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.

A long-read draft assembly of the Chinese mantis (Mantodea: Mantidae: Tenodera sinensis) genome reveals patterns of ion channel gain and loss across Arthropoda.

Praying mantids (Mantodea: Mantidae) are iconic insects that have captivated biologists for decades, especially the species with cannibalistic copulatory behavior. This behavior has been cited as evidence that insects lack nociceptive capacities and cannot feel pain; however, this behaviorally driven hypothesis has never been rigorously tested at the genetic or functional level. To enable future studies of nociceptive capabilities in mantids, we sequenced and assembled a draft genome of the Chinese praying mantis (Tenodera sinensis) and identified multiple classes of nociceptive ion channels by comparison to orthologous gene families in Arthropoda. Our assembly-produced using PacBio HiFi reads-is fragmented (total size = 3.03 Gb; N50 = 1.8 Mb; 4,966 contigs), but is highly complete with respect to gene content (BUSCO complete = 98.7% [odb10_insecta]). The size of our assembly is substantially larger than that of most other insects, but is consistent with the size of other mantid genomes. We found that most families of nociceptive ion channels are present in the T. sinensis genome; that they are most closely related to those found in the damp-wood termite (Zootermopsis nevadensis); and that some families have expanded in T. sinensis while others have contracted relative to nearby lineages. Our findings suggest that mantids are likely to possess nociceptive capabilities and provide a foundation for future experimentation regarding ion channel functions and their consequences for insect behavior.

Outgrowing self-denial.

After hitting rock bottom a few months into a prestigious fellowship, a postdoc recounts how they found their way to ADHD medication, therapy, and better mental health.

A de novo long-read genome assembly of the sacred datura plant (Datura wrightii) reveals a role of tandem gene duplications in the evolution of herbivore-defense response.

The sacred datura plant (Solanales: Solanaceae: Datura wrightii) has been used to study plant-herbivore interactions for decades. The wealth of information that has resulted leads it to have potential as a model system for studying the ecological and evolutionary genomics of these interactions. We present a de novo Datura wrightii genome assembled using PacBio HiFi long-reads. Our assembly is highly complete and contiguous (N50 = 179Mb, BUSCO Complete = 97.6%). We successfully detected a previously documented ancient whole genome duplication using our assembly and have classified the gene duplication history that generated its coding sequence content. We use it as the basis for a genome-guided differential expression analysis to identify the induced responses of this plant to one of its specialized herbivores (Coleoptera: Chrysomelidae: Lema daturaphila). We find over 3000 differentially expressed genes associated with herbivory and that elevated expression levels of over 200 genes last for several days. We also combined our analyses to determine the role that different gene duplication categories have played in the evolution of Datura-herbivore interactions. We find that tandem duplications have expanded multiple functional groups of herbivore responsive genes with defensive functions, including UGT-glycosyltranserases, oxidoreductase enzymes, and peptidase inhibitors. Overall, our results expand our knowledge of herbivore-induced plant transcriptional responses and the evolutionary history of the underlying herbivore-response genes.

A high-quality, long-read genome assembly of the whitelined sphinx moth (Lepidoptera: Sphingidae: Hyles lineata) shows highly conserved melanin synthesis pathway genes.

The sphinx moth genus Hyles comprises 29 described species inhabiting all continents except Antarctica. The genus diverged relatively recently (40-25 MYA), arising in the Americas and rapidly establishing a cosmopolitan distribution. The whitelined sphinx moth, Hyles lineata, represents the oldest extant lineage of this group and is one of the most widespread and abundant sphinx moths in North America. Hyles lineata exhibits the large body size and adept flight control characteristic of the sphinx moth family (Sphingidae), but it is unique in displaying extreme larval color variation and broad host plant use. These traits, in combination with its broad distribution and high relative abundance within its range, have made H. lineata a model organism for studying phenotypic plasticity, plant-herbivore interactions, physiological ecology, and flight control. Despite being one of the most well-studied sphinx moths, little data exist on genetic variation or regulation of gene expression. Here, we report a high-quality genome showing high contiguity (N50 of 14.2 Mb) and completeness (98.2% of Lepidoptera BUSCO genes), an important first characterization to facilitate such studies. We also annotate the core melanin synthesis pathway genes and confirm that they have high sequence conservation with other moths and are most similar to those of another, well-characterized sphinx moth, the tobacco hornworm (Manduca sexta).

