A pooled-sample draft genome assembly provides insights into host plant-specific transcriptional responses of a Solanaceae-specializing pest, Tupiocoris notatus (Hemiptera: Miridae).

The assembly of genomes from pooled samples of genetically heterogenous samples of conspecifics remains challenging. In this study, we show that high-quality genome assemblies can be produced from samples of multiple wild-caught individuals. We sequenced DNA extracted from a pooled sample of conspecific herbivorous insects (Hemiptera: Miridae: Tupiocoris notatus) acquired from a greenhouse infestation in Tucson, Arizona (in the range of 30-100 individuals; 0.5 mL tissue by volume) using PacBio highly accurate long reads (HiFi). The initial assembly contained multiple haplotigs (>85% BUSCOs duplicated), but duplicate contigs could be easily purged to reveal a highly complete assembly (95.6% BUSCO, 4.4% duplicated) that is highly contiguous by short-read assembly standards (N 50 = 675 kb; Largest contig = 4.3 Mb). We then used our assembly as the basis for a genome-guided differential expression study of host plant-specific transcriptional responses. We found thousands of genes (N = 4982) to be differentially expressed between our new data from individuals feeding on Datura wrightii (Solanaceae) and existing RNA-seq data from Nicotiana attenuata (Solanaceae)-fed individuals. We identified many of these genes as previously documented detoxification genes such as glutathione-S-transferases, cytochrome P450s, and UDP-glucosyltransferases. Together our results show that long-read sequencing of pooled samples can provide a cost-effective genome assembly option for small insects and can provide insights into the genetic mechanisms underlying interactions between plants and herbivorous pests.

Females translate male mRNA transferred during mating.

Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. Here, we develop an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. We find strong evidence for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance our understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction.

First Report of Tomato Spotted Wilt Virus infecting Lettuce in Yuma, Arizona.

Tomato spotted wilt virus (TSWV, family Tospoviridae, genus Orthotospovirus) is a thrips-vectored pathogen that infects lettuce (Lactuca sativa) and many vegetable crops (Kuo et al. 2014, Hasegawa et al. 2022). Another thrips-borne pathogen of lettuce, impatiens necrotic spot virus (INSV, Tospoviridae, Orthotospovirus), was first reported in 2021 in Yuma, Arizona (Hasegawa et al. 2022). Symptoms of both viruses in lettuce are similar and include necrotic spotting, leaf chlorosis and plant stunting (Kuo et al. 2014). Beginning February through April of 2022, lettuce displaying symptoms of orthotospovirus infection was collected from romaine lettuce (var. longifolia) fields in three regions of Yuma County. A total of 96 plants were collected (5 from Tacna on 2/21, 5 from Wellton on 2/21, 15 from Wellton on 3/23, 30 from Tacna on 4/4, 20 from Wellton on 4/4, and 21 from Yuma Valley on 4/4). The area of the fields ranged from 10 to 18 acres, and the percent disease incidence ranged from 0.8% (Tacna on 4/4) to 2.75% (Tacna on 2/21). Thrips vector were present in all fields were symptomatic plants were observed. One leaf disk per plant (8 mm in diameter) was sampled with a cork borer and grounded individually with a micro pestle in a 1.7 ml microcentrifuge tube with 150 ul of Tri-reagent (Molecular Research Center). Total RNA was extracted from each sample using the Zymo Direct-zol-96 kit (Zymo Research). Samples were diluted with water to a ratio of 1:10 after RNA extraction. RT-qPCR was performed in 20 ul reactions with 5 ul of input RNA using the PCR Biosystems qPCRBIO Probe 1-Step Go No-ROX for the cDNA/qPCR master mix. RT-qPCR assays were carried out in multiplex reactions using primers specific for TSWV and INSV, in addition to a lettuce internal control gene (LOC111918243), along with negative controls. Primer and probe sequence details are reported in supplemental Table 1. We used a cycle threshold (ct) < 40 to indicate a positive result for both INSV and TSWV (Chen et al. 2013; Boonham et al. 2002). RT-qPCR successfully amplified INSV in 90 out of 96 samples and TSWV in 8 out of 96 samples. These 8 samples tested positive for both TSWV and INSV, showing that INSV and TSWV co-infected lettuce plants. Thus overall, ∼ 95% of symptomatic plants were infected with INSV alone, and ∼ 8% were co-infected with TSWV and INSV. Amplicons of 4 samples testing positive for TSWV were sent for Sanger sequencing (Eurofins Genomics, Louisville, KY). All were identified as TSWV. One amplicon with TSWV was sequenced for INSV and double infection was confirmed. BLAST results from the NCBI nt database show 100% (138 bp) identity to TWSV (MW519211) for the 4 TWSV amplicons and 99.22% (137 bp) identity to INSV (KX790323) for the INSV amplicon. Sanger sequence data are in the GenBank (accession: OQ685940-OQ685944). Based on RT-qPCR results, all TSWV infected plants were also infected with INSV. INSV may have been introduced to Yuma by infected plants or thrips from lettuce transplants produced in California (Hasegawa et al. 2022). TSWV could have been introduced similarly. To our knowledge, this is the first report of TSWV infecting lettuce in Yuma and the first report of INSV and TSWV co-infecting lettuce. TSWV and INSV infections have remained low since their discovery in Yuma, in part due to effective cultural and chemical management by lettuce growers (Palumbo, 2022). However, an increase in disease incidence and severity in the future could have a significant negative impact on production of romaine lettuce in the region.

