Toshiyuki Takano-Shimizu-Kouno

Sperm display remarkable diversity in size and morphology, especially in insects. Sperm length varies by several hundred-fold within Drosophila alone. In the model species Drosophila melanogaster, sperm are approximately 1.7 times longer than those of its close relatives Drosophila simulans and Drosophila mauritiana. However, the developmental mechanisms underlying such rapid divergence in sperm traits remain poorly understood. Somatic hub and cyst cells are essential for the maintenance of germline stem cells in Drosophila spermatogenesis, yet their contribution to cell growth later in spermatogenesis remains unclear. Here, we used primordial germ cell (PGC) xenotransplantation to determine whether sperm length regulation is cell autonomous or influenced by the somatic environment of the testes. When D. simulans PGCs were transplanted into agametic D. melanogaster embryos, the resulting sperm were indistinguishable in length from those of D. simulans donor males and remained significantly shorter than the sperm of D. melanogaster males. Our results suggest that the dramatic diversification in sperm length shaped by postcopulatory sexual selection in the Drosophila lineage has been largely driven by cell-autonomous modifications in the germ line.

Polyethylene terephthalate (PET) is one of the most widely used plastics, yet recycling relies on energy-intensive chemical processes with environmental burdens. As an alternative, PET hydrolytic enzymes such as PET hydrolase (PETase) from Piscinibacter sakaiensis have been proposed for biological PET recycling under mild conditions. Inspired by recent studies on plastic degradation by insects, here we explored genetically engineered insects as a platform for PET biodegradation. We heterologously expressed PETase in the fruit fly Drosophila melanogaster , targeting a subset of the intestinal tract and the salivary glands. PETase-expressing flies degraded both a water-soluble PET copolymer in vivo and solid PET in the surrounding environment under alkaline conditions. Notably, the secreted PETase was glycosylated, reducing catalytic activity but enhancing stability and enabling prolonged PET degradation. These findings demonstrate a proof of concept for the application of engineered insects in plastic biodegradation and suggest new directions for sustainable, biology-based recycling technologies.

Sperm storage in the female reproductive tract is essential for efficient progeny production in animals with internal fertilization. This is particularly so for insects, especially females that have specialized organs for long-term storage. Nep4 mutant males of Drosophila melanogaster produce an average number of morphologically normal sperm, but they cannot fertilize eggs because Nep4 sperm nuclei fail to decondense after entry into the egg. Furthermore, although Nep4 mutant sperm enter the sperm storage organs in a typical manner, they are largely lost from the seminal receptacle within 24 h. It is potentially advantageous for females to discard such defective sperm. However, whether females are actively involved in this rapid sperm loss is not known. First, this study characterized NEP4 expression in wild-type testes. Between the 2 isoforms, the soluble isoform (Nep4-PB) but not the membrane-bound isoform (Nep4-PA) was essential for male fertility. NEP4 protein was localized to the acrosome of mature sperm and remained so in female storage organs. Second, we performed rescue experiments; the rapid sperm-loss phenotype of Nep4 sperm could not be rescued by mating to an eggless female nor by insemination by a wild-type male beforehand. This indicates that Nep4 sperm are likely unable to stay in and autonomously exit the seminal receptacle. This study provides evidence that sperm retention and maintenance in female sperm storage organs involve different mechanisms from those for sperm entry into the storage organs and that the acrosome of Drosophila sperm plays an important role in this process.

G protein-coupled receptors (GPCRs) are essential cell surface proteins involved in transducing extracellular signals into intracellular responses, regulating various physiological processes. This study validated the use of the Tango assay, a sensitive method for detecting GPCR activation, in Drosophila Schneider 2 (S2) cells, focusing on the human Dopamine Receptor D4 (DRD4). Plasmids encoding the LexA-tagged human DRD4 receptor and a luciferase reporter were co-transfected into Drosophila S2 cells and stimulated with dopamine. Receptor activation was measured by quantifying the luciferase activity. The system showed high specificity for dopamine, with no activation in response to octopamine, a non-ligand for DRD4. Furthermore, the system effectively detects activation by a novel compound. These results demonstrate that Drosophila S2 cells, coupled with the Tango assay, provide a viable model for studying human GPCR function and ligand specificity. This system enables the rapid screening of potential GPCR ligands in a cost-effective cellular model.