Observational evidence of herbivore-specific associational effects between neighboring conspecifics in natural, dimorphic populations of Datura wrightii.

Associational effects-in which the vulnerability of a plant to herbivores is influenced by its neighbors-have been widely implicated in mediating plant-herbivore interactions. Studies of associational effects typically focus on interspecific interactions or pest-crop dynamics. However, associational effects may also be important for species with intraspecific variation in defensive traits. In this study, we observed hundreds of Datura wrightii-which exhibits dimorphism in its trichome phenotype-from over 30 dimorphic populations across California. Our aim was to determine whether a relationship existed between the trichome phenotype of neighboring conspecifics and the likelihood of being damaged by four species of herbivorous insects. We visited plants at three timepoints to assess how these effects vary both within and between growing seasons. We hypothesized that the pattern of associational effects would provide rare morphs (i.e., focal plants that are a different morph than their neighbors) with an advantage in the form of reduced herbivory, thereby contributing to the negative frequency-dependent selection previously documented in this system. We found the best predictor of herbivory/herbivore presence on focal plants was the phenotype of the focal plant. However, we also found some important neighborhood effects. The total number of plants near a focal individual predicted the likelihood and/or magnitude of herbivory by Tupiochoris notatus, Lema daturaphila, and Manduca sexta. We also found that velvety focal plants with primarily sticky neighbors are more susceptible to infestation by Tupiochoris notatus and Lema daturaphila. This does not align with the hypothesis that associational effects at the near-neighbor scale contribute to a rare-morph advantage in this system. Overall, the results of our study show that the number and trichome-morph composition of neighboring conspecifics impact interactions between D. wrightii and insect herbivores.

Volatile fatty acid and aldehyde abundances evolve with behavior and habitat temperature in Sceloporus lizards.

Animal signals evolve by striking a balance between the need to convey information through particular habitats and the limitations of what types of signals can most easily be produced and perceived. Here, we present new results from field measures of undisturbed behavior and biochemical analyses of scent marks from 12 species of Sceloporus lizards to explore whether evolutionary changes in chemical composition are better predicted by measures of species behavior, particularly those associated with visual displays, chemoreception, and locomotion, or by measures of habitat climate (precipitation and temperature). We found that more active lizard species used fewer compounds in their volatile scent marks, perhaps conveying less specific information about individual and species identity. Scent marks from more active lizard species also had higher proportions of saturated fatty acids, and the evolution of these compounds has been tracking the phylogeny closely as we would expect for a metabolic byproduct. In contrast, the proportions of unsaturated fatty acids were better explained by evolutionary shifts in habitat temperature (and not precipitation), with species in warmer climates using almost no volatile unsaturated fatty acids. The proportion of aldehydes was explained by both behavior and environment, decreasing with behavioral activity and increasing with habitat temperature. Our results highlight the evolutionary flexibility of complex chemical signals, with different chemical compounds responding to different elements of the selective landscape over evolutionary time.

Herbivore-mediated negative frequency-dependent selection underlies a trichome dimorphism in nature.

Negative frequency-dependent selection (NFDS) has been shown to maintain polymorphism in a diverse array of traits. The action of NFDS has been confirmed through modeling, experimental approaches, and genetic analyses. In this study, we investigated NFDS in the wild using morph-frequency changes spanning a 20-year period from over 30 dimorphic populations of Datura wrightii. In these populations, plants either possess glandular (sticky) or non-glandular (velvety) trichomes, and the ratio of these morphs varies substantially among populations. Our method provided evidence that NFDS, rather than drift or migration, is the primary force maintaining this dimorphism. Most populations that were initially dimorphic remained dimorphic, and the overall mean and variance in morph frequency did not change over time. Furthermore, morph-frequency differences were not related to geographic distances. Together, these results indicate that neither directional selection, drift, or migration played a substantial role in determining morph frequencies. However, as predicted by negative frequency-dependent selection, we found that the rare morph tended to increase in frequency, leading to a negative relationship between the change in the frequency of the sticky morph and its initial frequency. In addition, we found that morph-frequency change over time was significantly correlated with the damage inflicted by two herbivores: Lema daturaphila and Tupiochoris notatus. The latter is a specialist on the sticky morph and damage by this herbivore was greatest when the sticky morph was common. The reverse was true for L. daturaphila, such that damage increased with the frequency of the velvety morph. These findings suggest that these herbivores contribute to balancing selection on the observed trichome dimorphism.