Physiological and life history changes associated with seasonal adaptation in the cactophilic Drosophila mojavensis.

Many insects inhabiting temperate climates are faced with changing environmental conditions throughout the year. Depending on the species, these environmental fluctuations can be experienced within a single generation or across multiple generations. Strategies for dealing with these seasonal changes vary across populations. Drosophila mojavensis is a cactophilic Drosophila species endemic to the Sonoran Desert. The Sonoran Desert regularly reaches temperatures of 50°C in the summer months. As individuals of this population are rare to collect in the summer months, we simulated the cycling temperatures experienced by D. mojavensis in the Sonoran Desert from April to July (four generations) in a temperature- and light-controlled chamber, to understand the physiological and life history changes that allow this population to withstand these conditions. In contrast to our hypothesis of a summer aestivation, we found that D. mojavensis continue to reproduce during the summer months, albeit with lower viability, but the adult survivorship of the population is highly reduced during this period. As expected, stress resistance increased during the summer months in both the adult and the larval stages. This study examines several strategies for withstanding the Sonoran Desert summer conditions which may be informative in the study of other desert endemic species.

Divergent evolutionary trajectories shape the postmating transcriptional profiles of conspecifically and heterospecifically mated cactophilic Drosophila females.

Postmating-prezygotic (PMPZ) reproductive isolation is hypothesized to result from divergent coevolutionary trajectories of sexual selection and/or sexual conflict in isolated populations. However, the genetic basis of PMPZ incompatibilities between species is poorly understood. Here, we use a comparative framework to compare global gene expression in con- and heterospecifically mated Drosophila mojavensis and D. arizonae female reproductive tracts. We find striking divergence between the species in the female postmating transcriptional response to conspecific mating, including differences in differential expression (DE), alternative splicing (AS), and intron retention (IR). As predicted, heterospecific matings produce disrupted transcriptional profiles, but the overall patterns of misregulation are different between the reciprocal crosses. Moreover, we find a positive correlation between postmating transcriptional divergence between species and levels of transcriptional disruption in heterospecific crosses. This result indicates that mating responsive genes that have diverged more in expression also have more disrupted transcriptional profiles in heterospecifically mated females. Overall, our results provide insights into the evolution of PMPZ isolation and lay the foundation for future studies aimed at identifying specific genes involved in PMPZ incompatibilities and the evolutionary forces that have contributed to their divergence in closely related species.

Propagation of seminal toxins through binary expression gene drives could suppress populations.

