Drosophila Gene Research Articles

Local translatome sustains synaptic function in impaired Wallerian degeneration.

After injury, severed axons separated from their somas activate programmed axon degeneration, a conserved pathway to initiate their degeneration within a day. Conversely, severed projections deficient in programmed axon degeneration remain morphologically preserved with functional synapses for weeks to months after axotomy. How this synaptic function is sustained remains currently unknown. Here, we show that dNmnat overexpression attenuates programmed axon degeneration in distinct neuronal populations. Severed projections remain morphologically preserved for weeks. When evoked, they elicit a postsynaptic behavior, a readout for preserved synaptic function. We used ribosomal pulldown to isolate the translatome from these projections 1 week after axotomy. Translatome candidates of enriched biological classes identified by transcriptional profiling are validated in a screen using a novel automated system to detect evoked antennal grooming as a proxy for preserved synaptic function. RNAi-mediated knockdown reveals that transcripts of themTORC1 pathway, a mediator of protein synthesis, and of candidate genes involved in protein ubiquitination and Ca2+ homeostasis are required for preserved synaptic function. Our translatome dataset also uncovers several uncharacterized Drosophila genes associated with human disease. It may offer insights into novel avenues for therapeutic treatments.

An image-based RNAi screen identifies the EGF.R signaling pathway as a regulator of Imp/ IGF2BP RNP granules.

Biomolecular condensates have recently retained much attention since they provide a fundamental mechanism of cellular organization. Among those, cytoplasmic RNP granules selectively and reversibly concentrate RNA molecules and regulatory proteins, thus contributing to the spatio-temporal regulation of associated RNAs. Extensive in vitro work has unraveled the molecular and chemical bases of RNP granule assembly. The signaling pathways controlling this process in a cellular context are however still largely unknown. Here, we aimed at identifying regulators of cytoplasmic RNP granules characterized by the presence of the evolutionarily conserved IGF2BP/Imp/ZBP1 RNA binding protein. We performed a high-content image-based RNAi screen targeting all Drosophila genes encoding RNA binding proteins, phosphatases and kinases. This led to the identification of dozens of genes regulating the number of Imp+ RNP granules in S2R+ cells, among which components of the MAPK pathway. Combining functional approaches, phospho-mapping and generation of phospho-variants, we further showed that the EGF.R signaling inhibits Imp+ RNP granule assembly through activation of MAPK/Rolled and Imp S15 phosphosite. This work illustrates how signaling pathways can regulate cellular condensate assembly by post-translational modifications of specific components.

Peroxisome inter-organelle cooperation in Drosophila

Within many cellular organelles biochemical functions are compartmentalized, which facilitates optimized enzymatic environments. However, processing and or storage of metabolites in the same pathway can occur in multiple organelles. Thus, spatially separated organelles would need to cooperate functionally. Coordination would also be needed between organelles in different specialized cells, with shared metabolites passed via circulation. Peroxisomes are membrane-bounded organelles responsible for cellular redox and lipid metabolism in eukaryotic cells. Studies using single cells suggest peroxisomes coordinate with other organelles including mitochondria, ER (endoplasmic reticulum), lysosomes, and lipid droplets. Some of these coordinated functions require, or are at least enhanced by, direct contact between peroxisomes and other organelles. Peroxisome dysfunction in humans leads to multiorgan effects including neurological, metabolic, developmental, and age-related diseases. Thus, increased understanding of peroxisome coordination with other organelles, especially those specialized cells in various organs is essential. Drosophila melanogaster (fruit fly) has emerged recently as an effective animal model for understanding peroxisomes. Here we review current knowledge of genetic pathways regulating coordination between peroxisomes with other organelles in flies, speculating about analogous roles for conserved Drosophila genes encoding proteins with known organelle coordinating roles in other species.

Making sense of chromosome polymorphisms in two leptysmine grasshoppers.

