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Abstract Drosophila glue, a bioadhesive produced by fly larvae to attach themselves to a substrate for several days, has recently gained attention for its peculiar adhesive and mechanical properties. Although Drosophila glue production was described more than 50 years ago, a general survey of the adhesive and mechanical properties of this proteinaceous gel across Drosophila species is lacking. To measure adhesion, we present here a protocol that is robust to variations in protocol parameters, pupal age and calculation methods. We find that the glue, which covers the entire pupal surface, increases the animal rigidity and plasticity when bound to a glass slide. Our survey of pupal adhesion in 25 Drosophilidae species reveals a wide range of phenotypes, from species that produce no or little glue and adhere little, to species that produce high amounts of glue and adhere strongly. One species, D. hydei, stands out from the rest and emerges as a promising model for the development of future bioadhesives, as it has the highest detachment force per glue area and produces relatively large amounts of glue relative to its size. We also observe that species that invest more in glue tend to live in more windy and less rainy climates, suggesting that differences in pupal adhesion properties across species are shaped by ecological factors. Our present survey provides a basis for future biomimetic studies based on Drosophila glue.

Abstract Background During evolution, genes can experience duplications, losses, inversions and gene conversions. Why certain genes are more dynamic than others is poorly understood. Here we examine how several Sgs genes encoding glue proteins, which make up a bioadhesive that sticks the animal during metamorphosis, have evolved in Drosophila species. Results We examined high-quality genome assemblies of 24 Drosophila species to study the evolutionary dynamics of four glue genes that are present in D. melanogaster and are part of the same gene family - Sgs1, Sgs3, Sgs7 and Sgs8 - across approximately 30 millions of years. We annotated a total of 102 Sgs genes and grouped them into 4 subfamilies. We present here a new nomenclature for these Sgs genes based on protein sequence conservation, genomic location and presence/absence of internal repeats. Two types of glue genes were uncovered. The first category (Sgs1, Sgs3x, Sgs3e) showed a few gene losses but no duplication, no local inversion and no gene conversion. The second group (Sgs3b, Sgs7, Sgs8) exhibited multiple events of gene losses, gene duplications, local inversions and gene conversions. Our data suggest that the presence of short “new glue” genes near the genes of the latter group may have accelerated their dynamics. Conclusions Our comparative analysis suggests that the evolutionary dynamics of glue genes is influenced by genomic context. Our molecular, phylogenetic and comparative analysis of the four glue genes Sgs1, Sgs3, Sgs7 and Sgs8 provides the foundation for investigating the role of the various glue genes during Drosophila life.

Abstract Background To explore the evolutionary history of sequences, a sequence alignment is a first and necessary step, and its quality is crucial. In the context of the study of the proximal origins of SARS-CoV-2 coronavirus, we wanted to construct an alignment of genomes closely related to SARS-CoV-2 using both coding and non-coding sequences. To our knowledge, there is no tool that can be used to construct this type of alignment, which motivated the creation of CNCA. Results CNCA is a web tool that aligns annotated genomes from GenBank files. It generates a nucleotide alignment that is then updated based on the protein sequence alignment. The output final nucleotide alignment matches the protein alignment and guarantees no frameshift. CNCA was designed to align closely related small genome sequences up to 50 kb (typically viruses) for which the gene order is conserved. Conclusions CNCA constructs multiple alignments of small genomes by integrating both coding and non-coding sequences. This preserves regions traditionally ignored in conventional back-translation methods, such as non-coding regions.

The model organism Drosophila melanogaster has become a focal system for investigations of rapidly evolving genital morphology as well as the development and functions of insect reproductive structures. To follow up on a previous paper outlining unifying terminology for the structures of the male terminalia in this species, we offer here a detailed description of the female terminalia of D. melanogaster. Informative diagrams and micrographs are presented to provide a comprehensive overview of the external and internal reproductive structures of females. We propose a collection of terms and definitions to standardize the terminology associated with the female terminalia in D. melanogaster and we provide a correspondence table with the terms previously used. Unifying terminology for both males and females in this species will help to facilitate communication between various disciplines, as well as aid in synthesizing research across publications within a discipline that has historically focused principally on male features. Our efforts to refine and standardize the terminology should expand the utility of this important model system for addressing questions related to the development and evolution of animal genitalia, and morphology in general.