Sexual selection drives rapid evolution of morphological, physiological, and behavioral traits, especially in males, and it may also drive the rapid evolution of hybrid male sterility. Indeed, the faster male theory of speciation was once viewed as a major cause of Haldane's rule in male-heterogametic XY taxa, but is increasingly being replaced by the genetic conflict hypothesis partly because it cannot explain the faster evolution of hybrid female sterility in female-heterogametic ZW taxa. The theory nonetheless predicts that there should be more genes for hybrid male sterility than for hybrid female sterility even in such taxa, but this remains untested. Thus, finding evidence for the faster male theory of reproductive isolation beyond the F1 generation in ZW systems still represents a challenge to studying the impact of sexual selection. In this study, we examined F2 hybrids between the domesticated silkworm Bombyx mori and the wild silk moth Bombyx mandarina, which have ZW sex determination. We found that although only females showed reduced fertility in the F1 generation, the F2 hybrid males had a significant reduction in fertility compared with the parental and F1 males. Importantly, 27% of the F2 males and 15% of the F2 females were completely sterile, suggesting the presence of recessive incompatibilities causing male sterility in female-heterogametic taxa.

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15 agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 ± 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 ± 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

There is an urgent need to cryopreserve Drosophila stocks that have been maintained as living cultures for a long time. Long-term culture increases the risk of accidental loss and of unwanted genetic alteration. Here, we report that cryopreserved primordial germ cells (PGCs) can produce F1 progeny when transplanted into hosts. The cryopreserved donor PGCs could form germline stem cells in host gonads and contributed to continuous offspring production. Furthermore, the ability to produce offspring did not appear to vary with either differences between donor strains or cryopreservation duration. Therefore, we propose that our cryopreservation method is feasible for long-term storage of various Drosophila strains. These results underscore the potential usefulness of our cryopreservation method for backing up living stocks to avoid either accidental loss or genetic alteration.

Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing our understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. We focused on one such gene, Rack 1, and found that it plays an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful.

Nuclear factor I A (NFIA) is a transcription factor that belongs to the NFI family. Truncating variants or intragenic deletion of the NFIA gene are known to cause the human neurodevelopmental disorder known as NFIA‐related disorder, but no patient heterozygous for a missense mutation has been reported. Here, we document two unrelated patients with typical phenotypic features of the NFIA‐related disorder who shared a missense variant p.Lys125Glu (K125E) in the NFIA gene. Patient 1 was a 6‐year‐old female with global developmental delay, corpus callosum anomaly, macrocephaly, and dysmorphic facial features. Patient 2 was a 14‐month‐old male with corpus callosum anomaly and macrocephaly. By using Drosophila and zebrafish models, we functionally evaluated the effect of the K125E substitution. Ectopic expression of wild‐type human NFIA in Drosophila caused developmental defects such as eye malformation and premature death, while that of human NFIA K125E variant allele did not. nfia‐deficient zebrafish embryos showed defects of midline‐crossing axons in the midbrain/hindbrain boundary. This impairment of commissural neurons was rescued by expression of wild‐type human NFIA, but not by that of mutant variant harboring K125E substitution. In accordance with these in vivo functional analyses, we showed that the K125E mutation impaired the transcriptional regulation of HES1 promoter in cultured cells. Taken together, we concluded that the K125E variant in the NFIA gene is a loss‐of‐function mutation.

The completion of whole-genome sequences has greatly broadened our understanding of genes and genomes. The availability of model organism databases facilitates the sharing of information. However, it is still challenging to predict the pathogenicity of missense mutations, and it is more difficult to evaluate the functional impact of noncoding variants. What is more, it is a primary question to understand what variants interact to express phenotypes. Powerful genetic tools and resources available in Drosophila now make it much easier to replace endogenous genes with exogenous DNA. This allows us to directly investigate and compare the functions of orthologs, variants, and fragments in a single genetic background, the value of which should be widely appreciated. To take one example, we are currently studying so-called ultra-conserved elements, which have been conserved over hundreds of millions of years of vertebrate evolution. Many highly conserved elements are in noncoding regions and are thought to play a pivotal role in gene regulation. We generated transgenic fly lines carrying human ultra-conserved elements for enhancer reporter assay and indeed observed the reporter expression in one or more tissues of embryos and larvae in all elements tested. Currently, transgenic human-ORF lines expressing human genes under the control of GAL4/UAS system are also been developed, which will greatly facilitate the cross-species in Drosophila. In this chapter, I introduce useful tools and resources available in Drosophila to nonspecialists, encouraging their further use in many applications.