Insect Molecular Biology

Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is a globally invasive pest of soft-skinned fruits that is currently controlled largely through the use of broad-spectrum insecticides. Increasing resistance to pesticides and regulatory pressures have motivated the development of genetic control strategies. We previously developed a CRISPR/Cas9-based homing gene drive targeting the coding sequence of the female-specific exon of the sex-determination gene doublesex, achieving highly efficient inheritance (94-99%) in both male and female germlines. A major limitation of homing gene drives is the formation of resistant alleles that evade cleavage yet retain gene function. Multiplexing guide RNAs (gRNAs) could reduce the formation of such functional resistance alleles. Here, we generated and tested homing constructs expressing one, two, or three gRNAs targeting different regions of the female-specific exon of doublesex, including a splice-junction target site. A single gRNA targeting the splice junction supported high inheritance in males but showed reduced efficiency in females. Combining this gRNA with a coding sequence-targeting guide further reduced drive efficiency, particularly in the female germline. Constructs expressing two gRNAs performed similarly whether guides were linked by transfer RNA (tRNA) sequences or expressed from independent promoters. Constructs expressing three gRNAs using tRNA processing showed consistently low drive inheritance in both sexes, likely a consequence of reduced cleavage efficiency due to inefficient gRNA production. Inheritance was significantly higher in male than female germlines for several constructs, indicating that germline context strongly influences drive performance. Our findings highlight that the approach used for multi-gRNA expression, target site choice and sex-specific germline environments can impact gene drive efficiency, and emphasize the need to optimize construct design within the biological context of the target species.

RNA interference (RNAi) is a widely exploited reverse-genetics tool with potential uses for disease control. Successful RNAi has been reported in trematode-vectoring snails, but the composition of RNAi effector-encoding gene complements, a key driver for RNAi efficiency, remain unstudied in these species. Using bioinformatics and comparative genomics, we searched for orthologues of 115 RNAi effector sequences in genomes or transcriptomes of four snail vectors: Biomphalaria glabrata, B. pfeifferi, Bulinus truncatus, and Lymnaea staginalis. Gene expression patterns of selected RNAi effectors were then examined across developmental stages and tissues of the model B. glabrata snail. At least 74 RNAi-related proteins were conserved across all four species, including core components known to be essential for gene silencing. Classical systemic RNAi-deficient (SID) genes that facilitate systemic RNAi in other systems were absent, suggesting that alternative pathways may compensate for dsRNA uptake and transport. Core effectors of secondary RNAi amplification and heritable RNAi were not detected. Expressions of Dicer-1, Argonaute-2, and the exonuclease Eri-1 did not vary significantly with snail size. A putative RNAi-inhibiting Staufen orthologue showed elevated expression in the ovotestis, while another putative cholesterol-interacting gene was overexpressed in the trunk tissue and may partly contribute to RNAi import. Altogether, our results present the most comprehensive overview of RNAi pathway effectors in major intermediate snail hosts for trematodes. The findings underscore the likely broad potential for RNAi use in trematode intermediate hosts as an experimental tool and potential control method.

Gene model for the ortholog of tango (tgo) in the Sep. 2015 (UC Berkeley ASM127793v1/DbusGB1) Genome Assembly (GenBank Accession: GCA_001277935.1) of Drosophila busckii. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