The touchstone of the 'New Synthesis' was population cytogenetics -rather than genetics - due to the abundant polymorphic inversions in the genus Drosophila. Grasshoppers were not a material of choice because of their conservative karyotypes. However, nowadays seven species of Acrididae were described for polymorphic centric fusions, five of them in South-America. Leptysma argentina and the likely biocontrol of water-hyacinth Cornops aquaticum are semiaquatic Leptysminae (Acrididae: Orthoptera), polymorphic for centric fusions, supernumerary segments and a B-chromosome. We sought to demonstrate the operation of natural selection on them, by detecting: (I) latitudinal clines; (II) regression on environmental variables; (III) deviation from null models, such as linkage equilibrium; (IV) seasonal variation; (V) comparison between age classes and (VI) selection component analyses. All of them were confirmed in L. argentina, just (I) and (II) in C. aquaticum. Furthermore, the relationship between karyotype, phenotype and recombination was confirmed in both species. Karyotype-phenotype relationship may be due to the body enlargement the fusions are associated with, along with a latitudinal transition in voltinism. Karyotype-related recombination reduction in both species may help explain all fusion clines, although there is probably more than one factor at work. No effects were noticed for a supernumerary segment in L. argentina, but it is ubiquitous and certainly non-neutral. C. aquaticum is poised for introduction in South-Africa as a biocontrol of water-hyacinths; the recent discovery of four more segment polymorphisms may imply more chromosomal markers to make sense of its genetic system.

Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs.

Retinoblastoma tumor suppressor proteins (Rb) are highly conserved metazoan transcriptional corepressors involved in regulating the expression of thousands of genes. The vertebrate lineage and the Drosophila genus independently experienced an Rb gene duplication event, leading to the expression of several Rb paralogs whose unique and redundant roles in gene regulation remain to be fully explored. Here, we used a novel CRISPRi system in Drosophila to identify the significance of paralogy in the Rb family. We engineered dCas9 fusions to the fly Rbf1 and Rbf2 paralogs and deployed them to gene promoters in vivo, studying them in their native chromatin context. By directly querying the in vivo response of dozens of genes to Rbf1 and Rbf2 targeting, using both transcriptional as well as sensitive developmental readouts, we find that Rb paralogs function as "soft repressors" and have highly context-specific activities. Our comparison of targeting endogenous genes to reporter genes in cell culture identified striking differences in activity, underlining the importance of using CRISPRi effectors in a physiologically relevant context to identify paralog-specific activities. Our study uncovers the complexity of Rb-mediated transcriptional regulation in a living organism, and serves as a stepping stone for future CRISPRi development in Drosophila.

From genes to color: Evolutionary insights and mechanistic pathways of the yellow gene in insect pigmentation

Insect pigmentation plays a vital role in ecology and evolution, influencing survival, mate attraction, and communication. This review examines the complex mechanisms governing insect coloration, focusing on the roles of pigments (melanins and carotenoids) and structural colors. Specifically, the yellow gene in Drosophila is highlighted for its critical function in melanin biosynthesis and its evolutionary implications. Variations in the yellow gene's expression and regulation, influenced by both cis- and trans-regulatory elements, contribute to the phenotypic diversity of pigmentation patterns across insect species. The interplay between developmental genes and the yellow gene further illustrates how subtle genetic changes drive morphological diversity. While advances in CRISPR and other genetic tools have enhanced our understanding of pigmentation, challenges remain in deciphering the gene’s complex interactions and the influence of environmental factors. Future research should focus on refining genomic techniques, exploring environmental impacts on pigmentation, and utilizing single-cell omics to gain deeper insights into the regulatory networks governing the yellow gene. This comprehensive understanding of insect pigmentation will not only enrich our knowledge of biodiversity but also inform conservation and biotechnological innovations.

The Drosophila maternal-effect gene abnormal oocyte (ao) does not repress histone gene expression.