Predatory lizards perceive plant-derived volatile odorants.

Many lizards are olfactory foragers and prey upon herbivorous arthropods, yet their responses to common herbivore-associated plant volatiles remain unknown. As such, their role in mediating plant indirect defenses also remains largely obscured. In this paper, we use a cotton-swab odor presentation assay to ask whether lizards respond to two arthropod-associated plant-derived volatile compounds: 2-(E)-hexenal and hexanoic acid. We studied the response of two lizard species, Sceloporus virgatusand Aspidoscelis exsanguis, because they differ substantially in their foraging behavior. We found that the actively foraging A. exsanguisresponded strongly to hexanoic acid, whereas the ambush foraging S. virgatus responded to 2-(E)-hexenal-an herbivore-associated plant volatile involved in indirect defense against herbivores. These findings indicate that S. virgatus may contribute to plant indirect defense and that a species' response to specific odorants is linked with foraging mode. Future studies can elucidate how lizards use various compounds to locate prey and how these responses impact plant-herbivore interactions.

Herbivory elicits changes in green leaf volatile production via jasmonate signaling and the circadian clock.

The timing of plant volatile emissions is important for a robust indirect defense response. Green leaf volatiles (GLVs) are emitted by plants upon damage but can be suppressed by herbivore-associated elicitors, and the abundance and composition of GLVs vary depending on the timing of herbivore attack. We show that the GLV biosynthetic enzyme HYDROPEROXIDE LYASE (HPL) is transcriptionally regulated by the circadian clock in Nicotiana attenuata. In accordance with transcript abundance of NaHPL, GLV aldehyde pools in intact leaves peaked at night and at subjective night under diurnal and continuous light conditions, respectively. Moreover, although the basal abundance of NaHPL transcripts is upregulated by jasmonate (JA) signaling, JA does not regulate the reduction of NaHPL transcript abundance in damaged leaves by simulated herbivore treatment. Unexpectedly, the plant circadian clock was strongly altered when Manduca sexta larvae fed on N. attenuata, and this was also independent of JA signaling. Lastly, the temporal dynamics of NaHPL transcripts and total GLV emissions were strongly altered by M. sexta larval feeding. Our data suggest that the temporal dynamics of emitted GLV blends result from a combination of damage, JA signaling, herbivore-associated elicitors, and the plant circadian clock.

The circadian clock contributes to diurnal patterns of plant indirect defense in nature.

The plant circadian clock regulates the rhythms of plant metabolism. Many herbivore-induced plant volatiles (HIPVs) fluctuate, diurnally, but the role of the circadian clock in the emission of HIPVs and their ecological consequences remains largely unknown. Here, we show that the timing of herbivore attack can alter the outcome of tri-trophic interactions, and this is mediated by the circadian clock, under both field and glasshouse conditions. Although most HIPV emissions did not have a circadian rhythm, the circadian clock modulated HIPV emissions in a time-dependent manner. HIPVs mediate tri-trophic interactions, and the circadian clock may affect these interactions by modulating HIPV emission in nature.

Skin lipids of the striped plateau lizard (Sceloporus virgatus) correlate with female receptivity and reproductive quality alongside visual ornaments.

Sex pheromones can perform a variety of functions ranging from revealing the location of suitable mates to being honest signals of mate quality, and they are used in the mate selection process by many species of reptile. In this study, we determined whether the skin lipids of female striped plateau lizards (Sceloporus virgatus) can predict the reproductive quality of females, thereby having the potential to serve as pheromones. Using gas chromatography/mass spectrometry, we identified 17 compounds present in skin lipids of female lizards. Using principal component analysis to compare the skin lipid profile of receptive and non-receptive females, we determined that an uncharacterized compound may allow for chemical identification of receptive mates. We also compared extracted principal components to measures of female fitness and reproductive qualities and found that the level of two 18 carbon fatty acids present in a female's skin lipids may indicate her clutch size. Finally, we compared the information content of the skin lipids to that of female-specific color ornaments to assess whether chemical and visual cues transmit different information or not. We found that the chroma of a female's orange throat patch is also related to her clutch size, suggesting that chemical signals may reinforce the information communicated by visual ornamentation in this species which would support the "backup signals" hypothesis for multiple signals.