Drosophila suzukii (Matsumura) is a species native to Western Asia that is able to pierce intact fruit during egg laying, causing it to be considered a fruit crop pest in many countries. Drosophila suzukii have a rapid expansion worldwide; occurrences were recorded in North America and Europe in 2008, and South America in 2013. Due to this rapid expansion, we modeled the potential distribution of this species using the Maximum Entropy Modeling (MaxEnt) algorithm and the Genetic Algorithm for Ruleset Production (GARP) using 407 sites with known occurrences worldwide and 11 predictor variables. After 1000 replicates, the value of the average area under the curve (AUC) of the model predictions with 1000 replicates was 0.97 for MaxEnt and 0.87 for GARP, indicating that both models had optimal performances. The environmental variables that most influenced the prediction of the MaxEnt model were the annual mean temperature, the maximum temperature of the warmest month, the mean temperature of the coldest quarter and the annual precipitation. The models indicated high environmental suitability, mainly in temperate and subtropical areas in the continents of Asia, Europe and North and South America, where the species has already been recorded. The potential for further invasions of the African and Australian continents is predicted due to the environmental suitability of these areas for this species.

Phylogenetic relationships of the subgenus Oxyphortica were reconstructed based on two mitochondrial genes (COI and ND2). The results revealed the paraphyly of Oxyphortica and supported high levels of cryptic diversity within this subgenus. By integrating morphological characteristics and molecular evidence, we identified 17 new species as members of Oxyphortica: S. (O.) amphigya sp. nov., S. (O.) armillata sp. nov., S. (O.) ashima sp. nov., S. (O.) bawo sp. nov., S. (O.) crypta sp. nov., S. (O.) gelea sp. nov., S. (O.) hengduanmontana sp. nov., S. (O.) jinmingi sp. nov., S. (O.) mengbalanaxi sp. nov., S. (O.) mouig sp. nov., S. (O.) setipes sp. nov., S. (O.) shangrila sp. nov., S. (O.) tsauri sp. nov., S. (O.) valleculata sp. nov., S. (O.) wanhei sp. nov., S. (O.) yangjin sp. nov. and S. (O.) hypophaia sp. nov. To test the early morphological identifications and confirm the species boundaries, different species delimitation methods, including Automatic Barcode Gap Discovery (ABGD) and Bayesian Phylogenetics and Phylogeography (BP&P), were used, together with traditional distance. All species boundaries were clearly defined. As Oxyphortica species are mainly distributed across Southwest China (e.g., 20 spp. from the Hengduan Mountains), the complex climate and topographic landforms of the area may be responsible for the high levels of species diversity and endemism.

Drosophila melanogaster is an important laboratory model for studies of antiviral immunity in invertebrates, and Drosophila species provide a valuable system to study virus host range and host switching. Here, we use metagenomic RNA sequencing of about 1600 adult flies to discover 25 new RNA viruses associated with six different drosophilid hosts in the wild. We also provide a comprehensive listing of viruses previously reported from the Drosophilidae. The new viruses include Iflaviruses, Rhabdoviruses, Nodaviruses, and Reoviruses, and members of unclassified lineages distantly related to Negeviruses, Sobemoviruses, Poleroviruses, Flaviviridae, and Tombusviridae. Among these are close relatives of Drosophila X virus and Flock House virus, which we find in association with wild Drosophila immigrans. These two viruses are widely used in experimental studies but have not been previously reported to naturally infect Drosophila. Although we detect no new DNA viruses, in D. immigrans and Drosophila obscura, we identify sequences very closely related to Armadillidium vulgare iridescent virus (Invertebrate iridescent virus 31), bringing the total number of DNA viruses found in the Drosophilidae to three.