The abnormal oocyte (ao) gene of Drosophila melanogaster is a maternal-effect lethal gene previously identified as encoding a transcriptional regulator of core histones. However, background genetic mutations in existing ao mutant strains could compromise their utility in manipulating histone levels. To distinguish the true ao phenotype from background effects, we created two new ao reagents: a CRISPR/Cas9-mediated knockout of the ao allele for genetic and molecular analyses and an epitope-tagged ao allele for cytological experiments. Using these reagents, we confirm previous findings that ao exhibits maternal-effect lethality, which can be rescued by either a decrease in the histone gene copy number or by Y chromosome heterochromatin. We also confirm that the Ao protein localizes to the histone locus bodies in ovaries. Our data also suggest that ao genetically interacts with the histone genes and heterochromatin, as previously suggested. However, contrary to prior findings, we find that ao does not repress core histone transcript levels. Thus, the molecular basis for ao-associated maternal-effect lethality remains unknown.

Molecular cloning, functional characterization and differential expression of two novel GABAAR-like subunits from red swamp crayfish Procambarus clarkii.

In this work, we cloned and functionally expressed two novel GABAA receptor subunits from Procambarus clarkii crayfish. These two new subunits, PcGABAA-α and PcGABAA-β2, revealed significant sequence homology with the PcGABAA-β subunit, previously identified in our laboratory. In addition, PcGABAA-α subunit also shared a significant degree of identity with the Drosophila melanogaster genes DmGRD (GABA and glycine-like receptor subunits of Drosophila) as well as PcGABAA-β2 subunit with DmLCCH3 (ligand-gated chloride channel homolog 3). Electrophysiological recordings showed that the expression in HEK cells of the novel subunits, either alone or in combination, failed to form functional homo- or heteromeric receptors. However, the co-expression of PcGABAA-α with PcGABAA-β evoked sodium- or chloride-dependent currents that accurately reproduced the time course of the GABA-evoked currents in the X-organ neurons from crayfish, suggesting that these GABA subunits combine to form two types of GABA receptors, one with cationic selectivity filter and the other preferentially permeates anions. On the other hand, PcGABAA-β2 and PcGABAA-β co-expression generated a chloride current that does not show desensitization. Muscimol reproduced the time course of GABA-evoked currents in all functional receptors, and picrotoxin blocked these currents; bicuculline did not block any of the recorded currents. Reverse transcription polymerae chain reaction (RT-PCR) amplifications and FISH revealed that PcGABAA-α and PcGABAA-β2 are predominantly expressed in the crayfish nervous system. Altogether, these findings provide the first evidence of a neural GABA-gated cationic channel in the crayfish, increasing our understanding of the role of these new GABAA receptor subunits in native heteromeric receptors.

Drosophila are hosts to the first described parasitoid wasp of adult flies

Parasitoid wasps are exceptionally diverse and use specialized adaptations capable of manipulating the physiology and behaviour of host organisms1. In more than two centuries since the first records of Drosophila-parasitizing wasps, nearly 200 described and provisional parasitoid species of drosophilids have been identified2. These include endoparasitoids and ectoparasitoids, as well as species attacking larval and pupal hosts3. Despite a deep history of research attention and remarkable biodiversity, a wasp species that attacks and develops inside the adult stage of a fly host has not been described previously. Here we report the discovery of a wasp species that infects the adult stage of fruit flies in the genus Drosophila, including one of the most deeply studied model organisms in biology, Drosophila melanogaster. Notably, this wasp can be easily collected from backyard fly baits and has a broad geographic distribution throughout the eastern USA. We document its life history and unique host interactions, including egg-laying into and larval emergence from adult flies, and provide protocols to raise wasps from wild-caught host flies. Our results emphasize the need for ongoing research investment in insect biodiversity and systematics. As parasitoid research continues to uncover unusual biology and supports fundamental mechanistic insights into immunity4, metabolism5, ecology6, evolution7–9 and behaviour10–12, we anticipate that this wasp’s association with the laboratory model organism, D. melanogaster, will provide new research opportunities across the life sciences.

Drosophila model to clarify the pathological significance of OPA1 in autosomal dominant optic atrophy.

Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase (OPA1) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 (hOPA1) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1. This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies.