Abstract Male genitalia are usually extremely divergent between closely related species, but relatively constant within one species. Here we examine the effect of temperature on the shape of the ventral branches, a male genital structure involved in reproductive isolation, in the sister species Drosophila santomea and Drosophila yakuba. We designed a semi‐automatic measurement machine learning pipeline that can reliably identify curvatures and landmarks based on manually digitized contours of the ventral branches. With this method, we observed that temperature does not affect ventral branches in D. yakuba but that in D. santomea ventral branches tend to morph into a D. yakuba‐like shape at lower temperature. We found that male genitalia structures involved in reproductive isolation can be relatively variable within one species and can resemble the shape of closely related species’ genitalia through plasticity to temperature. Our results suggest that reproductive isolation mechanisms can be dependent on the environmental context.

Abstract Introduction Male genitalia are thought to ensure transfer of sperm through direct physical contact with female during copulation. However, little attention has been given to their pre-copulatory role with respect to sexual selection and sexual conflict. Males of the fruitfly Drosophila pachea have a pair of asymmetric external genital lobes, which are primary sexual structures and stabilize the copulatory complex of female and male genitalia. We wondered if genital lobes in D. pachea may have a role before or at the onset of copulation, before genitalia contacts are made. Results We tested this hypothesis with a D. pachea stock where males have variable lobe lengths. In 92 mate competition trials with a single female and two males, females preferentially engaged into a first copulation with males that had a longer left lobe and that displayed increased courtship vigor. In 53 additional trials with both males having partially amputated left lobes of different lengths, we observed a weaker and non-significant effect of left lobe length on copulation success. Courtship durations significantly increased with female age and when two males courted the female simultaneously, compared to trials with only one courting male. In addition, lobe length did not affect sperm transfer once copulation was established. Conclusion Left lobe length affects the chance of a male to engage into copulation. The morphology of this primary sexual trait may affect reproductive success by mediating courtship signals or by facilitating the establishment of genital contacts at the onset of copulation.

CRISPR-based homing gene drive is a genetic control technique aiming to modify or eradicate natural populations. This technique is based on the release of individuals carrying an engineered piece of DNA that can be preferentially inherited by the progeny. The development of countermeasures is important to control the spread of gene drives, should they result in unanticipated damages. One proposed countermeasure is the introduction of individuals carrying a brake construct that targets and inactivates the drive allele but leaves the wild-type allele unaffected. Here we develop models to investigate the efficiency of such brakes. We consider a variable population size and use a combination of analytical and numerical methods to determine the conditions where a brake can prevent the extinction of a population targeted by an eradication drive. We find that a brake is not guaranteed to prevent eradication and that characteristics of both the brake and the drive affect the likelihood of recovering the wild-type population. In particular, brakes that restore fitness are more efficient than brakes that do not. Our model also indicates that threshold-dependent drives (drives that can spread only when introduced above a threshold) are more amenable to control with a brake than drives that can spread from an arbitrary low introduction frequency (threshold-independent drives). Based on our results, we provide practical recommendations and discuss safety issues.

Abstract The probability D that a given clustered regularly interspaced short palindromic repeats (CRISPR)‐based gene drive element contaminates another, nontarget species can be estimated by the following Drive Risk Assessment Quantitative Estimate (DRAQUE) Equation: D=hyb+transf×express×cut×flank×immune×nonextinct with hyb = probability of hybridization between the target species and a nontarget species; transf = probability of horizontal transfer of a piece of DNA containing the gene drive cassette from the target species to a nontarget species (with no hybridization); express = probability that the Cas9 and guide RNA genes are expressed; cut = probability that the CRISPR‐guide RNA recognizes and cuts at a DNA site in the new host; flank = probability that the gene drive cassette inserts at the cut site; immune = probability that the immune system does not reject Cas9‐expressing cells; nonextinct = probability of invasion of the drive within the population. We discuss and estimate each of the seven parameters of the equation, with particular emphasis on possible transfers within insects, and between rodents and humans. We conclude from current data that the probability of a gene drive cassette to contaminate another species is not insignificant. We propose strategies to reduce this risk and call for more work on estimating all the parameters of the formula.