The causes of high biological diversity in biodiversity hotspots have long been a major subject of study in conservation biology. To investigate this matter, we conducted a phylogeographic study of five Drosophila (Diptera: Drosophilidae) species from East and Southeast Asia: Drosophila albomicans Duda, D. formosana Duda, D. immigrans Sturtevant, D. melanogaster Meigen, and D. simulans Sturtevant. We collected 185 samples from 28 localities in eight countries. From each collected individual, we sequenced the autosomal extra sex comb gene (esc) and seven mitochondrial genes, including nicotinamide adenine dinucleotide hydrate-reductase dehydrogenase subunit 4 (ND4), ND4L, tRNA-His, tRNA-Pro, tRNA-Thr, partial ND5, and partial ND6. Phylogenetic analyses using maximum- likelihood and Bayesian methods revealed interesting population structure and identified the existence of two distinct D. formosana lineages (Southeast Asian and Taiwanese populations). Genetic differentiation among groups of D. immigrans suggests the possibility of endemic speciation in Taiwan. In contrast, D. melanogaster remained one extensively large population throughout East and Southeast Asia, including nearby islets. A molecular clock was used to estimate divergence times, which were compared with past geographical events to infer evolutionary scenarios. Our findings suggest that interglacial periods may have caused population isolation, thus enhancing population differentiation more strongly for some of the Drosophila species. The population structure of each Drosophila species in East and Southeast Asia has been influenced by past geographic events.

Since the early phase of the intercontinental dispersal of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), fermentation baits have been used for monitoring. Self-made lures and commercial products are often based on wine and vinegar. From an ecological perspective, the formulation of these baits is expected to target especially vinegar flies associated with overripe fruit, such as Drosophila melanogaster (Meigen) (Diptera: Drosophilidae). Hanseniaspora uvarum (Niehaus) (Ascomycota: Saccharomyceta) is a yeast closely associated with D. suzukii and fruit, and furthermore attractive to the flies. Based on this relation, H. uvarum might represent a suitable substrate for the development of lures that are more specific than vinegar and wine. In the field, we therefore, compared H. uvarum to a commercial bait that was based on vinegar and wine with respect to the number of trapped D. suzukii relative to other drosophilids and arthropods. Trap captures were higher with the commercial bait but specificity for D. suzukii was greater with H. uvarum. Moreover, H. uvarum headspace extracts, as well as a synthetic blend of H. uvarum volatiles, were assayed for attraction of D suzukii in a wind tunnel and in the field. Headspace extracts and the synthetic blend induced strong upwind flight in the wind tunnel and confirmed attraction to H. uvarum volatiles. Furthermore, baited with H. uvarum headspace extract and a drowning solution of aqueous acetic acid and ethanol, 74% of field captured arthropods were D. suzukii. Our findings suggest that synthetic yeast headspace formulations might advance the development of more selective monitoring traps with reduced by-catch.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genomic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families-genes directly mediating interactions with plant chemical defenses-underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many insect lineages are ancient (>150 million years ago (mya)), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several nonherbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza has among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant-binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on living plants (bitter or electrophilic phytotoxins) or their ancestral diet (fermenting plant volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight gene candidates that have also been linked to other dietary transitions in Drosophila.

Three Stigmatomyces species were detected on five drosophilid species from Japan. We report Stigmatomyces majewskii on Drosophila rufa and Drosophila suzukii, Stigmatomyces scaptodrosophilae on Scaptodrosophila coracina and Scaptodrosophila subtilis, and Stigmatomyces sacaptomyzae on Scaptomyza graminum. Except for Scaptomyza graminum, each of these species is a newly identified Stigmatomyces host. Our discovery that D. suzukii is a host of S. majewskii may provide new pest management approaches for this global agricultural pest insect.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genetic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families - genes directly mediating interactions with plant chemical defenses - underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many lineages are ancient (>150 million years ago [mya]), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several non-herbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza have among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on plants (bitter or electrophilic phytotoxins) or their ancestral diet (yeast and fruit volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight strong gene candidates that have also been linked to other dietary transitions in Drosophila .