Mitochondrial dysfunction and NDUFS3: Insights from a PINK1B9 Drosophila model in Parkinson’s disease pathogenesis

PTEN-induced kinase1 (PINK1) mutation is the main cause of autosomal recessive inheritance and early-onset Parkinson’s disease. Mitochondrial respiratory chain complex I (CI) functional impairment has been considered to be an important factor in the pathogenesis of PD in recent years. In addition, NDUFS3 (nicotinamide adenine dinucleotide deoxylase iron-thionein 3) is one of the core subunits of mitochondrial CI. Therefore, this study explored the role of NDUFS3 gene in PINK1B9 transgenic Drosophila and its possible related mechanisms. In this study, the PD transgenic Drosophila model of MHC-Gal4/UAS system was selected to specifically activate the expression of PINK1B9 gene in the chest muscle tissue of Drosophila melanogaster. NDUFS3 RNAi interference was used to interfere with PINK1B9 transgenic Drosophila melanogaster and its effect on PD transgenic flies was studied. The results suggest that down-regulation of NDUFS3 gene expression may have a protective effect on PINK1B9 transgenic Drosophila melanogaster, and we speculate that down-regulation of NDUFS3 gene expression to reduce oxidative stress and restore mitochondrial function may be related to mitochondrial stress response.

Highly regenerative species-specific genes improve age-associated features in the adult Drosophila midgut

BackgroundThe remarkable regenerative abilities observed in planarians and cnidarians are closely linked to the active proliferation of adult stem cells and the precise differentiation of their progeny, both of which typically deteriorate during aging in low regenerative animals. While regeneration-specific genes conserved in highly regenerative organisms may confer regenerative abilities and long-term maintenance of tissue homeostasis, it remains unclear whether introducing these regenerative genes into low regenerative animals can improve their regeneration and aging processes.ResultsHere, we ectopically express highly regenerative species-specific JmjC domain-encoding genes (HRJDs) in Drosophila, a widely used low regenerative model organism. Surprisingly, HRJD expression impedes tissue regeneration in the developing wing disc but extends organismal lifespan when expressed in the intestinal stem cell lineages of the adult midgut under non-regenerative conditions. Notably, HRJDs enhance the proliferative activity of intestinal stem cells while maintaining their differentiation fidelity, ameliorating age-related decline in gut barrier functions.ConclusionsThese findings together suggest that the introduction of highly regenerative species-specific genes can improve stem cell functions and promote a healthy lifespan when expressed in aging animals.

Transmembrane and coiled-coil 2 associates with Alzheimer's disease pathology in the human brain.

Transmembrane and coiled-coil 2 (TMCC2) is a human orthologue of the Drosophila gene dementin, mutant alleles of which cause neurodegeneration with features of Alzheimer's disease (AD). TMCC2 and Dementin further have an evolutionarily conserved interaction with the amyloid protein precursor (APP), a protein central to AD pathogenesis. To investigate if human TMCC2 might also participate in mechanisms of neurodegeneration, we examined TMCC2 expression in late onset AD human brain and age-matched controls, familial AD cases bearing a mutation in APP Val717, and Down syndrome AD. Consistent with previous observations of complex formation between TMCC2 and APP in the rat brain, the dual immunocytochemistry of control human temporal cortex showed highly similar distributions of TMCC2 and APP. In late onset AD cases stratified by APOE genotype, TMCC2 immunoreactivity was associated with dense core senile plaques and adjacent neuronal dystrophies, but not with Aβ surrounding the core, diffuse Aβ plaques or tauopathy. In Down syndrome AD, we observed in addition TMCC2-immunoreactive and methoxy-X04-positive pathological features that were morphologically distinct from those seen in the late onset and familial AD cases, suggesting enhanced pathological alteration of TMCC2 in Down syndrome AD. At the protein level, western blots of human brain extracts revealed that human brain-derived TMCC2 exists as at least three isoforms, the relative abundance of which varied between the temporal gyrus and cerebellum and was influenced by APOE and/or dementia status. Our findings thus implicate human TMCC2 in AD via its interactions with APP, its association with dense core plaques, as well as its alteration in Down syndrome AD.