Gene drives can be highly effective in controlling a target population by disrupting a female fertility gene. To spread across a population, these drives require that disrupted alleles be largely recessive so as not to impose too high of a fitness penalty. We argue that this restriction may be relaxed by using a double gene drive design to spread a split binary expression system. One drive carries a dominant lethal/toxic effector alone and the other a transactivator factor, without which the effector will not act. Only after the drives reach sufficiently high frequencies would individuals have the chance to inherit both system components and the effector be expressed. We explore through mathematical modeling the potential of this design to spread dominant lethal/toxic alleles and suppress populations. We show that this system could be implemented to spread engineered seminal proteins designed to kill females, making it highly effective against polyandrous populations.

Novel genetic basis of resistance to Bt toxin Cry1Ac in Helicoverpa zea.

Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis have advanced pest management, but their benefits are diminished when pests evolve resistance. Elucidating the genetic basis of pest resistance to Bacillus thuringiensis toxins can improve resistance monitoring, resistance management, and the design of new insecticides. Here, we investigated the genetic basis of resistance to Bacillus thuringiensis toxin Cry1Ac in the lepidopteran Helicoverpa zea, one of the most damaging crop pests in the United States. To facilitate this research, we built the first chromosome-level genome assembly for this species, which has 31 chromosomes containing 375 Mb and 15,482 predicted proteins. Using a genome-wide association study, fine-scale mapping, and RNA-seq, we identified a 250-kb quantitative trait locus on chromosome 13 that was strongly associated with resistance in a strain of Helicoverpa zea that had been selected for resistance in the field and lab. The mutation in this quantitative trait locus contributed to but was not sufficient for resistance, which implies alleles in more than one gene contributed to resistance. This quantitative trait locus contains no genes with a previously reported role in resistance or susceptibility to Bacillus thuringiensis toxins. However, in resistant insects, this quantitative trait locus has a premature stop codon in a kinesin gene, which is a primary candidate as a mutation contributing to resistance. We found no changes in gene sequence or expression consistently associated with resistance for 11 genes previously implicated in lepidopteran resistance to Cry1Ac. Thus, the results reveal a novel and polygenic basis of resistance.

Gene expression and alternative splicing dynamics are perturbed in female head transcriptomes following heterospecific copulation.

BACKGROUND: Despite the growing interest in the female side of copulatory interactions, the roles played by differential expression and alternative splicing mechanisms of pre-RNA on tissues outside of the reproductive tract have remained largely unknown. Here we addressed these questions in the context of con- vs heterospecific matings between Drosophila mojavensis and its sister species, D. arizonae. We analyzed transcriptional responses in female heads using an integrated investigation of genome-wide patterns of gene expression, including differential expression (DE), alternative splicing (AS) and intron retention (IR). RESULTS: Our results indicated that early transcriptional responses were largely congruent between con- and heterospecific matings but are substantially perturbed over time. Conspecific matings induced functional pathways related to amino acid balance previously associated with the brain's physiology and female postmating behavior. Heterospecific matings often failed to activate regulation of some of these genes and induced expression of additional genes when compared with those of conspecifically-mated females. These mechanisms showed functional specializations with DE genes mostly linked to pathways of proteolysis and nutrient homeostasis, while AS genes were more related to photoreception and muscle assembly pathways. IR seems to play a more general role in DE regulation during the female postmating response. CONCLUSIONS: We provide evidence showing that AS genes substantially perturbed by heterospecific matings in female heads evolve at slower evolutionary rates than the genome background. However, DE genes evolve at evolutionary rates similar, or even higher, than those of male reproductive genes, which highlights their potential role in sexual selection and the evolution of reproductive barriers.

Mate discrimination among subspecies through a conserved olfactory pathway.

Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner.

Contributions of cis- and trans-Regulatory Evolution to Transcriptomic Divergence across Populations in the Drosophila mojavensis Larval Brain.