Transposable elements (TEs) have the main role in shaping the evolution of genomes and host species, contributing to the creation of new genes and promoting rearrangements frequently associated with new regulatory networks. Support for these hypotheses frequently results from studies with model species, and Drosophila provides a great model organism to the study of TEs. Micropia belongs to the Ty3/Gypsy group of long terminal repeats (LTR) retroelements and comprises one of the least studied Drosophila transposable elements. In this study, we assessed the evolutionary history of Micropia within Drosophilidae, while trying to assist in the classification of this TE. At first, we performed searches of Micropia presence in the genome of natural populations from several species. Then, based on searches within online genomic databases, we retrieved Micropia-like sequences from the genomes of distinct Drosophilidae species. We expanded the knowledge of Micropia distribution within Drosophila species. The Micropia retroelements we detected consist of an array of divergent sequences, which we subdivided into 20 subfamilies. Even so, a patchy distribution of Micropia sequences within the Drosophilidae phylogeny could be identified, with incongruences between the species phylogeny and the Micropia phylogeny. Comparing the pairwise synonymous distance (dS) values between Micropia and three host nuclear sequences, we found several cases of unexpectedly high levels of similarity between Micropia sequences in divergent species. All these findings provide a hypothesis to the evolution of Micropia within Drosophilidae, which include several events of vertical and horizontal transposon transmission, associated with ancestral polymorphisms and recurrent Micropia sequences diversification.

The genus Dettopsomyia was established by Lamb in 1914 for a single species, De.formosa described therein. It contains 13 known species recorded from the Old World (the Oriental, Australasian, Palearctic and Afrotropical regions). In the present paper, five new species discovered from southern China are described as members of Dettopsomyia: De.acutipenis Wang & Gao, sp. nov., De.serripenis Wang & Gao, sp. nov., De.discontinua Wang & Gao, sp. nov., De.camelonota Wang, Li & Gao, sp. nov. and De.paranigrovittata Wang, Li & Gao, sp. nov. The new species were delimitated, based on not only morphological characters but also molecular data.

The survival of insect larvae often depends on the mother's choice of oviposition substrate, and thus, this choice is an essential part of an insect species' ecology. Especially species with narrow substrate preferences may suffer from changes in substrate availability triggered by, for example, climate change. Recent climate warming is affecting species directly (e.g., physiology) but also indirectly (e.g., biological interactions) leading to mismatching phenologies and distributions. However, the preferred oviposition substrate is still unknown for many drosophilid species, especially for those at higher elevations. In this study, we investigated the oviposition-substrate preference of the montane-alpine fly Drosophila nigrosparsa in rearing and multiple-choice experiments using natural substrates in the laboratory. Insect emergence from field-collected substrates was tested. More than 650 insects were reared from natural substrates, among them 152 drosophilids but no individual of D. nigrosparsa. In the multiple-choice experiments, D. nigrosparsa preferred ovipositing on mushrooms (> 93% of eggs); additionally, a few eggs were laid on berries but none on other substrates such as cow faeces, rotten plant material, and soil. The flies laid 24 times more eggs per day when mushrooms were included in the substrates than when they were excluded. We infer that D. nigrosparsa is a mushroom breeder with some variation in oviposition choice. The flies favoured some mushrooms over others, but they were not specialised on a single fungal taxon. Although it is unclear if and how climate change will affect D. nigrosparsa, our results indicate that this species will not be threatened by oviposition-substrate limitations in the near future because of the broad altitudinal distribution of the mushrooms considered here, even if the flies will have to shift upwards to withstand increasing temperatures.

The origin, diversification, and secondary loss of sexually dimorphic characters are common in animal evolution. In some cases, structurally and functionally similar traits have evolved independently in multiple lineages. Prominent examples of such traits include the male-specific grasping structures that develop on the front legs of many dipteran insects. In this report, we describe the evolution and development of one of these structures, the male-specific "sex brush." The sex brush is composed of densely packed, irregularly arranged modified bristles and is found in several distantly related lineages in the family Drosophilidae. Phylogenetic analysis using 250 genes from over 200 species provides modest support for a single origin of the sex brush followed by many secondary losses; however, independent origins of the sex brush cannot be ruled out completely. We show that sex brushes develop in very similar ways in all brush-bearing lineages. The dense packing of brush hairs is explained by the specification of bristle precursor cells at a near-maximum density permitted by the lateral inhibition mechanism, as well as by the reduced size of the surrounding epithelial cells. In contrast to the female and the ancestral male condition, where bristles are arranged in stereotypical, precisely spaced rows, cell migration does not contribute appreciably to the formation of the sex brush. The complex phylogenetic history of the sex brush can make it a valuable model for investigating coevolution of sex-specific morphology and mating behavior.