BACKGROUNDThe soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a pest of soybean in North America that can cause significant yield loss when outbreaks are not managed. Current management tactics primarily rely on inexpensive pyrethroids, but the sustainability of this option is threatened by insecticide-resistance in A. glycines populations across the Upper-Midwest United States. Field-evolved resistance is associated with mutations in the voltage-gated sodium channel subunit h1 (vgsc-h1) gene. RESULTSFour double-stranded RNA (dsRNA) molecules, each matching the sequence of a vgsc-h1 transcript variant ("Specific dsRNAs"), were topically applied to aphids with a genotype carrying the corresponding allele. The mortality of pyrethroid resistant aphids exposed to a Specific dsRNA increased in a dose-dependent manner when applied alone or with a constant concentration of lambda-cyhalothrin, plateauing at 1000 ng ul-1. Synergism was detected between two of four combinations of the Specific dsRNAs and lambda-cyhalothrin. These results were mirrored by the topical application of a single dsRNA with the consensus sequence of all vgsc-h1 variants ("Combined dsRNA"). Mortality was consistently higher in aphids treated with either Specific dsRNA or the Combined dsRNA, alone or with lambda-cyhalothrin, compared to insecticide alone. The number of nymphs produced per female treated with the Specific or Combined dsRNA alone decreased significantly compared to untreated controls. CONCLUSIONThis study demonstrates that the topical application of dsRNAs targeting vgsc-h1 increases the susceptibility and reduces the reproductive capacity of pyrethroid resistant soybean aphids, potentially providing a novel tool for the management of insecticide-resistant aphid populations.

Reporters are widely used in Drosophila genetics to visualize gene expression and cell lineages. However, uncharacterized limitations in specific reporter lines can lead to data misinterpretation. Here, we identify a consistent, driver-independent tdTomato signal in the adult proventriculus from the widely used lexAop-tdTomato.nls reporter line. This signal was observed across multiple lexA driver combinations and was directly detectable in lexAop-tdTomato.nls responder-alone adult proventriculi lacking any lexA driver and without antibody staining. In contrast, no comparable native red fluorescence was detected in larval proventriculi under the same no-antibody imaging condition. Mouse and rabbit anti-RFP immunostaining further supported the presence of proventriculus-associated tdTomato/RFP antigen in adult responder-alone animals. In larval responder-alone proventriculi, antibody-amplified staining was antibody-source-dependent: a detectable signal was observed only with rabbit anti-RFP, whereas mouse and rat anti-RFP produced no reliable detectable signal under the same staining condition. A driver-matched comparison using lexAop-RFP.nls did not reproduce the proventricular signal, arguing against detectable ectopic activity of the tested lexA driver in this tissue. However, because lexAop-tdTomato.nls and lexAop-RFP.nls differ in reporter/transgene architecture and possibly genomic insertion context, the underlying cause cannot be assigned specifically to the lexAop sequence. Our findings highlight the necessity of including driver-negative and no-antibody controls when using this reporter line in adult Drosophila proventriculus and gut studies.

Gene model for the ortholog of Density regulated protein (DENR) in the Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly (GenBank Accession: GCA_000236325.2) of D. eugracilis. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Gene model for the ortholog of raptor in the D. grimshawi May 2011 (Agencourt dgri_caf1/DgriCAF1) Genome Assembly (GenBank Accession: GCA_000005155.1) of Drosophila grimshawi. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Gene model for the ortholog of raptor in the May 2011 (Agencourt Dere_CAF1/DereCAF1) Genome Assembly (GenBank Accession: GCA_000005135.1) of Drosophila erecta. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

The migratory endoparasitic pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, causing significant economic and ecological losses in conifer forest ecosystems in Europe and Asia. Understanding the molecular mechanisms regulating PWN parasitism-related genes may lead to new sustainable solutions for control. Based on previous PWN transcriptomic datasets from the pre-parasitic and parasitic stages and from the pharyngeal gland cells (GC), an in silico analysis was performed to identify transcription factors (TF) highly expressed in the GC. Seven candidates TF genes were selected, and their spatial expression validated by in situ hybridisation. From those, two GC-expressed TFs, BXY_079 and BXY_022, each encoding zinc finger domains, were successfully knocked down by RNA interference. Transcriptomic data from silenced BXY_079 and BXY_022 TFs, analysed with existing life cycle specific transcriptomic data, showed that both TFs control genes expressed at similar times, by repressing male-related genes while activating genes expressed during the J3 and D3 stages, yet each represents the extreme of the others minor function. In addition to these common roles, BXY_079 also activates parasitism-related genes in the J2 stage. These BXY_079-activated parasitism-related genes predominantly encode proteins with lytic functions, including secreted peptidases and glycoside hydrolases. Consistent with their proposed role in parasitism, these genes are highly expressed during the parasitic juvenile stages and are likely involved in nematode feeding, tissue penetration, and migration within the host. In contrast, BXY_022 also represses the expression of several genes related to the reproduction system, such as major sperm proteins and cytosolic motility proteins, particularly in the adult male stage. Taken together, both dual-functional TFs work together, non-redundantly, to regulate gene expression across the life cycle, while each is additionally specialised to regulate diverse and distinct gene sets: ranging from genes implicated in lytic parasitic functions to sexual dimorphism.

