Genome editing retraces the evolution of toxin resistance in the monarch butterfly.

Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness1. Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits2-4, and new genome editing technology is facilitating functional validation of these mutations in whole organisms1,5. We combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins6-11. Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na+/K+-ATPase)7-11, the physiological target of cardiac glycosides12. Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization13,14. We then performed CRISPR-Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage11,15. We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides16, culminating in triple mutant 'monarch flies' that were as insensitive to cardiac glycosides as monarch butterflies. 'Monarch flies' retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators17-19. The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis13,14,20-22. Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities2,6-11,15,17-19.

A Reference Genome Assembly of Hybrid-Derived California Wild Radish (Raphanus sativus × raphanistrum).

For agriculturally important plants, pollination and herbivory are 2 ecological factors that play into the success of crop yields. Each is also important in natural environments where invasive plants and their effect on species interactions may alter the native ecology. The California Wild Radish (Raphanus sativus × raphanistrum), a hybrid derived from an agriculturally important crop and a nonnative cultivar, is common in California. Remarkably, it has recently replaced wild populations of both progenitor species. Experiments on phenotypic variation for petal color and antiherbivore defenses suggest both pairs of polymorphisms are maintained as a result of pollinator- and herbivore-mediated natural selection. This species provides an opportunity to understand how natural selection shapes the evolution of ecologically important traits when traits are constrained by 2 opposing forces. Here we provide the genome assembly of the California Wild Radish displaying improvement to currently existing genomes for agronomically important crucifers. This genome sequence provides the tools to dissect the genomic architecture of traits related to herbivory and pollination using natural variation in the wild as well as the ability to infer demographic and selective history in the context of hybridization. Study systems like these will improve our understanding and predictions of evolutionary change for correlated traits.

Life-History Plasticity and Water-Use Trade-Offs Associated with Drought Resistance in a Clade of California Jewelflowers.

Water limitation is a primary driver of plant geographic distributions and individual plant fitness. Drought resistance is the ability to survive and reproduce despite limited water, and numerous studies have explored its physiological basis in plants. However, it is unclear how drought resistance and trade-offs associated with drought resistance evolve within plant clades. We quantified the relationship between water availability and fitness for 13 short-lived plant taxa in the Streptanthus clade that vary in their phenology and the availability of water in the environments where they occur. We derived two parameters from these relationships: plant fitness when water is not limiting and the water inflection point (WIF), the watering level at which additional water is most efficiently turned into fitness. We used phylogenetic comparative methods to explore trade-offs related to drought resistance and trait plasticity and the degree to which water relationship parameters are conserved. Taxa from drier climates produced fruits at the lowest water levels, had a lower WIF, flowered earlier, had shorter life spans, had greater plastic water-use efficiency (WUE), and had lower fitness at nonlimiting water. In contrast, later-flowering Streptanthus taxa from less xeric climates experienced high fitness at nonlimiting water but had no fitness at the lowest water levels. Across the clade, we found a trade-off between drought resistance and fitness at high water, though a single ruderal species was an outlier in this relationship. Our results suggest that drought escape trades off with maximal fitness under nonlimiting water, and both are tied to phenology. We also found that variation in trait plasticity determines how different plant species produce fitness over a water gradient.

Behavior Problems Following Childhood TBI: The Role of Sex, Age, and Time Since Injury.

OBJECTIVE: To examine the frequency of behavioral problems after childhood traumatic brain injury (TBI) and their associations with injury severity, sex, and social environmental factors. SETTING: Children's hospitals in the Midwestern/Western United States. PARTICIPANTS: 381 boys and 210 girls with moderate (n = 359) and severe (n = 227) TBI, with an average age at injury of 11.7 years (range 0.3-18) who were injured ≤3 years ago. DESIGN: Secondary data analysis of a multistudy cohort. MAIN MEASURES: Child Behavior Checklist (CBCL) administered pretreatment. RESULTS: Thirty-seven percent had borderline/clinical elevations on the CBCL Total Problem Scale, with comparable rates of Internalizing and Externalizing problems (33% and 31%, respectively). Less parental education was associated with higher rates of internalizing, externalizing, and total problems. Time since injury had a linear association with internalizing symptoms, with greater symptoms at longer postinjury intervals. Younger boys had significantly higher levels of oppositional defiant symptoms than girls, whereas older girls had significantly greater attention-deficit hyperactivity disorder symptoms than boys. CONCLUSIONS: Pediatric TBI is associated with high rates of behavior problems, with lower socioeconomic status predicting substantially elevated risk. Associations of higher levels of internalizing symptoms with greater time since injury highlight the importance of tracking children over time.