Phenylacetaldehyde (PAH), an aromatic odorant, exists in varied fruits including overripe bananas and prickly pear cactus, the 2 major host fruits of Drosophila melanogaster. It acts as a potent ligand for the Ionotropic receptor 84a (IR84a) and the Odorant receptor 67a (OR67a), serving as an important food and courtship cue for adult fruit flies. Drosophila melanogaster larvae respond robustly to diverse feeding odorants, such as ethyl acetate (EA), an aliphatic ester. Since the chemical identity and concentration of an odorant are vital neural information handled by the olfactory system, we studied how larvae respond to PAH, an aromatic food odorant with aphrodisiac properties for adult flies. Our findings revealed that PAH attracted larvae significantly in a dose-dependent manner. Larvae could also be trained with PAH associated to appetitive and aversive reinforcers. Thus, like EA, PAH might serve as an important odorant cue for larvae, aiding in food tracking and survival in the wild. Since IR84a/IR8a complex primarily governs PAH response in adult flies, we examined expression of Ir84a and Ir8a in early third-instar larvae. Our experiments showed the presence of Ir8a, a novel finding. However, contrary to adult flies, PAH-responsive Ir84a was not found. Our behavioral experiments with Ir8a1 mutant larvae exhibited normal chemotaxis to PAH, whereas Orco1 mutant showed markedly reduced chemotaxis, indicating an OR-mediated neural circuitry for sensing of PAH in larvae. The results obtained through this study are significantly important as information on how larvae perceive and process PAH odorant at the neuronal level is lacking.

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.

The genus Rhinoleucophenga Hendel (1917) encompasses 21 described species but is still not well known as a taxon. Even the currently described species lack full descriptions, i.e., illustrations of the male genitalia are absent, which makes their identification difficult. This study describes two new species of Rhinoleucophenga with the illustration of their male genitalia.

The plasma membrane of Drosophila (Sophophora) melanogaster spermatozoa contains an alpha-l-fucosidase that might be involved in fertilization by interacting with alpha-l-fucose residues on the micropyle of the eggshell. D. (S.) melanogaster has a single gene called CG6128 or Fuca encoding for a putative alpha-l-fucosidase. Two transcripts have been annotated, RA of 3514 bp, and RB of 1673 bp. While both transcripts encode an alpha-l-fucosidase, RA contains an upstream open reading frame, translated into a polypeptide containing a predicted BTB/POZ domain. We demonstrate that Fuca is expressed in male and female germ lines. RT-PCR analysis indicated a broader tissue expression. Homologous genes are expressed in the same tissues in several drosophilid flies belonging to the genera Drosophila and Scaptodrosophila. However, the long transcript is restricted to species belonging to the subgenus Sophophora. The presence of two transcripts in species of the subgenus Sophophora and only one in species belonging to the subgenus Drosophila might be related to the phylogenetic relationships of these subgenera. Immunofluorescence demonstrated that the gene product, localized to the sperm plasma membrane, is absent from Scaptodrosophila lebanonensis spermatozoa. These findings support the hypothesis that the enzyme is involved in the molecular events of primary gamete interactions that are conserved among drosophilids belonging to Drosophila genus.