The electrophysiological process of auditory transduction in insects remains largely conjecture due to the unknown role of ion channels localised to the cilia, but experimental evidence supports either NompC or Nan-Iav as the auditory mechanotransduction ion channel. Here, we knocked down two key genes that code for the two candidate sound-activated ion channels using dsRNA-mediated RNA interference. We measured sound-evoked activity of the auditory nerve and intracellular electrical currents from the ciliated ending of individual auditory receptors. We found that the sound-evoked nerve activity was reduced in nompC, nan and ift88 knockdown. Using whole-cell patch clamp recordings we found that nompC and nan knockdown resulted in reduced sound-evoked transduction current. Stochastic depolarisations hypothesised to be mediated from one of the candidate mechanotransduction ion channels, either NompC or Nan-Iav, where not affected by knockdown of either channel. The discrete depolarisations are therefore mediated through another unidentified ion channel. We test the hypothesis that discrete depolarisations are graded action potentials that travel toward the soma through noise analysis of the transduction current and analysis of discrete depolarisations to voltage-steps. As a positive control we also knocked down ift88, a protein essential for transporting proteins, including ion channels, along the cilium and found both the transduction current and the discrete depolarisations decreased. Key pointsO_LIInjection of dsRNA decreased RNA of nompC and nan C_LIO_LISound-evoked nerve activity is reduced for RNAi-mediated knockdown of nompC and nan C_LIO_LINompC and Nan both contribute to the transduction current C_LIO_LIThe stochastic discrete depolarisations are not due to NompC or Nan-Iav ion channel but to a third unidentified ion channel. C_LIO_LINoise analysis of the transduction current and the discrete depolarisations suggests they are graded action potentials that travel in the direction of the soma. C_LIO_LIKnockdown of ift88 reduced both the transduction current and discrete depolarisations. C_LI Significance StatementInsects are important to understand, economically, agriculturally and medically. However, we still do not understand fundamental aspects of how insects detect their own body movements, vibrations and sound. These senses are detected by insect chordotonal organs, specialised miniaturised mechanoreceptors that convert movements into electrical signals through specialised ion channels. Previous experimental work has advocated either NompC or Nan-Iav as the mechanosensitive ion channel. Here, for the first time, we reduced the expression of both nompC and nan and measured the sound-evoked transduction current directly from neurons in a specialised auditory chordotonal organ. In contradiction to previous studies, we show that both ion channels contribute to the transduction current and find that electrical signals termed "discrete depolarisations" travel toward the soma.

The zebra mussel (Dreissena polymorpha) is an invasive species that causes extensive economic and ecological damage. Here, we identify and characterize the key components of the small RNA (sRNA) and RNA interference (RNAi) pathways in zebra mussels. Like other mollusks, zebra mussels have extensive microRNA (miRNA) and Piwi-interacting RNA (piRNA) machinery but lack or have modified canonical factors needed to produce small interfering RNA (siRNA). Specifically, the zebra mussel Dicer sequence displays substitutions in the conserved DEAD box motif that is required for substrate processivity, and this organism also lacks some attendant accessory factors such as R2D2. We sequenced the small RNA found in both isolated somatic tissue (adductor muscle) and whole animals (including germline), and identified both conserved and novel miRNA and diverse piRNA sequences, but few endogenous siRNAs. To determine whether their remaining sRNA machinery could still be co-opted to initiate gene silencing, we injected dsRNA targeting several genes into zebra mussel adductor muscle. The injected rpn8-targeting dsRNA reduced rpn8 mRNA levels and was processed into sRNA that resemble endogenous miRNAs and piRNAs. The levels of both sRNA types correlated with mRNA knockdown, suggesting that they may act together to initiate RNAi as seen elsewhere. dsRNA targeting other genes produced variable results suggesting that particular criteria may be needed to trigger an RNAi response in this assay. Our results characterize endogenous sRNA pathways in zebra mussels, establish that dsRNA can induce RNAi, and lay the groundwork for further optimizations to establish RNAi-based genetic manipulation tools for this damaging invasive species.

Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K1-10). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG211, the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (~130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.

Homing endonuclease genes spread by cleaving homologous chromosomes that lack the endonuclease cassette, after which repair from the endonuclease-containing chromosome converts the cut allele into a copy of the drive allele. This mechanism has provided a conceptual foundation for synthetic gene drive systems, including CRISPR-based drives, that represent promising strategies for the genetic control of insect pests. However gene drive performance depends critically on the repair pathways available in the germline of the target organism. Here, we report a set of transgenic assays originally developed as part of an attempt to establish gene targeting in the malaria mosquito Anopheles gambiae using an in vivo-generated linear targeting molecule. Although the intended FLP-mediated excision step was not achieved in the mosquito germline, analysis of the component strains revealed efficient germline activity of the rare-cutting homing endonuclease I-SceI and a striking bias towards homology-based repair of I-SceI-induced double-strand breaks. Across reporter and donor configurations, cleavage outcomes were dominated by single-strand annealing, microhomology-mediated repair, synthesis-dependent strand annealing and gene conversion-like events, with comparatively limited evidence for classical non-homologous end joining. In reciprocal crosses designed to distinguish gene conversion from gamete loss, I-SceI cleavage also produced inheritance distortion consistent with both conversion of the cleaved allele and reduced recovery of gametes carrying extensively damaged donor alleles. These findings indicate that the An. gambiae germline can strongly favour homology-dependent repair following homing endonuclease cleavage and that cleavage can also generate meiotic drive-like distortion through selective loss of damaged gametes. The results have direct relevance for the design and interpretation of homing endonuclease and CRISPR-based gene drives in malaria mosquitoes, where the balance between homology-directed repair, end joining and gamete viability will determine drive efficiency, resistance formation and transmission bias. Author summaryGene drives depend on a simple but demanding principle: a nuclease cuts one chromosome, and the cell repairs the break using the homologous chromosome as a template, copying the drive element in the process. Before CRISPR, this type of system was explored using naturally occurring homing endonucleases such as I-SceI. We attempted to develop a gene targeting system in Anopheles gambiae based on the Rong and Golic strategy, in which FLP recombinase would excise a donor molecule and I-SceI would linearise it to stimulate recombination. The full knockout technology did not work because FLP-mediated excision was not detected in the mosquito germline. However, the component tests revealed something more broadly important: I-SceI-induced breaks were repaired predominantly through homology-based pathways rather than simple end joining. We also observed inheritance distortion consistent with both gene conversion and loss of damaged gametes. These results help explain why homing-based systems can work in mosquitoes, while also highlighting why repair pathway choice and gamete viability need to be measured directly in any new drive configuration.

Hearing in Lepidoptera mediates a range of ecologically important behaviours, including mate communication, predator avoidance, and acoustic signalling. In moths, the evolution of predator-prey interactions with bats has further shaped hearing through a sensory arms race, with repeated co-option of auditory organs to detect and evade echolocating predators. Despite significant prior characterization of the neurophysiology and behaviour of hearing in moths, the genetic basis of hearing is poorly understood in most insects. In this study, we identify a core set of putative auditory genes in Lepidoptera using a combination of homology-based searches from Drosophila and evolutionary rate analyses. We find 56 genes present across all species and investigate whether gene copy number varies among non-hearing and hearing lineages and among 3 different ear types. We discovered seven genes associated with ear type and one with ear presence, but did not find significant losses in gene copy number in non-hearing species. We identified three genes (btv, Dnai2, and nompB) with strong evidence of selection in hearing clades and five genes with weaker evidence of selection. We discuss the potential roles of btv, nompB, and Dnai2 in ciliary transport and the aging of hair cells, as well as the possibility of actively amplified hearing. Our study serves as a primer and resource for further gene mining and functional testing of auditory genes in moths and other insects.

