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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 

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. 

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 

Abstract Decaying wood, while an abundant and stable resource, presents considerable nutritional challenges due to its structural rigidity, chemical recalcitrance, and low nitrogen content. Despite these challenges, certain insect lineages have successfully evolved saproxylophagy (consuming and deriving sustenance from decaying wood), impacting nutrient recycling in ecosystems and carbon sequestration dynamics. This study explores the uneven phylogenetic distribution of saproxylophagy across insects and delves into the evolutionary origins of this trait in disparate insect orders. Employing a comprehensive analysis of gut microbiome data, from both saproxylophagous insects and their non‐saproxylophagous relatives, including new data from unexplored wood‐feeding insects, this Hypothesis paper discusses the broader phylogenetic context and potential adaptations necessary for this dietary specialization. The study proposes the “Detritivore‐First Hypothesis,” suggesting an evolutionary pathway to saproxylophagy through detritivory, and highlights the critical role of symbiotic gut microbiomes in the digestion of decaying wood. 

IntroductionWood digestion in insects relies on the maintenance of a mosaic of numerous microhabitats, each colonized by distinct microbiomes. Understanding the division of digestive labor between these microhabitats- is central to understanding the physiology and evolution of symbiotic wood digestion. A microhabitat that has emerged to be of direct relevance to the process of lignocellulose digestion is the surface of ingested plant material. Wood particles in the guts of some termites are colonized by a specialized bacterial fiber-digesting microbiome, but whether this represents a widespread strategy among insect lineages that have independently evolved wood-feeding remains an open question. MethodsIn this study, we investigated the bacterial communities specifically associated with wood fibers in the gut of the passalid beetleOdontotaenius disjunctus. We developed a Percoll-based centrifugation method to isolate and enrich the wood particles from the anterior hindgut, allowing us to access the wood fibers and their associated microbiome. We then performed assays of enzyme activity and used short-read and long-read amplicon sequencing of the 16S rRNA gene to identify the composition of the fiber-associated microbiome. ResultsOur assays demonstrated that the anterior hindgut, which houses a majority of the bacterial load, is an important site for lignocellulose digestion. Wood particles enriched from the anterior hindgut contribute to a large proportion of the total enzyme activity. The sequencing revealed thatO. disjunctus, like termites, harbors a distinct fiber-associated microbiome, but notably, its community is enriched in insect-specific groups ofLactococcusandTuricibacter. DiscussionOur study underscores the importance of microhabitats in fostering the complex symbiotic relationships between wood-feeding insects and their microbiomes. The discovery of distinct fiber-digesting symbionts inO. disjunctus, compared to termites, highlights the diverse evolutionary paths insects have taken to adapt to a challenging diet. 

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. 

Abstract The Miltogramminae (Diptera: Sarcophagidae) includes ~600 species across >40 genera, which constitute ~20% of global Sarcophagidae. While molecular phylogenetic hypotheses have been produced for this group, critical problems persist, including the presence of paraphyletic genera, uncertain relationships between genera, a bias of sampling towards Palaearctic taxa, and low support for many branches. The present study remedies these issues through the application of Anchored Hybrid Enrichment (AHE) to a sample including ~60% of the currently recognised genera (16% of known species) representing all biogeographic regions except the Neotropical. An alignment of 1,281 concatenated loci was analysed with maximum likelihood (RAxML, IQ‐TREE), Bayesian inference (ExaBayes) and coalescent‐based approaches (ASTRAL, SVDquartets), which resulted in highly supported and concordant topologies, providing unprecedented insight into the relationships of this subfamily of flesh flies, allowing a major update to miltogrammine classification. The AHE phylogenetic hypothesis supports the monophyly of a large proportion of genera. The monophyly ofMetopiaMeigen is restored by synonymy withAenigmetopiaMalloch,syn.n.To achieve monophyly ofMiltogrammaMeigen, eight species are transferred fromPterellaRobineau‐Desvoidy. The genusPterellais shown to be paraphyletic in its current circumscription, and to restore generic monophylyPterellais restricted to contain onlyPt. grisea(Meigen).ErioproctaEnderlein,stat.rev., is resurrected. The genusSenotainiaMacquart is reconstructed as paraphyletic. The monotypic genusMetopodiaBrauer & Bergenstamm is synonymised withTaxigrammaMacquart,syn.n.In light of our phylogenetic hypotheses, a new Miltogramminae tribal classification is proposed, composed of six tribes. 