Natural selection on gene expression was originally predicted to result primarily in cis- rather than trans-regulatory evolution, due to the expectation of reduced pleiotropy. Despite this, numerous studies have ascribed recent evolutionary divergence in gene expression predominantly to trans-regulation. Performing RNA-seq on single isofemale lines from genetically distinct populations of the cactophilic fly Drosophila mojavensis and their F1 hybrids, we recapitulated this pattern in both larval brains and whole bodies. However, we demonstrate that improving the measurement of brain expression divergence between populations by using seven additional genotypes considerably reduces the estimate of trans-regulatory contributions to expression evolution. We argue that the finding of trans-regulatory predominance can result from biases due to environmental variation in expression or other sources of noise, and that cis-regulation is likely a greater contributor to transcriptional evolution across D. mojavensis populations. Lastly, we merge these lines of data to identify several previously hypothesized and intriguing novel candidate genes, and suggest that the integration of regulatory and population-level transcriptomic data can provide useful filters for the identification of potentially adaptive genes.

Chromosome-level hybrid de novo genome assemblies as an attainable option for nonmodel insects.

The emergence of third-generation sequencing (3GS; long-reads) is bringing closer the goal of chromosome-size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of nonmodel organisms. However, long-read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short-reads and long-reads, provide an alternative efficient and cost-effective approach to generate de novo, chromosome-level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation are constantly being expanded and improved. This makes it difficult for nonexperts to find efficient, fast and tractable computational solutions for genome assembly, especially in the case of nonmodel organisms lacking a reference genome or one from a closely related species. In this study, we review and test the most recent pipelines for hybrid assemblies, comparing the model organism Drosophila melanogaster to a nonmodel cactophilic Drosophila, D. mojavensis. We show that it is possible to achieve excellent contiguity on this nonmodel organism using the dbg2olc pipeline.

Genomic analysis of the four ecologically distinct cactus host populations of Drosophila mojavensis.

BACKGROUND: Relationships between an organism and its environment can be fundamental in the understanding how populations change over time and species arise. Local ecological conditions can shape variation at multiple levels, among these are the evolutionary history and trajectories of coding genes. This study examines the rate of molecular evolution at protein-coding genes throughout the genome in response to host adaptation in the cactophilic Drosophila mojavensis. These insects are intimately associated with cactus necroses, developing as larvae and feeding as adults in these necrotic tissues. Drosophila mojavensis is composed of four isolated populations across the deserts of western North America and each population has adapted to utilize different cacti that are chemically, nutritionally, and structurally distinct. RESULTS: High coverage Illumina sequencing was performed on three previously unsequenced populations of D. mojavensis. Genomes were assembled using the previously sequenced genome of D. mojavensis from Santa Catalina Island (USA) as a template. Protein coding genes were aligned across all four populations and rates of protein evolution were determined for all loci using a several approaches. CONCLUSIONS: Loci that exhibited elevated rates of molecular evolution tend to be shorter, have fewer exons, low expression, be transcriptionally responsive to cactus host use and have fixed expression differences across the four cactus host populations. Fast evolving genes were involved with metabolism, detoxification, chemosensory reception, reproduction and behavior. Results of this study give insight into the process and the genomic consequences of local ecological adaptation.

Assessing the Architecture of Drosophila mojavensis Locomotor Evolution with Bulk Segregant Analysis.

Behavior is frequently predicted to be especially important for evolution in novel environments. If these predictions are accurate, there might be particular patterns of genetic architecture associated with recently diverged behaviors. Specifically, it has been predicted that behaviors linked to population divergence should be underpinned by a few genes of relatively large effect, compared to architectures of intrapopulation behavioral variation, which is considered to be highly polygenic. More mapping studies of behavioral variation between recently diverged populations are needed to continue assessing the generality of these predictions. Here, we used a bulk segregant mapping approach to dissect the genetic architecture of a locomotor trait that has evolved between two populations of the cactophilic fly Drosophila mojavensis We created an F8 mapping population of 1,500 individuals from advanced intercross lines and sequenced the 10% of individuals with the highest and lowest levels of locomotor activity. Using three alternative statistical approaches, we found strong evidence for two relatively large-effect QTL that is localized in a region homologous to a region of densely packed behavior loci in Drosophila melanogaster, suggesting that clustering of behavior genes may display relatively deep evolutionary conservation. Broadly, our data are most consistent with a polygenic architecture, though with several loci explaining a high proportion of variation in comparison to similar behavioral traits. We further note the presence of several antagonistic QTL linked to locomotion and discuss these results in light of theories regarding behavioral evolution and the effect size and direction of QTL for diverging traits in general.

