Brian Wiegmann

Flies are diverse models for the study of the evolution of the parasitic habit. Multiple repeated origins of specialized feeding in flies can reveal the molecular mechanisms underlying adaptive changes that both produce and sustain the parasitic lifestyle. With 1525 described species the blow flies (family Calliphoridae), are an ideal candidate for comparative study of the genetic and behavioral determinants of feeding adaptations. In this family of flies, different species have been recorded feeding on living tissues of a vertebrates, living tissues of invertebrates; decaying organic matter-- especially carrion or wound tissue; and blood. Considering the feeding habits of their larvae, blow flies are classified in: necrosaprophagous species, which feed on decomposing tissue; facultative ectoparasites, which feed on dead organic matter (necrophagous), or infest necrotic tissues of living vertebrates; and obligate parasites, which feed only on the living tissues of their hosts. For this reason, they are important as indicator organisms in forensic cases, are major agricultural pests of livestock, and they play a major role in nearly all terrestrial ecosystems as decomposers of organic material. The origin and evolutionary history of diverse specialized feeding habits in Calliphoridae are still vastly understudied, and are limited by a lack of phylogenetic, genetic and ecological information. With this project, an established team of evolutionary, ecological and genomic scientists will collaboratively investigate the causes and consequences of trophic specialization as a driver of species diversity across three integrated dimensions of blow fly research: (1) phylogenetic: reconstructing the history of blow fly evolutionary relationships and mapping the transitions of feeding habits that occurred repeatedly in the family; (2) functional, quantifying the index of preference for different food sources, and testing the effects of selection on feeding preferences; and investigating the role of the blow fly gut microbiome in mediating diet adaptations and maintaining diverse biotic interactions; and (3) genetic/genomic, identifying genes and gene regions associated with specific specialized feeding habits and using an established CRISPR Cas9 gene editing system to test hypotheses about the lability, maintenance, and control of feeding strategies in diverse blow fly models. The project includes a novel cross laboratory training and data collection for graduate students and postdocs between NCSU and Sao Paulo, Brazil, the development of an on-line data resource of genomic, taxonomic and ecological information on blow flies, and participation in multi-institutional training and research opportunities that include resources and outreach to individuals and communities that are underrepresented in science.

This study focuses on phylogenomic sampling and analysis to address evolutionary questions at the species-level and above in mosquitoes (Insecta: Diptera: Culicidae). Phylogenetic understanding of mosquitoes lags well behind most other major economically or biomedically important insects. Four primary objectives guide our efforts to reconstruct the evolutionary history of mosquito species and associated aetiological agents through phylogenomics: 1) To generate DNA sequences from field collected mosquitoes and museum holdings using anchored enrichment (AHE) processes to identify monophyletic lineages and establish relationships within and among all higher Culicid taxa, from specific to family level; 2) Use comprehensively sampled phylogenetic estimates for Aedine clades that contain taxa that have crossed ecological boundaries to address evolutionary questions about domestication, and accompanying habitat- and host-shifts that appear to drive the evolution of major disease vectors; 3) To anchor these changes within the biogeographic and temporal context of mosquito diversification, and test the effects of ecological change on diversification rates in multiple areas of the Culicid tree, and 4) To further expand the development of an existing species and pathogen biodiversity database (www.VectorMap.si.edu), to include a data-enabled, interactive phylogeny of World mosquitoes that provides a contextualized interface for evolutionary studies of mosquito ecology, evolution, species interactions and taxonomic identification. Intellectual Merit: Phylogeny estimates will be used as a comparative and temporal framework to test hypotheses involving ecological specialization, microorganism interactions, and host use in mosquitoes. Phylogenetic findings will be used to test hypotheses about ecological associations on rates and patterns of speciation. The proposed research will contribute to the understanding of the evolution of domestication in mosquitoes and their impact across the human/nature interface integrating taxonomic, phylogenetic, and molecular genetic methods with an emphasis on basic mosquito research that spans evolutionary, ecological and behavioral disciplines. Broader Impacts: The broader impacts of our proposed work are far-reaching by providing new basic research findings on a little known clade with enormous human impact. Mosquitoes, and the visceral reactions they incite, are an incredibly effective group to use to engage the public on the impact of biodiversity and evolution on human society and health. Our work will be disseminated through various educational, citizen science, and public outreach channels through the the National Museum of Natural History, California Academy of Sciences and the North Carolina Museum of Natural Sciences (NCMNS).