Learning from the Codon Table: Convergent Recoding Provides Novel Understanding on the Evolution of A-to-I RNA Editing.

Adenosine-to-inosine (A-to-I) RNA editing recodes the genetic information. Apart from diversifying the proteome, another tempting advantage of RNA recoding is to correct deleterious DNA mutation and restore ancestral allele. Solid evidences for beneficial restorative editing are very rare in animals. By searching for "convergent recoding" under a phylogenetic context, we proposed this term for judging the potential restorative functions of particular editing site. For the well-known mammalian Gln>Arg (Q>R) recoding site, its ancestral state in vertebrate genomes was the pre-editing Gln, and all 470 available mammalian genomes strictly avoid other three equivalent ways to achieve Arg in protein. The absence of convergent recoding from His>Arg, or synonymous mutations on Gln codons, could be attributed to the strong maintenance on editing motif and structure, but the absence of direct A-to-G mutation is extremely unexpected. With similar ideas, we found cases of convergent recoding in Drosophila genus, reducing the possibility of their restorative function. In summary, we defined an interesting scenario of convergent recoding, the occurrence of which could be used as preliminary judgements for whether a recoding site has a sole restorative role. Our work provides novel insights to the natural selection and evolution of RNA editing.

A distinct isoform of Msp300/Nesprin organizes the perinuclear microtubule organizing center in adipose cells.

In many cell types, disparate non-centrosomal microtubule-organizing centers (ncMTOCs) replace functional centrosomes and serve the unique needs of the cell types in which they are formed. In Drosophila fat body cells (adipocytes), an ncMTOC is organized on the nuclear surface. This perinuclear ncMTOC is anchored by Msp300, encoded by one of two Nesprin-encoding genes in Drosophila. Msp300 and the spectraplakin short stop (shot) are co-dependent for localization to the nuclear envelope to generate the ncMTOC where they recruit the microtubule minus-end stabilizer Patronin (CAMSAP). The fat body perinuclear ncMTOC requires Patronin, Ninein, and Msps (ortholog of ch-TOG), but does not require γ-tubulin for MT assembly. The Msp300 gene is complex, encoding at least eleven isoforms. Here we show that two Msp300 isoforms, Msp300-PE and -PG, are required and only one, Msp300-PE, appears sufficient for generation of the ncMTOC. Loss of Msp300-PE,-PG retains the presence of the other isoforms at the nuclear surface, indicating that they are not sufficient to generate the ncMTOC. Loss of Msp300-PE,-PG results in severe loss of localization of shot and Patronin, and disruption of the MT array. This results in nuclear mispositioning and loss of endosomal trafficking. Msp300-PE has an unusual domain structure including a lack of a KASH domain and very few spectrin repeats and appears therefore to have a highly derived function suited to generating an ncMTOC on the nuclear surface.

Molecular and genetics inquiry program using Drosophila eyes absent gene.

The aim of this study was to develop molecular genetics inquiry programs using the eyes absent gene of Drosophila melanogaster. The program was composed of various molecular genetics experiments, including mutation observation, cross-breeding, searching for genetic information in web databases, gDNA extraction, and PCR. Each experiment was designed to include a reasoning process, thus aligning the program closely with the structure of authentic scientific research. This program was also developed with a modular design to provide flexibility in its implementation. The program was implemented for middle school students affiliated with a university science education institute for the gifted, and surveys indicated that students had positive experiences with the program. Our findings suggest that the program provides students with a contextual understanding of how authentic research is conducted. Finally, we suggest ways to implement the program effectively.

ZW10: an emerging orchestrator of organelle dynamics during the cell division cycle.

Zeste white 10 (ZW10) was first identified as a centromere/kinetochore protein encoded by the ZW10 gene in Drosophila. ZW10 guides the spindle assembly checkpoint signaling during mitotic chromosome segregation in metazoans. Recent studies have shown that ZW10 is also involved in membranous organelle interactions during interphase and plays a vital role in membrane transport between the endoplasmic reticulum and Golgi apparatus. Despite these findings, the precise molecular mechanisms by which ZW10 regulates interactions between membranous organelles in interphase and the assembly of membraneless organelle kinetochore in mitosis remain elusive. Here, we highlight how ZW10 forms context-dependent protein complexes during the cell cycle. These complexes are essential for mediating membrane trafficking in interphase and ensuring the accurate segregation of chromosomes in mitosis.