A Comparison of 2 Online Parent Skills Training Interventions for Early Childhood Brain Injury: Improvements in Internalizing and Executive Function Behaviors.

OBJECTIVE: To examine the effectiveness of a web-based parenting intervention (Internet-Based Interacting Together Everyday: Recovery After Childhood TBI [I-InTERACT]) and an abbreviated version (Express) in reducing executive dysfunction and internalizing problems among young children following traumatic brain injury (TBI). METHOD: Parents of 113 children (ages 3-9 years) who had sustained a TBI were randomized to 1 of 3 treatment groups: I-InTERACT, Express, or an Internet Resource Comparison (IRC) group. Parents who participated in either I-InTERACT or Express completed self-guided web sessions and received live coaching of their parenting skills via videoconferencing with a therapist. I-InTERACT included additional psychoeducation, stress management, and family communication skills (eg, marriage, grief, pain, and sleep). Analyses of covariance were utilized to compare the groups on parent-reported executive function behaviors (ie, Behavior Rating Inventory of Executive Function) and internalizing symptoms (ie, Child Behavior Checklist) at baseline and 6 months. RESULTS: Parents who participated in Express reported significantly lower levels of executive dysfunction than those in I-InTERACT, ß = -0.49; t(2, 73) = -2.47, P = .048, and significantly lower levels of withdrawal than those in the IRC group, ß = -0.44; t(2, 74) = -2.22, P = .03. The Express group did not significantly differ from the IRC group on executive function behaviors or the I-InTERACT group on internalizing problems, all P > .05. Children with more problems at baseline, families with lower education levels, and parents with more symptoms of depression benefited most. CONCLUSION: A brief, online parent training intervention may be efficacious in improving executive dysfunction and internalizing problems following early TBI, particularly among children of lower socioeconomic status or with existing behavioral concerns.

Plant species within Streptanthoid Complex associate with distinct microbial communities that shift to be more similar under drought.

Prolonged water stress can shift rhizoplane microbial communities, yet whether plant phylogenetic relatedness or drought tolerance predicts microbial responses is poorly understood. To explore this question, eight members of the Streptanthus clade with varying affinity to serpentine soil were subjected to three watering regimes. Rhizoplane bacterial communities were characterized using 16S rRNA gene amplicon sequencing and we compared the impact of watering treatment, soil affinity, and plant species identity on bacterial alpha and diversity. We determined which taxa were enriched among drought treatments using DESeq2 and identified features of soil affinity using random forest analysis. We show that water stress has a greater impact on microbial community structure than soil affinity or plant identity, even within a genus. Drought reduced alpha diversity overall, but plant species did not strongly differentiate alpha diversity. Watering altered the relative abundance of bacterial genera within Proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes, and Acidobacteria, which responded similarly in the rhizoplane of most plant species. In addition, bacterial communities were more similar when plants received less water. Pseudarthrobacter was identified as a feature of affinity to serpentine soil while Bradyrhizobium, Chitinophaga, Rhodanobacter, and Paenibacillus were features associated with affinity to nonserpentine soils among Streptanthus. The homogenizing effect of drought on microbial communities and the increasing prevalence of Gram-negative bacteria across all plant species suggest that effects of water stress on root-associated microbiome structure may be predictable among closely related plant species that inhabit very different soil environments. The functional implications of observed changes in microbiome composition remain to be studied.

Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genomic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families-genes directly mediating interactions with plant chemical defenses-underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many insect lineages are ancient (>150 million years ago (mya)), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several nonherbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza has among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant-binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on living plants (bitter or electrophilic phytotoxins) or their ancestral diet (fermenting plant volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight gene candidates that have also been linked to other dietary transitions in Drosophila.

Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genetic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families - genes directly mediating interactions with plant chemical defenses - underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many lineages are ancient (>150 million years ago [mya]), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several non-herbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza have among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on plants (bitter or electrophilic phytotoxins) or their ancestral diet (yeast and fruit volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight strong gene candidates that have also been linked to other dietary transitions in Drosophila .

The clinical utility of the Children's Communication Checklist-2 in children with early childhood traumatic brain injury.

ObjectivePediatric traumatic brain injury (TBI) is associated with long-term cognitive and behavioral deficits. Social communication impairments are common and impact functional outcomes, such as social engagement and academic performance. There are many barriers to identifying social communication deficits following TBI, including the absence of a standardized parent-reported communication measure for use in this population. The Children's Communication Checklist-Second Edition (CCC-2) has demonstrated utility in identifying communication deficits in diagnoses other than TBI. This study investigated the clinical utility of the CCC-2's social communication scales in children with TBI. Method: 203 children who sustained TBI or orthopedic injuries between the ages of 36 and 83 months were recruited as part of a larger, longitudinal study. We analyzed social communication subscale scores from the CCC-2 an average of 3.5 years postinjury. We used binary logistic regression analyses to examine the measure's accuracy in classifying children with and without social communication deficits on other measures of pragmatic language and social competence. Correlation analyses and linear mixed models were used to examine the construct validity of the CCC-2. Results: The CCC-2 was able to accurately classify those with and without pragmatic language impairments on the Comprehensive Assessment of Spoken Language 92% of the time (sensitivity = 55%) and 96% of the time on the Home and Community Social Behavior scale (sensitivity = 72%). The CCC-2 demonstrated strong correlations with and predictive validity for measures of social communication and competence. Conclusions: The findings offer support for the clinical utility of the CCC-2 in the pediatric TBI population.

Caregiver and Child Behavioral Health Service Utilization Following Pediatric Traumatic Brain Injury.

Given sparse literature examining receipt of behavioral health service in children and caregivers following traumatic brain injury (TBI), we sought to identify predictors of unmet need. We performed an individual participant data meta-analysis using generalized linear mixed-effect models to examine predictors of behavioral health service use and unmet need. We included 572 children, ages 3 to 18, who were hospitalized overnight following complicated mild to severe TBI between 2002 and 2015. Caregivers completed ratings of depression and distress, child behavior problems, family functioning, and behavioral health service utilization. For children, unmet behavioral health service need was defined as an elevation on one or more child behavior problem scales without receipt of behavioral health services. For caregivers, unmet need was defined as an elevation on either a depression or distress scale without behavioral health service utilization. Among those with behavioral health needs, rates of unmet need were high for both children (77.8%) and caregivers (71.4%). Poorer family functioning was related to more unmet need in children (F(1, 497) = 6.57, p = 0.01; OR = 1.8) and caregivers (F(1, 492) = 17.54, p < 0.001; OR = 2.7). Children with unmarried caregivers also had more unmet behavioral health service need than those with married caregivers (F(1, 497) = 12.14, p < 0.001; OR = 2.2). In conclusion, unmet needs are common after pediatric TBI and relate to family factors. The findings underscore the importance of monitoring service needs following pediatric TBI and point to disparities in service use.

Highly contiguous assemblies of 101 drosophilid genomes.

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

Long term effects of early childhood traumatic brain injury on narrative discourse gist and psychosocial functioning.