The primary purpose of this agreement is to further develop methods for the accurate identification of pest species in support of USA production of fruit and specialty crops by limiting the spread of exotic fruit flies and disease spread by insect vectors that damage agricultural commodities. Anastrepha and related genus Toxotrypana form the largest group of fruit flies within Latin America and the Caribbean, containing more than 300 species, many of which are undescribed. They include the most economically important pest species in the region, some that have invaded the US and others that remain serious threats to U.S. agriculture. Better tools are needed to support detection and eradication programs. Current identification tools for Anastrepha species are mainly based on adult morphology, particularly of females. For many species, there is inadequate knowledge of morphological variation due to incomplete sampling or lack of comprehensive study. Males of numerous species have not been described, and many cannot be identified. Taxonomic tools for the immature stages are even more limited. Although some molecular methods have been developed, only a few DNA regions have been investigated, and their effectiveness is limited by inadequate sampling or poor resolution among closely-related species. To address these issues, our team will use phylogenomic strategies with Next Generation Sequencing (NGS) methods to investigate a broad range of DNA regions for their diagnostic potential. These data will be valuable in providing detailed understanding of species limits, allow us to track and evaluate the rate and spread of specific pests and genes, and develop a framework for rapid identification based on multiple genomic markers. We will investigate regions of high potential by analyzing genetic variation in sequences from numerous species as well as a broad range of fly populations recently gathered and identified reference material as well as new collections from poorly sampled areas.

This suggestion addresses two important goals: 1) improving the capacity to diagnose fruit fly pests and 2) developing rapid and effective diagnostic tools for their identification regardless of life stage. The methods developed in this suggestion could be implemented beyond these taxa to other pests of economic concern which is an added value to the work. These tools are needed for the timely and accurate identification of fruit fly pests captured in the United States. Diagnosis to species-level is difficult because the current taxonomy is based mainly on morphological characters of the adult female and generally requires dissection of the female ovipositor; males of many species and the immature stages of most species are difficult to impossible to identify. Some species, which belong to cryptic species complexes, cannot be distinguished using morphological characters alone. Molecular data are limited; prior to this project less than half of the known species of Anastrepha had been studied even for the most commonly used DNA regions. Fresh samples suitable for the latest molecular methods were limited or unavailable for most species. Their morphological and molecular variation also has been poorly understood. Widespread sampling of pest species as well as close relatives is needed to construct informative and effective keys and molecular diagnostic methods. In the first two years of this project, our team has obtained a significant portion of the samples needed to develop effective diagnostic methods. Although some molecular diagnostic methods have been developed, only a few DNA regions have been investigated, and their effectiveness is limited by inadequate sampling or poor resolution among closely-related species, especially those of the fraterculus species complex. Our team is currently utilizing Next Generation Sequencing (NGS) methods to investigate a broad range of DNA regions for their diagnostic potential. In Year 4 we will test the diagnostic usefulness of these regions on an expanded taxonomic and geographic range of fly collections that include pest species from our current reference material as well as new collections in Year 4 from poorly sampled areas. Implementing single nucleotide polymorphism (SNP) panel procedures to the major Anastrepha pest species and others of economic concern should help develop a new approach to species identification and source estimation where many pests are examined using a common laboratory set-up and instruments. For Year 4 wWe will finalize all are also updating and expandsion of ing online identification tools that rely on morphological characters for both adult and larval stages, describing new species, and compiling new geographic and host plant data for these flies.

The primary purpose of this agreement is to further develop methods for the accurate identification of pest species in support of USA production of fruit and specialty crops by limiting the spread of exotic fruit flies and disease spread by insect vectors that damage agricultural commodities. Anastrepha and related genus Toxotrypana form the largest group of fruit flies within Latin America and the Caribbean, containing more than 300 species, many of which are undescribed. They include the most economically important pest species in the region, some that have invaded the US and others that remain serious threats to U.S. agriculture. Better tools are needed to support detection and eradication programs. Current identification tools for Anastrepha species are mainly based on adult morphology, particularly of females. For many species, there is inadequate knowledge of morphological variation due to incomplete sampling or lack of comprehensive study. Males of numerous species have not been described, and many cannot be identified. Taxonomic tools for the immature stages are even more limited. Although some molecular methods have been developed, only a few DNA regions have been investigated, and their effectiveness is limited by inadequate sampling or poor resolution among closely-related species. To address these issues, our team will use phylogenomic strategies with Next Generation Sequencing (NGS) methods to investigate a broad range of DNA regions for their diagnostic potential. These data will be valuable in providing detailed understanding of species limits, allow us to track and evaluate the rate and spread of specific pests and genes, and develop a framework for rapid identification based on CO1 gene and multiple genomic markers. We will investigate regions of high potential by analyzing genetic variation in sequences from numerous species as well as a broad range of fly populations recently gathered and identified reference material as well as new collections from poorly sampled areas. Using these data, we will develop rapid real-time PCR assays that provide reproducible results among different laboratories. Adapting these procedures to economically important pests will help develop a new approach to species identification where many pests are examined using a common laboratory set up and instruments.