Identification and genetic analysis of a pervasive 'needle-eye' sperm phenotype in Drosophila sterile hybrid males.

Interspecies hybrid sterility has been extensively studied, especially in the genus Drosophila. Hybrid sterility is more often found in the heterogametic (XY or ZW) sex, a trend called Haldane's rule. Although this phenomenon is pervasive, identification of a common genetic mechanism remains elusive, with modest support found for a range of potential theories. Here, we identify a single precise morphological phenotype, which we call 'needle-eye sperm', that is associated with hybrid sterility in three separate species pairs that span the Drosophila genus. The nature of the phenotype indicates a common point of meiotic failure in sterile hybrid males. We used 10 generations of backcross selection paired with whole-genome pooled sequencing to genetically map the regions underlying the needle-eye (NE) sperm phenotype. Surprisingly, the sterility phenotype was present in ~50% of males even after 10 generations of backcrossing, and only a single region of the X chromosome was associated with sterility in one direction of backcross. Owing to the common phenotype among sterile male hybrids, and the strong effect of individual loci, further exploration of these findings may identify a universal mechanism for the evolution of hybrid sterility.

Genomic context-dependent histone H3K36 methylation by three Drosophila methyltransferases and implications for dedicated chromatin readers.

Methylation of histone H3 at lysine 36 (H3K36me3) marks active chromatin. The mark is interpreted by epigenetic readers that assist transcription and safeguard the integrity of the chromatin fiber. The chromodomain protein MSL3 binds H3K36me3 to target X-chromosomal genes in male Drosophila for dosage compensation. The PWWP-domain protein JASPer recruits the JIL1 kinase to active chromatin on all chromosomes. Unexpectedly, depletion of K36me3 had variable, locus-specific effects on the interactions of those readers. This observation motivated a systematic and comprehensive study of K36 methylation in a defined cellular model. Contrasting prevailing models, we found that K36me1, K36me2 and K36me3 each contribute to distinct chromatin states. A gene-centric view of the changing K36 methylation landscape upon depletion of the three methyltransferases Set2, NSD and Ash1 revealed local, context-specific methylation signatures. Set2 catalyzes K36me3 predominantly at transcriptionally active euchromatin. NSD places K36me2/3 at defined loci within pericentric heterochromatin and on weakly transcribed euchromatic genes. Ash1 deposits K36me1 at regions with enhancer signatures. The genome-wide mapping of MSL3 and JASPer suggested that they bind K36me2 in addition to K36me3, which was confirmed by direct affinity measurement. This dual specificity attracts the readers to a broader range of chromosomal locations and increases the robustness of their actions.

Molecular Insights into Female Hybrid Sterility in Interspecific Crosses between Drosophila melanogaster and Drosophila simulans.

Species of the genus Drosophila have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are under-investigated. Here, we performed an analysis of hybrid female sterility by crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis and molecular, cellular, and genetic approaches, we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis, and functional defects of oogenesis in hybrids. Premature germline stem cell loss was the most prominent defect of oogenesis in hybrid ovaries. Because of the differential expression of genes encoding piRNA pathway components, rhino and deadlock, the functional RDCmel complex in hybrid ovaries was not assembled. However, the activity of the RDCsim complex was maintained in hybrids independent of the genomic origin of piRNA clusters. Despite the identification of a cohort of overexpressed TEs in hybrid ovaries, we found no evidence that their activity can be considered the main cause of hybrid sterility. We revealed a complicated pattern of Vasa protein expression in the hybrid germline, including partial AT-chX piRNA targeting of the vasasim allele and a significant zygotic delay in vasamel expression. We arrived at the conclusion that the hybrid sterility phenotype was caused by intricate multi-locus differences between the species.