Long-distance movement in insects has crucial impacts on agriculture, human health, and biodiversity. Although it was long assumed that only large, specialist insects had the navigation capacity to support long-distance dispersal, recent studies have demonstrated that smaller insects, such as the tiny fruit fly Drosophila melanogaster, can maintain extended, straight paths while flying or walking. This raises the question of whether other Drosophila species possess the navigation capacity to support extended dispersal. Resolving this question is particularly important for Drosophila suzukii(spotted-wing drosophila), a potent pest species that causes enormous damage worldwide to ripe fruit and berries. Spotted-wing drosophila has been thought to lack a capacity for long-distance dispersal, as prior studies have estimated maximal daily dispersal distances of less than 90 m. We developed a system to continuously track the flight trajectories of magnetically tethered D. suzukii relative to a discrete, overhead LED that mimicked the sun. We found that flies maintained remarkably straight flight headings that varied unpredictably across individuals. Male and female D. suzukii exhibited a similar navigation capacity; both sexes responded to rotation of a discrete sun stimulus with compensatory turns to maintain a stable relative heading. Our results suggest that D. suzukiihas an underappreciated capacity for rapid, radial dispersal, which could exceed 250 m in 15 min. This capacity may contribute to the pest species' invasiveness and its reliable, annual re-establishment in seasonally intolerable climates. Our findings highlight the importance of developing area-wide, regional strategies to manage the impacts of D. suzukii.

A parasitic mite of honey bee brood (Tropilaelaps mercedesae), is spreading through populations of Apis mellifera honey bees in new regions and poses a major threat to honey bee health. Despite its clear threat, the biology of this mite is poorly understood, with gaps on such fundamental issues as how fast its populations can grow. This leaves the beekeeping world underprepared to plan for its arrival and management. In this study, we documented the growth of T. mercedesae populations in untreated A. mellifera colonies in Thailand and South Korea, and did the same for another parasitic mite (Varroa destructor) when possible. We found that the population growth of T. mercedesae was variable but could reach high levels (daily r of 0.010, 0.036, and 0.057), while the population growth of V. destructor (r = 0.021) matched previous estimates. Our results indicate that T. mercedesae populations can grow rapidly but they do not always attain this potential. Based on our results, humidity should be studied as a potential driver of population growth. If future work can reveal key drivers of T. mercedesae population growth, this would help predict infestations and help design management strategies that exploit the pest's biological vulnerabilities.

The kynurenine pathway metabolizes tryptophan into 3-hydroxykynurenine (3-HK), a precursor for ommochrome eye pigments synthesized via the cardinal (cd) gene in mosquitoes. While cd disruption was presumed neutral, we observed fitness costs in Anopheles stephensi knock-in but not knock-out cd mutants. Here we investigated this anomaly further by assessing survival, fecundity, and midgut integrity across multiple cd mutant lines. Heterozygous knock-in lines, expressing a fluorescent marker and guide RNA for CRISPR/Cas9, exhibited reduced survival post-blood feeding, larva-to-adult survival deficits, and midgut barrier dysfunction, whereas knock-outs showed no such costs. Oral supplementation with xanthurenic acid partially rescued knock-in mortality, implicating oxidative stress linked to 3-HK metabolism. Expression analyses suggest transgene insertion effects, rather than cd disruption, underlie these fitness costs. These findings highlight the importance of evaluating insertional effects in gene drive target selection and support cd as a viable target for genetic control strategies in An. stephensi.