Abstract The genusLiriomyzaMik (Diptera: Agromyzidae) is a diverse and globally distributed group of acalyptrate flies. Phylogenetic relationships amongLiriomyzaspecies have remained incompletely investigated and have never been fully addressed using molecular data. Here, we reconstruct the phylogeny of the genusLiriomyzausing various phylogenetic methods (maximum likelihood, Bayesian inference, and gene tree coalescence) on target‐capture‐based phylogenomic datasets (nucleotides and amino acids) obtained from anchored hybrid enrichment (AHE). We have recovered tree topologies that are nearly congruent across all data types and methods, and individual clade support is strong across all phylogenetic analyses. Moreover, defined morphological species groups and clades are well‐supported in our best estimates of the molecular phylogeny.Liriomyza violivora(Spencer) is a sister group to all remaining sampledLiriomyzaspecies, and the well‐known polyphagous vegetable pests [L. huidobrensis(Blanchard),L. langeiFrick,L. bryoniae.(Kaltenbach),L. trifolii(Burgess),L. sativaeBlanchard, andL. brassicae(Riley)]. belong to multiple clades that are not particularly closely related on the trees. Often, closely relatedLiriomyzaspecies feed on distantly related host plants. We reject the hypothesis that cophylogenetic processes betweenLiriomyzaspecies and their host plants drive diversification in this genus. Instead,Liriomyzaexhibits a widespread pattern of major host shifts across plant taxa. Our new phylogenetic estimate forLiriomyzaspecies provides considerable new information on the evolution of host‐use patterns in this genus. In addition, it provides a framework for further study of the morphology, ecology, and diversification of these important flies. 

Blow flies (Lucilia sericataandPhormia regina) are necrophagous insects that interact with dense microbial reservoirs and are opportunistic vectors of human and animal pathogens. Despite constant exposure to diverse environmental microbes, it is unclear whether their bacterial communities are primarily acquired stochastically or shaped by host factors that could influence pathogen carriage. We conducted a systematic comparison of wildL. sericataandP. reginacollected from seven cities across an urban-rural gradient to determine whether microbiome composition is structured by host species identity or environmental variables. Using 16S rRNA gene sequencing of individual flies, we profiled bacterial communities and applied alpha- and beta-diversity analyses, PERMANOVA, and Random Forest classification to quantify species-level microbiome differentiation. Species identity was the strongest predictor of microbiome composition (PERMANOVA,p = 0.001), while location, land cover type, sampling month, and sex had no significant effects. Random Forest modeling identified multiple bacterial taxa that consistently distinguished the two species, includingIgnatzschineriaandDysgonomonas, which were enriched inP. regina, andVagococcusandEscherichia-Shigella, which were enriched inL. sericata. These taxa are of clinical relevance, withIgnatzschineriain particular increasingly reported from human myiasis and soft-tissue infections, sometimes exhibiting antimicrobial resistance. Our findings demonstrate that wild blow flies maintain species-specific microbiomes despite shared environments, suggesting that host identity strongly filters microbial communities. The presence of opportunistic pathogens within these structured microbiomes underscores the need to understand how blow fly–microbe associations contribute to pathogen persistence and dissemination. By revealing predictable, species-dependent microbiome patterns, this study highlights potential targets for microbiome-based strategies aimed at mitigating blow fly–associated disease risks.