Positive selection at sites of chemosensory genes is associated with the recent divergence and local ecological adaptation in cactophilic Drosophila.

BACKGROUND: Adaptation to new hosts in phytophagous insects often involves mechanisms of host recognition by genes of sensory pathways. Most often the molecular evolution of sensory genes has been explained in the context of the birth-and-death model. The role of positive selection is less understood, especially associated with host adaptation and specialization. Here we aim to contribute evidence for this latter hypothesis by considering the case of Drosophila mojavensis, a species with an evolutionary history shaped by multiple host shifts in a relatively short time scale, and its generalist sister species, D. arizonae. RESULTS: We used a phylogenetic and population genetic analysis framework to test for positive selection in a subset of four chemoreceptor genes, one gustatory receptor (Gr) and three odorant receptors (Or), for which their expression has been previously associated with host shifts. We found strong evidence of positive selection at several amino acid sites in all genes investigated, most of which exhibited changes predicted to cause functional effects in these transmembrane proteins. A significant portion of the sites identified as evolving positively were largely found in the cytoplasmic region, although a few were also present in the extracellular domains. CONCLUSIONS: The pattern of substitution observed suggests that some of these changes likely had an effect on signal transduction as well as odorant recognition and protein-protein interactions. These findings support the role of positive selection in shaping the pattern of variation at chemosensory receptors, both during the specialization onto one or a few related hosts, but as well as during the evolution and adaptation of generalist species into utilizing several hosts.

Behavioral evolution accompanying host shifts in cactophilic Drosophila larvae.

For plant utilizing insects, the shift to a novel host is generally accompanied by a complex set of phenotypic adaptations. Many such adaptations arise in response to differences in plant chemistry, competitive environment, or abiotic conditions. One less well-understood factor in the evolution of phytophagous insects is the selective environment provided by plant shape and volume. Does the physical structure of a new plant host favor certain phenotypes? Here, we use cactophilic Drosophila, which have colonized the necrotic tissues of cacti with dramatically different shapes and volumes, to examine this question. Specifically, we analyzed two behavioral traits in larvae, pupation height, and activity that we predicted might be related to the ability to utilize variably shaped hosts. We found that populations of D. mojavensis living on lengthy columnar or barrel cactus hosts have greater activity and pupate higher in a laboratory environment than populations living on small and flat prickly pear cactus cladodes. Crosses between the most phenotypically extreme populations suggest that the genetic architectures of these behaviors are distinct. A comparison of activity in additional cactophilic species that are specialized on small and large cactus hosts shows a consistent trend. Thus, we suggest that greater motility and an associated tendency to pupate higher in the laboratory are potential larval adaptations for life on a large plant where space is more abundant and resources may be more sparsely distributed.

Transcriptional variation associated with cactus host plant adaptation in Drosophila mettleri populations.

Although the importance of host plant chemistry in plant-insect interactions is widely accepted, the genetic basis of adaptation to host plants is not well understood. Here, we investigate transcriptional changes associated with a host plant shift in Drosophila mettleri. While D. mettleri is distributed mainly throughout the Sonoran Desert where it specializes on columnar cacti (Carnegiea gigantea and Pachycereus pringleii), a population on Santa Catalina Island has shifted to chemically divergent coastal prickly pear cactus (Opuntia littoralis). We compared gene expression of larvae from the Sonoran Desert and Santa Catalina Island when reared on saguaro (C. gigantea), coastal prickly pear and laboratory food. Consistent with expectations based on the complexity and toxicity of cactus relative to laboratory food, within-population comparisons between larvae reared on these food sources revealed transcriptional differences in detoxification and other metabolic pathways. The majority of transcriptional differences between populations on the cactus hosts were independent of the rearing environment and included a disproportionate number of genes involved in processes relevant to host plant adaptation (e.g. detoxification, central metabolism and chemosensory pathways). Comparisons of transcriptional reaction norms between the two populations revealed extensive shared plasticity that likely allowed colonization of coastal prickly pear on Santa Catalina Island. We also found that while plasticity may have facilitated subsequent adaptive divergence in gene expression between populations, the majority of genes that differed in expression on the novel host were not transcriptionally plastic in the presumed ancestral state.