Purpose: To examine the long-term consequences of early childhood traumatic brain injury (TBI), approximately 6.8 years post-injury, on cognitive communication competency within a complex interplay of individual, contextual, and psychosocial factors. Additionally, we were interested in looking at the association of communication competence with social competence and overall functioning.Methods: 64 children with orthopedic injury, 40 children with moderate traumatic brain injury, and 14 children with severe traumatic brain injury who were between 3 years and 6 years 11 months at injury completed a narrative discourse task and clinical measures in a single visit at a longer term follow up in early adolescence, an average of 6.8 years post injury. Analyses of covariance were conducted to compare groups on the discourse task, and hierarchical linear regressions were conducted to examine the association of discourse measures with clinical measures of cognitive and psychosocial functioning.Results: Children with severe traumatic brain injury performed worse than children with moderate traumatic brain injury and orthopedic injury on all discourse indices and clinical measures. Injury severity, pragmatic skills, and socioeconomic status were associated with discourse gist. Discourse gist was the most sensitive measure of communication competence, and it was significantly associated with psychosocial outcomes independent of group.Conclusion: Children who sustain a severe traumatic brain injury in early childhood are at risk for long-term cognitive communication impairments that may be related to a complex interplay of injury, individual, and social factors.Implications for rehabilitationThe assessment and treatment of cognitive communication impairments after traumatic brain injury are complex and require consideration of individual, contextual, and psychosocial factors.Discourse, in general, and gist reasoning specifically, is a sensitive measure for detecting cognitive communication competence several years after pediatric traumatic brain injury.Treatments to address gist reasoning deficits may support improvements in global and social functioning.

Exemplar Variability Facilitates Retention of Word Learning by Children With Specific Language Impairment.

Purpose: Variability in the input plays an important role in language learning. The current study examined the role of object variability for new word learning by preschoolers with specific language impairment (SLI). Method: Eighteen 4- and 5-year-old children with SLI were taught 8 new words in 3 short activities over the course of 3 sessions. Half of the children saw 3 identical objects corresponding to each new word during training (No Variability group); the other half of the children saw 3 different objects corresponding to each new word during training (High Variability group). Children completed vocabulary learning tests for objects seen during training and for new within-category objects that were never seen during training as a test of category generalization. Learning was assessed the day after each training activity, and retention was assessed 3 weeks after the last training session. Results: There were no group differences on trained or generalization items immediately following training sessions. However, children in the High Variability group demonstrated significantly better retention 3 weeks after experimental training. Conclusion: These findings demonstrate that object variability facilitates retention of new word learning by children with SLI. Supplemental Material: https://doi.org/10.23641/asha.5583979.

Technology-assisted rehabilitation interventions following pediatric brain injury.

INTRODUCTION: Following traumatic brain injury (TBI), children experience a variety of physical, motor, speech, and cognitive deficits that can have a long-term detrimental impact. The emergence and popularity of new technologies has led to research into the development of various apps, gaming systems, websites, and robotics that might be applied to rehabilitation. The objective of this narrative review was to describe the current literature regarding technologically-assisted interventions for the rehabilitation of motor, neurocognitive, behavioral, and family impairments following pediatric TBI. EVIDENCE ACQUISITION: We conducted a series of searches for peer-reviewed manuscripts published between 2000 and 2017 that included a technology-assisted component in the domains of motor, language/communication, cognition, behavior, social competence/functioning, family, and academic/school-based functioning. EVIDENCE SYNTHESIS: Findings suggested several benefits of utilizing technology in TBI rehabilitation including facilitating engagement/adherence, increasing access to therapies, and improving generalizability across settings. There is fairly robust evidence regarding the efficacy of online family problem-solving therapy in improving behavior problems, executive functioning, and family functioning. There was less compelling, but still promising, evidence regarding the efficacy other technology for motor deficits, apps for social skills, and computerized programs for cognitive skills. Overall, many studies were limited in the rigor of their methodology due to small heterogeneous samples and lack of control groups. CONCLUSIONS: Technology-assisted interventions have the potential to enhance pediatric rehabilitation after TBI. Future research is needed to further support their efficacy with larger controlled trials and to identify characteristics of children who are most likely to benefit.

Learning of grammar-like visual sequences by adults with and without language-learning disabilities.

