Arjen E. Van'T Hof

Moths and butterflies (Lepidoptera) are the largest group of organisms with female heterogamety and the sex chromosome system WZ/ZZ (female/male) or exceptionally Z0/ZZ. However, the genetic basis of sex determination in Lepidoptera remained unknown for a long time until the sex‐determining pathway was discovered in 2014 in the silkworm Bombyx mori . In this species, the dominant W chromosome carries a Feminizer ( Fem ) gene encoding a precursor of a Fem piRNA that promotes femaleness by downregulating the expression of a Z‐linked gene, Masculinizer ( Masc ). In the W chromosome absence, Masc promotes male development and controls dosage compensation. In the 10 years since this discovery, significant progress has been made in understanding the molecular mechanisms of sex determination in Lepidoptera. Data from recent studies discussed in this review suggest a conserved role for Masc in male sex determination and dosage compensation in the clade Ditrysia, which comprises the majority of Lepidoptera. Although the primary sex‐determining signals are not conserved, the presence of feminizing piRNAs of different origins in distantly related species suggests convergent evolution of a similar mechanism of female sex determination. A unique exception is zygosity‐based sex determination in the butterfly Bicyclus anynana , where the primary signal is the state of the hypervariable Masc gene. In other species with a dispensable W chromosome, such as the silkmoth Samia cynthia , sex is determined by the Z:A ratio, but a molecular mechanism is not yet known. Overall, the available data suggest considerable diversity in the upstream molecular mechanisms of sex determination in Lepidoptera.

Nature has devised many ways of producing males and females. Here, we report on a previously undescribed mechanism for Lepidoptera that functions without a female-specific gene. The number of alleles or allele heterozygosity in a single Z-linked gene ( BaMasc ) is the primary sex-determining switch in Bicyclus anynana butterflies. Embryos carrying a single BaMasc allele develop into WZ (or Z0) females, those carrying two distinct alleles develop into ZZ males, while (ZZ) homozygotes initiate female development, have mismatched dosage compensation, and die as embryos. Consequently, selection against homozygotes has favored the evolution of spectacular allelic diversity: 205 different coding sequences of BaMasc were detected in a sample of 246 females. The structural similarity of a hypervariable region (HVR) in BaMasc to the HVR in Apis mellifera csd suggests molecular convergence between deeply diverged insect lineages. Our discovery of this primary switch highlights the fascinating diversity of sex-determining mechanisms and underlying evolutionary drivers.

The evolution of sibling cannibalism as a maternal strategy is particularly challenging to explain when nurseries are shared among multiple females. Such is the case for the damselfly, Megaloprepus caerulatus, whose females lay eggs in bark above the water line in large, water-filled tree holes. Asynchronous egg hatching appears to be a maternal bet-hedging strategy to increase the chances that cannibalistic offspring hatch during windows of opportunity, which occur after the remaining large larvae emerge, having eaten all others. We investigated the proximate causes of asynchronous hatching. By monitoring the pattern of egg hatching under ambient temperature in an insectary, we found that egg hatching co-occurred with lower ambient temperatures, which decreased with increasing rainfall. Treating fully developed eggs to a lower temperature for two hours triggered increased hatching relative to controls at ambient temperature. Dissection of control clutches indicated that embryonic development of siblings was asynchronous. Results suggested that the hatching trigger is adaptive. Rainfall assures a recharge of the larval habitat with water and provides wet conditions essential for neonate mobility on bark. Only 40% of neonates in a 4-day drying treatment survived; none survived the 8- and 14-day treatments. This novel hatching trigger should increase the number of neonates entering the nursery after rains, constraining a mother’s control over the timing of egg hatch, while increasing the competition among related and unrelated offspring for limited windows of opportunity in the shared nursery.

Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of malaria control, but the genetics of resistance are only partially understood. We performed a large scale multi-country genome-wide association study of resistance to two widely used insecticides: deltamethrin and pirimiphos-methyl, using sequencing data from An. gambiae and An. coluzzii from ten locations in West Africa. Resistance was highly multi-genic, multi-allelic and variable between populations. While the strongest and most consistent association with deltamethrin resistance came from Cyp6aa1, this was based on several independent copy number variants (CNVs) in An. coluzzii, and on a non-CNV haplotype in An. gambiae. For pirimiphos-methyl, signals included Ace1, cytochrome P450s, glutathione S-transferases and the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes showed evidence of cross-resistance to both insecticides. These locally-varying, multi-allelic patterns highlight the challenges involved in genomic monitoring of resistance, and may form the basis for improved surveillance methods.