Connecting genotypes, phenotypes and fitness: harnessing the power of CRISPR/Cas9 genome editing.

One of the fundamental goals in evolution and ecology is to identify the genetic basis of adaptive phenotypes. Unfortunately, progress towards this goal has been hampered by a lack of genetic tools available for nonmodel organisms. The exciting new development of the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9) genome-editing system now promises to transform the field of molecular ecology by providing a versatile toolkit for manipulating the genome of a wide variety of organisms. Here, we review the numerous applications of this groundbreaking technology and provide a practical guide to the creation of genetic knockouts, transgenics and other related forms of gene manipulation in nonmodel organisms. We also specifically discuss the potential uses of the CRISPR/Cas9 system in ecological and evolutionary studies, which will further advance the field towards the long-standing goal of connecting genotypes, phenotypes and fitness.

Ecological genomics of host shifts in Drosophila mojavensis.

Advances in next-generation sequencing technologies have liberated our dependency on model laboratory species for answering genomic and transcriptomic level questions. These new techniques have dramatically expanded our breadth of study organisms and have allowed the analysis of species from diverse ecological environments. One such species is the cactophilic Drosophila mojavensis that inhabits the deserts of western North America. These insects feed and develop in the necrotic cacti, feeding largely on the microflora of the necrotic plant tissues. Drosophila mojavensis is composed of four geographically and ecologically separated populations. Each population (Baja California peninsula, mainland Sonoran Desert, Mojave Desert and Santa Catalina Island) utilizes the necrotic tissues of distinct cactus species. The differences in the nutritional and chemical composition of the necroses include a set of toxic compounds to which resident population must adapt. These ecological differences have facilitated many of the life history, behavior, physiological and genetic differences between the cactus host populations. Genomic resources have allowed investigators to examine the genomic and transcriptional level changes associated with the local adaptation of the four D. mojavensis populations, thereby providing further understanding of the genetic mechanism of adaptation and its role in the divergence of ecologically distinct populations.

Preadult parental diet affects offspring development and metabolism in Drosophila melanogaster.

When Drosophila melanogaster larvae are reared on isocaloric diets differing in their amounts of protein relative to sugar, emerging adults exhibit significantly different development times and metabolic pools of protein, glycogen and trigylcerides. In the current study, we show that the influence of larval diet experienced during just one generation extends into the next generation, even when that subsequent generation had been shifted to a standard diet during development. Offspring of flies that were reared on high protein relative to sugar underwent metamorphosis significantly faster, had higher reproductive outputs, and different metabolic pool contents compared to the offspring of adults from low protein relative to sugar diets. In addition, isofemale lines differed in the degree to which parental effects were observed, suggesting a genetic component to the observed transgenerational influences.

Mutations in the neverland gene turned Drosophila pachea into an obligate specialist species.

Most living species exploit a limited range of resources. However, little is known about how tight associations build up during evolution between such specialist species and the hosts they use. We examined the dependence of Drosophila pachea on its single host, the senita cactus. Several amino acid changes in the Neverland oxygenase rendered D. pachea unable to transform cholesterol into 7-dehydrocholesterol (the first reaction in the steroid hormone biosynthetic pathway in insects) and thus made D. pachea dependent on the uncommon sterols of its host plant. The neverland mutations increase survival on the cactus's unusual sterols and are in a genomic region that faced recent positive selection. This study illustrates how relatively few genetic changes in a single gene may restrict the ecological niche of a species.