. Some species have evolved specialized parasitic associations with vertebrate or invertebrate hosts, which are behaviors with important implications for agriculture and for understanding evolutionary transitions between saprophagy and parasitism. This study presents a comprehensive phylogenetic analysis of the Calliphoridae, utilizing 711 of 736 analysed nuclear genes, using anchored hybrid enrichment, from a global collection of blow flies and their relatives. Our results provide a robust and novel reconstruction of the evolutionary history of this group, pinpointing major transitions in larval feeding habits. We argue that saprophagy evolved independently multiple times from invertebrate parasitic ancestors, with vertebrate parasitism emerging from a number of different feeding strategies. These findings challenge prior hypotheses and offer new insights into the adaptive traits driving trophic specialization and diversification in this group. 

The transformer ( tra ) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina . Sex-specific tra RNA splicing is controlled by Sex lethal ( Sxl ) in Drosophila melanogaster but is auto-regulated in L . cuprina . Sxl also represses X chromosome dosage compensation in female D . melanogaster . We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L . cuprina . In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L . cuprina . However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos. 

Abstract The evolution of obligate ectoparasitism in blowflies (Diptera: Calliphoridae) has intrigued scientists for over a century, and surprisingly, the genetics underlying this lifestyle remain largely unknown. Blowflies use odors to locate food and oviposition sites; therefore, olfaction might have played a central role in niche specialization within the group. In insects, the coreceptor Orco is a required partner for all odorant receptors (ORs), a major gene family involved in olfactory-evoked behaviors. Hence, we characterized the Orco gene in the New World screwworm, Cochliomyia hominivorax , a blowfly that is an obligate ectoparasite of warm-blooded animals. In contrast, most of the closely related blowflies are scavengers that lay their eggs on dead animals. We show that the screwworm Orco orthologue ( ChomOrco ) is highly conserved within Diptera, showing signals of strong purifying selection. Expression of ChomOrco is broadly detectable in chemosensory appendages, and is related to morphological, developmental, and behavioral aspects of the screwworm biology. We used CRISPR/Cas9 to disrupt ChomOrco and evaluate the consequences of losing the OR function on screwworm behavior. In two-choice assays, Orco mutants displayed an impaired response to floral-like and animal host-associated odors, suggesting that OR-mediated olfaction is involved in foraging and host-seeking behaviors in C . hominivorax . These results broaden our understanding of the chemoreception basis of niche occupancy by blowflies.