Sex chromosomes are predicted to harbour elevated levels of sexually antagonistic variation due to asymmetries in the heritability of recessive traits in the homogametic versus heterogametic sex. This evolutionary dynamic may manifest as high recessive load specifically affecting the homogametic sex, and the retention of haplotype diversity in small populations. We tested the hypothesis that the Z chromosome in the butterfly Bicyclus anynana carries a high inbred load for male fertility and viability. Homozygosity of Z chromosome blocks was produced by daughter–father backcrosses, and inferred from marker loci positioned via a linkage map. Male sterility was, in general, unrelated to homozygosity in any region of the Z, but there was an extreme deficit of homozygous males within a 2 cM interval in all families. In contrast, no corresponding skew in Z genotype was detected in their (hemizygous) sisters. The same pattern was observed in historically inbred lines, indicating a high frequency of recessive lethals in the ancestral population. Allele-frequency changes between 1993 and 2006 (70 generations at N_e ~ 160) show that, despite the loss of many haplotypes, diversity was retained significantly above the neutral expectation. Effective overdominance in the lethal region can account for this effect locally but not in other parts of the chromosome, that are also associated with persistent linkage disequilibrium. These unexpected patterns suggest the operation of other factors, such as epistatic selection, recombination suppression, assortative mating and meiotic drive. Our results highlight the role of balancing selection in maintaining the inbred load and linked genetic diversity.

The rise of dark (melanic) forms of many species of moth in heavily coal-polluted areas of nineteenth- and twentieth-century Britain, and their post-1970s fall, point to a common selective pressure (camouflage against bird predators) acting at the community level. The extent to which this convergent phenotypic response relied on similar genetic and developmental mechanisms is unknown. We examine this problem by testing the hypothesis that the locus controlling melanism inPhigalia pilosariaandOdontopera bidentata, two species of geometrid moth that showed strong associations between melanism and coal pollution, is the same as that controlling melanism inBiston betularia, previously identified as the genecortex. Comparative linkage mapping using family material supports the hypothesis for both species, indicating a deeply conserved developmental mechanism for melanism involvingcortex. However, in contrast to the strong selective sweep signature seen in BritishB. betularia, no significant association was detected betweencortex-region markers and melanic morphs in wild-caught samples ofP. pilosariaandO. bidentata, implying much older, or diverse, origins of melanic morph alleles in these latter species.

The spread of resistance to insecticides in disease-carrying mosquitoes poses a threat to the effectiveness of control programmes, which rely largely on insecticide-based interventions. Monitoring mosquito populations is essential, but obtaining phenotypic measurements of resistance is laborious and error-prone. High-throughput genotyping offers the prospect of quick and repeatable estimates of resistance, while also allowing resistance markers to be tracked and studied. To demonstrate the potential of highly-mulitplexed genotypic screening for measuring resistance-association of mutations and tracking their spread, we developed a panel of 28 known or putative resistance markers in the major malaria vector Anopheles gambiae, which we used to screen mosquitoes from a wide swathe of Sub-Saharan Africa (Burkina Faso, Ghana, Democratic Republic of Congo (DRC) and Kenya). We found resistance association in four markers, including a novel mutation in the detoxification gene Gste2 (Gste2-119V). We also identified a duplication in Gste2 combining a resistance-associated mutation with its wild-type counterpart, potentially alleviating the costs of resistance. Finally, we describe the distribution of the multiple origins of kdr resistance, finding unprecedented diversity in the DRC. This panel represents the first step towards a quantitative genotypic model of insecticide resistance that can be used to predict resistance status in An. gambiae.

Background Malaria vector control and research rely heavily on monitoring mosquito populations for the development of resistance to public health insecticides. One standard method for determining susceptibility in adult mosquito populations is the World Health Organization test (WHO bioassay). The WHO bioassay kit consists of several acrylic pieces that are assembled into a unit. Parts of the kit commonly break, reducing the capacity of insectaries to carry out resistance profiling. Since there is at present only a single supplier for the test kits, replacement parts can be hard to procure in a timely fashion. Here, we present 3D printable versions for all pieces of the WHO bioassay kit. Results Using widely available polylactic acid (PLA) filament as a printing material, we were able to design and print functional replacements for each piece of the WHO bioassay kit. We note no significant difference in mortality results obtained from PLA printed tubes and WHO acrylic tubes. Additionally, we observed no degradation of PLA in response to prolonged exposure times of commonly used cleaning solutions. Conclusions Our designs can be used to produce replacement parts for the WHO bioassay kit in any facility with a 3D printer, which are becoming increasingly widespread. 3D printing technologies can affordably and rapidly address equipment shortages and be used to develop bespoke equipment in laboratories.