Developmental processes depend on carefully coordinated gene expression. Expression is modulated by the binding of transcription factors (TFs) to cis-regulatory elements (CREs), like enhancers and promoters. Many computational and experimental approaches have been developed to find CREs, particularly enhancers, in the genome, each with strengths and caveats. Given the increasing availability of ATAC-seq data and methods to find TF binding therein, we hypothesized that we could use TF footprinting tools to find clusters of TF binding events within accessible chromatin that may act as CREs. Using Drosophila anterior-posterior patterning network as a test bed, we used a digital genomic footprinting tool (DGT), TOBIAS, on previously published early embryo ATAC-seq data to characterize the TF footprint landscape of 16 TFs essential for embryonic patterning. Even in this system, with its extensive enhancer annotation, most footprinted TF binding sites lie outside of known enhancers, with intergenic and intronic regions hosting the highest TF footprint count, albeit at low density. To find potential novel enhancers, we identified high-density TF footprint clusters that are highly conserved and overlap with active enhancer histone mark signals. Five high confidence candidates were selected for reporter assay validation and all five were found to drive spatially patterned expression in the embryo. This study shows that even in a highly characterized system, the analysis of footprinted TF binding sites in ATAC-seq data can uncover new regulatory regions and suggests this approach may be helpful in using existing ATAC-seq data to find novel CREs. ARTICLE SUMMARYGiven the increasing availability of ATAC-seq datasets, workflows to exploit the data to uncover new cis-regulatory elements (CREs), including enhancers, are valuable. Using early anterior-posterior patterning in the Drosophila embryo as a test case, we find that previously published transcription factor footprinting tools and ATAC-seq data can be analyzed to yield new candidate CREs. Experimental validation confirms the activity of selected candidate CREs, suggesting that existing data can be analyzed to find novel regulatory elements.

Animals fight for resources to obtain fitness benefits; most contests are intrasexual, and males tend to fight more than females. Although the genetic basis of male aggression is well studied, we know little about the genetic variation of female aggression. Female aggression varies with reproductive status and is potentially influenced not only by her genotype, but also by the genotype of her mate. Here we measured both male and female aggression in a set of Drosophila melanogaster inbred lines by competing each line against a standard competitor. Aggression varied among lines for both sexes, but male and female aggression were not correlated. Female aggression for many lines increased with mating, as expected, but not all lines changed aggression. However, when females were mated to males of different lines, male genotype did not affect the post-mating change in aggression, suggesting that ejaculate-mediated effects do not vary across these lines. The aggression level of the standard opponent was positively correlated with that of focal individuals indicating that individuals modulate their behavior according to the genotype of their opponent.

In search of recurrent patterns of postzygotic hybrid incompatibility, we investigated the Holarctic 'Old World Swallowtail' (Papilio machaon Linnaeus) butterfly complex, whose members can easily be crossed in the laboratory. How many species this model system comprises remains unclear, as taxa with highly distinctive larvae but uncertain status come into contact with authentic P. machaon subspecies in Southern parts of the Palearctic region. By determining mitochondrial and ITS2 haplotypes within and away from contact zones, we found that these neighboring populations do exchange genes, as expected from F1 hybrids being generally fertile in our laboratory crosses. Nevertheless, recurrent instances of dysregulation of diapause were uncovered in hybrid progenies. In keeping with Haldane's Rule, pupae of the heterogametic (female) sex were either unable to enter diapause or, in reciprocal crosses, unable to resume development after having initiated diapause, whereas F1 males experienced normal, photoperiod-regulated diapause, but passed on abnormal diapause regulation to part of their female offspring when backcrossed. Comparing male and female pupal weights in hybrid progenies provides estimates of parental incompatibility that allow to rank taxa and predict quantitatively the outcome of additional crosses, as expected if the same regulatory system were involved. A survey of the entomological literature confirms that diapausing pupae that cannot resume development ('perpetual nymphs') are a recurrent feature of interspecific lepidopteran crosses. Moreover, of the two parent species of a perpetual nymph, the paternal one generally has fewer broods per year. These observations are discussed in the light of models of the speciation process.