PURPOSE: Two studies examined learning of grammar-like visual sequences to determine whether a general deficit in statistical learning characterizes this population. Furthermore, we tested the hypothesis that difficulty in sustaining attention during the learning task might account for differences in statistical learning. METHOD: In Study 1, adults with normal language (NL) or language-learning disability (LLD) were familiarized with the visual artificial grammar and then tested using items that conformed or deviated from the grammar. In Study 2, a 2nd sample of adults with NL and LLD were presented auditory word pairs with weak semantic associations (e.g., groom + clean) along with the visual learning task. Participants were instructed to attend to visual sequences and to ignore the auditory stimuli. Incidental encoding of these words would indicate reduced attention to the primary task. RESULTS: In Studies 1 and 2, both groups demonstrated learning and generalization of the artificial grammar. In Study 2, neither the NL nor the LLD group appeared to encode the words presented during the learning phase. CONCLUSION: The results argue against a general deficit in statistical learning for individuals with LLD and demonstrate that both NL and LLD learners can ignore extraneous auditory stimuli during visual learning.

Variability in the language input to children enhances learning in a treatment context.

PURPOSE: Artificial language learning studies have demonstrated that learners exposed to many different nonword combinations representing a grammatical form demonstrate rapid learning of that form without explicit instruction. However, learners presented with few exemplars, even when they are repeated frequently, fail to learn the underlying grammar. This study translated this experimental finding in a therapeutic context. METHOD: Eighteen preschool children with language impairment received conversational recast treatment for morpheme errors. Over a 6-week period, half heard 12 unique verbs twice each during recasts (low-variability condition), and half heard 24 unique verbs (high-variability condition). Children's use of trained and untrained morphemes on generalization probes as well as spontaneous use of trained morphemes was tracked throughout treatment. RESULTS: The high-variability condition only produced significant change in children's use of trained morphemes, but not untrained morphemes. Data from individual children confirmed that more children in the high- than the low-variability condition showed a strong treatment effect. Children in the high-variability condition also produced significantly more unique utterances containing their trained morpheme than children in the low-variability condition. CONCLUSION: The results support the use of highly variable input in a therapeutic context to facilitate grammatical morpheme learning.

Exemplar variability facilitates rapid learning of an otherwise unlearnable grammar by individuals with language-based learning disability.

PURPOSE: Even without explicit instruction, learners are able to extract information about the form of a language simply by attending to input that reflects the underlying grammar. In this study, the authors explored the role of variability in this learning by asking whether varying the number of unique exemplars heard by the learner affects learning of an artificial syntactic form. METHOD: Learners with normal language (n = 16) and language-based learning disability (LLD; n = 16) were exposed to strings of nonwords that represented an underlying grammar. Half of the learners heard 3 exemplars 16 times each (low variability group), and the other half of the learners heard 24 exemplars twice each (high variability group). Learners were then tested for recognition of items heard and generalization of the grammar with new nonword strings. RESULTS: Only those learners with LLD who were in the high variability group were able to demonstrate generalization of the underlying grammar. For learners with normal language, both those in the high and the low variability groups showed generalization of the grammar, but relative effect sizes suggested a larger learning effect in the high variability group. CONCLUSION: The results demonstrate that the structure of the learning context can determine the ability to generalize from specific training items to novel cases.

Implicit language learning: Adults' ability to segment words in Norwegian.

Previous language learning research reveals that the statistical properties of the input offer sufficient information to allow listeners to segment words from fluent speech in an artificial language. The current pair of studies uses a natural language to test the ecological validity of these findings and to determine whether a listener's language background influences this process. In Study 1, the "guessibility" of potential test words from the Norwegian language was presented to 22 listeners who were asked to differentiate between true words and nonwords. In Study 2, 22 adults who spoke one of 12 different primary languages learned to segment words from continuous speech in an implicit language learning paradigm. The task consisted of two sessions, approximately three weeks apart, each requiring participants to listen to 7.2 minutes of Norwegian sentences followed by a series of bisyllabic test items presented in isolation. The participants differentially accepted the Norwegian words and Norwegian-like nonwords in both test sessions, demonstrating the capability to segment true words from running speech. The results were consistent across three broadly-defined language groups, despite differences in participants' language background.