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Conditional sex transformation systems could improve genetic control strategies against insect pests. Here, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly,Lucilia cuprina. Using Tet-Off–regulated expression of Cas9 and dCas9, we targeted the sex-determining genetransformerwith the goal of converting females into males. Conditional Cas9 expression enabled knockout of a visual marker gene, confirming inducible genome editing. However, strains expressingtransformer-targeting sgRNA arrays did not undergo sex transformation. Embryonic microinjection oftransformer-targeting sgRNAs into Cas9-expressing embryos produced intersex individuals, indicating that sgRNA expression from the integrated arrays was insufficient to disrupt the sex determination pathway. In contrast, high-level dCas9 expression was associated with developmental delays, reduced body weight, and lethality. These findings establish the first conditional CRISPR expression system inL. cuprinaand demonstrate that Cas9 is compatible with inducible gene editing, whereas dCas9 is poorly tolerated at high expression levels. 

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 

Abstract BackgroundLucilia cuprina(Wiedemann, 1830) (Diptera: Calliphoridae) is the main causative agent of flystrike of sheep in Australia and New Zealand. Female flies lay eggs in an open wound or natural orifice, and the developing larvae eat the host’s tissues, a condition called myiasis. To improve our understanding of host-seeking behavior, we quantified gene expression in male and female antennae based on their behavior. MethodsA spatial olfactometer was used to evaluate the olfactory response ofL. cuprinamated males and gravid females to fresh or rotting beef. Antennal RNA-Seq analysis was used to identify sensory receptors differentially expressed between groups. ResultsLucilia cuprinafemales were more attracted to rotten compared to fresh beef (> fivefold increase). However, males and some females did not respond to either type of beef. RNA-Seq analysis was performed on antennae dissected from attracted females, non-attracted females and males. Transcripts encoding sensory receptors from 11 gene families were identified above a threshold (≥ 5 transcript per million) including 49 ATP-binding cassette transporters (ABCs), two ammonium transporters (AMTs), 37 odorant receptors (ORs), 16 ionotropic receptors (IRs), 5 gustatory receptors (GRs), 22 odorant-binding proteins (OBPs), 9 CD36-sensory neuron membrane proteins (CD36/SNMPs), 4 chemosensory proteins (CSPs), 4 myeloid lipid-recognition (ML) and Niemann-Pick C2 disease proteins (ML/NPC2), 2pickpocketreceptors (PPKs) and 3 transient receptor potential channels (TRPs). Differential expression analyses identified sex-biased sensory receptors. ConclusionsWe identified sensory receptors that were differentially expressed between the antennae of both sexes and hence may be associated with host detection by female flies. The most promising for future investigations were as follows: an odorant receptor (LcupOR46) which is female-biased inL. cuprinaandCochliomyia hominivoraxCoquerel, 1858; an ABC transporter (ABC G23.1) that was the sole sensory receptor upregulated in the antennae of females attracted to rotting beef compared to non-attracted females; a female-biased ammonia transporter (AMT_Rh50), which was previously associated with ammonium detection inDrosophila melanogasterMeigen, 1830. This is the first report suggesting a possible role for ABC transporters inL. cuprinaolfaction and potentially in other insects. 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. 

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.