Specific ecological interactions between insects and microbes have potential in the development of targeted pest monitoring or control techniques for the spotted wing drosophilid, Drosophila suzukii (Matsumura), an exotic invasive pest of soft fruit. To evaluate D. suzukii attraction to yeast species from preferred types of fruit, three yeasts were isolated from blackberry fruit and two yeasts from raspberry fruit and used to bait simple plastic bottle traps. Saccharomyces cerevisiae and Hanseniaspora uvarum were identified from blackberries, whereas a different H. uvarum strain was identified from raspberry. Yeast identification was based on sequence analysis of the D1/D2 domain of the large subunit 26S rRNA gene. Commercial baker's yeast (S. cerevisiae) was similar or more effective for the capture of D. suzukii males and females than yeasts isolated from blackberry or raspberry when grown in sucrose. However, when grown in corn syrup, a strain of S. cerevisiae from blackberry captured the highest number of females and a strain of H. uvarum isolated from raspberry captured high numbers of males and females. Species of Candida, Hanseniaspora, and Pichia from a laboratory yeast collection did not outperform baker's yeast in pairwise tests when grown in sucrose solution or yeast-peptone-dextrose medium. The raspberry strain of H. uvarum grown in corn syrup outperformed S. cerevisiae grown in sucrose, in terms of captures in baited traps under laboratory conditions. We conclude that yeast species, strain, and growth medium can have a marked influence on D. suzukii attraction to baited traps, a finding that could assist in the development of yeast-related monitoring or control techniques targeted at this pest.

The invasion of the spotted wing drosophila, Drosophila suzukii Matsumura, across the Americas and Europe has led to increased insecticide applications to protect fruit crops. This insecticide usage conflicts with integrated pest management programs, as well as harvest, export, and pollination services in the affected crops. A novel management tool was assessed against D. suzukii that may mitigate these conflicts. HOOK SWD, an attract-and-kill (A&K) formulation applied as a sprayable bait, was evaluated for three growing seasons in two berry crops in New Jersey and California. In blueberry crops treated with HOOK SWD, fruit infestations by D. suzukii were 2-8 times lower than in untreated crops. In trials in commercial raspberry fields, weekly or biweekly HOOK SWD applications combined with a single grower standard D. suzukii-targeted cover spray resulted in nearly 2-5 times fewer fruit infestations compared to the grower standard cover spray alone. Assays of the residual activity of HOOK SWD resulted in more than 78-93% adult D. suzukii mortality when exposed to raspberry leaves after the formulation had aged for 35 d in the field under plastic hoop houses. These results suggest that this A&K strategy can be integrated in D. suzukii management programs.

The mitotic metaphases of five Andean species of genus Drosophila are described for the first time. The evolutionary and interspecific genetic relationships within three Neotropical Drosophila species groups are analyzed. The diploid chromosome number for each species is as follows: D.cashapamba Céspedes et Rafael, 2012 2n = 6 (2V, 1J) (X = J, Y = R), D.ecuatoriana Vela et Rafael, 2004 2n = 10 (3R, 2V) (X = V, Y = R), D.ninarumi Vela et Rafael, 2005 2n = 10 (3R, 1V, 1D) (X = V, Y = R), D.urcu Vela et Rafael, 2005 2n = 12 (4R, 2V) (X = V, Y = R), D.valenteae Llangarí-Arizo et Rafael, 2018 2n = 8 (3R, 1J) (X = J, Y = R).

Island radiations present natural laboratories for studying the evolutionary process. The Hawaiian Drosophilidae are one such radiation, with nearly 600 described species and substantial morphological and ecological diversification. These species are largely divided into a few major clades, but the relationship between clades remains uncertain. Here, we present new assembled transcriptomes from 12 species across these clades, and use these transcriptomes to resolve the base of the evolutionary radiation. We recover a new hypothesis for the relationship between clades, and demonstrate its support over previously published hypotheses. We then use the evolutionary radiation to explore dynamics of concordance in phylogenetic support, by analyzing the gene and site concordance factors for every possible topological combination of major groups. We show that high bootstrap values mask low evolutionary concordance, and we demonstrate that the most likely topology is distinct from the topology with the highest support across gene trees and from the topology with highest support across sites. We then combine all previously published genetic data for the group to estimate a time-calibrated tree for over 300 species of drosophilids. Finally, we digitize dozens of published Hawaiian Drosophilidae descriptions, and use this to pinpoint probable evolutionary shifts in reproductive ecology as well as body, wing, and egg size. We show that by examining the entire landscape of tree and trait space, we can gain a more complete understanding of how evolutionary dynamics play out across an island radiation.