Animal terminalia represent some of the most diverse and rapidly evolving structures in the animal kingdom, and for this reason have been a mainstay in the taxonomic description of species. The terminalia of Drosophila melanogaster, with its wide range of experimental tools, have recently become the focus of increased interest in the fields of development, evolution, and behavior. However, studies from different disciplines have often used discrepant terminologies for the same anatomical structures. Consequently, the terminology of genital parts has become a barrier to integrating results from different fields, rendering it difficult to determine what parts are being referenced. We formed a consortium of researchers studying the genitalia of D. melanogaster to help establish a set of naming conventions. Here, we present a detailed visual anatomy of male genital parts, including a list of synonymous terms, and suggest practices to avoid confusion when referring to anatomical parts in future studies. The goal of this effort is to facilitate interdisciplinary communication and help newcomers orient themselves within the exciting field of Drosophila genitalia.

Abstract Background Male genitals have repeatedly evolved left-right asymmetries, and the causes of such evolution remain unclear. The Drosophila nannoptera group contains four species, among which three exhibit left-right asymmetries of distinct genital organs. In the most studied species, Drosophila pachea, males display asymmetric genital lobes and they mate right-sided on top of the female. Copulation position of the other species is unknown. Results To assess whether the evolution of genital asymmetry could be linked to the evolution of one-sided mating, we examined phallus morphology and copulation position in D. pachea and closely related species. The phallus was found to be symmetric in all investigated species except D. pachea, which displays an asymmetric phallus with a right-sided gonopore, and D. acanthoptera, which harbors an asymmetrically bent phallus. In all examined species, males were found to position themselves symmetrically on top of the female, except in D. pachea and D. nannoptera, where males mated right-sided, in distinctive, species-specific positions. In addition, the copulation duration was found to be increased in the nannoptera group species compared to closely related outgroup species. Conclusion Our study shows that gains, and possibly losses, of asymmetry in genital morphology and mating position have evolved repeatedly in the nannoptera group. Current data does not allow us to conclude whether genital asymmetry has evolved in response to changes in mating position, or vice versa.

Abstract Background At the very end of the larval stage Drosophila expectorate a glue secreted by their salivary glands to attach themselves to a substrate while pupariating. The glue is a mixture of apparently unrelated proteins, some of which are highly glycosylated and possess internal repeats. Because species adhere to distinct substrates (i.e. leaves, wood, rotten fruits), glue genes are expected to evolve rapidly. Results We used available genome sequences and PCR-sequencing of regions of interest to investigate the glue genes in 20 Drosophila species. We discovered a new gene in addition to the seven glue genes annotated in D. melanogaster. We also identified a phase 1 intron at a conserved position present in five of the eight glue genes of D. melanogaster, suggesting a common origin for those glue genes. A slightly significant rate of gene turnover was inferred. Both the number of repeats and the repeat sequence were found to diverge rapidly, even between closely related species. We also detected high repeat number variation at the intrapopulation level in D. melanogaster. Conclusion Most conspicuous signs of accelerated evolution are found in the repeat regions of several glue genes.

The glue produced by Drosophila larvae to attach themselves to a substrate for several days and resist predation until the end of metamorphosis represents an attractive model to develop new adhesives for dry environments. The adhesive properties of this interesting material have been investigated recently, and it was found that it binds as well as strongly adhesive commercial tapes to various types of substrates. This glue hardens rapidly after excretion and is made of several proteins. In D. melanogaster, eight glue proteins have been identified: four are long glycosylated mucoproteins containing repeats rich in prolines, serines and threonines, and four others are shorter proteins rich in cysteines. This protein mix is produced by the salivary glands through a complex packaging process that is starting to be elucidated. Drosophila species have adapted to stick to various substrates in diverse environmental conditions and glue genes appear to evolve rapidly in terms of gene number, number of repeats and sequence of the repeat motifs. Interestingly, besides its adhesive properties, the glue may also have antimicrobial activities. We discuss future perspectives and avenues of research for the development of new bioadhesives mimicking Drosophila fly glue.