This project addresses an important general question: How is biodiversity generated and maintained? The project focuses on a species-rich tropical tritrophic community of plants (Cucurbitaceae), herbivores (newly discovered species of tephritid fruit flies in the genus Blepharoneura), predators (parasitic wasps)������������������and tests the hypothesis that highly specific lethal interactions between herbivores and predators may explain patterns of local community diversity and patterns of diversification through space and time. Interactions between Blepharoneura and one diverse group of braconid wasps (Bellopius) are highly specific: each wasp species can kill only one fly species. Wasps attacking the ����������������wrong��������������� species of fly die. These bi-directional lethal interactions may be mediated by microbes (in wasps, flies, or both), by traits of flies������������������ immune systems, or both. Some fly-lineages appear to be more vulnerable to wasps than other fly-lineages, and some wasp-lineages appear to be able to overcome defenses of many fly species. The project will test the hypothesis that defenses (prey vs predator, and vice versa) affect diversification rates. To discover and identify mechanisms of diversification, this project will use: 1) high-resolution genetic data, multiple nuclear loci, and mtCOI haplotypes to delineate species, and resolve deeper phylogenetic relationships; 2) microsatellites and RADtags to discover and quantify fine-scale genetic diversity within and among populations; 3) phylogenies and field experiments to test hypotheses about mechanisms generating and controlling diversity on ancient, recent, and contemporary timescales. Both selection and stochastic processes are likely to operate within lineages of flies and wasps. Lineage-specific mechanisms of defense and virulence (including those mediated by symbionts) may both drive and result from diversification in fly and wasp lineages. INTELLECTUAL MERIT: Plants and insects represent the majority of multicellular terrestrial species, and their diversity peaks in the tropics. Explaining the generation and maintenance of that diversity is a major goal of evolutionary and ecological sciences. Is diversity generated and maintained by ecological processes (e.g., interspecific interactions) or is diversification an ecologically neutral process related to time and space? This project will use methods designed to: detect and analyze mechanisms associated with ����������������hard selection��������������� (life or death); reveal fine-scale patterns of genetic divergence that are not associated with selection; reveal ancient patterns of divergence; identify mechanisms of detection and defense. Together, the methods will allow the team to quantify diversity (discover and delineate species) and to test hypotheses about mechanisms generating and controlling that diversity. BROADER IMPACTS: Undergraduates from six institutions (Cornell College, Elms College, NCSU, UG, UMBC, UI) will do research in the field (in the tropics) and in laboratories, and will report their findings at annual meetings and public venues (e.g., local museums), and in publications. Building on a successful track record, the project will recruit students from groups under-represented in science. The project focuses on agriculturally important organisms (Tephritidae- pests; Cucurbitaceae- squash, melons; parasitoids-- biocontrol) and on problems relevant to medicine (e.g., immunology) and conservation (e.g., tropical biodiversity). Undergraduates will design their own projects and test their own hypotheses. Undergraduates will share expertise at inter-institutional collaborative tropical workshops scheduled for winter break and summer months, and will be encouraged to carry-out projects for at least a month in the field. Students unable to participate in the field will generate specific hypotheses that can be tested in the lab, or through analyses of existing datasets. Each year, at least 20 undergraduates will participate in research. Members of the team will also engage K-12 teachers (and their students) here and abroad in ����������������citizen science��������������� projects. Burke will offer a BioInformatics workshop open to all participants and students. An online survey will be used to assess the impact and success of workshops and outreach following the published NSF framework for evaluating impacts of broadening participation projects.

Anastrepha and the closely related genus Toxotrypana form the largest group of fruit flies within Latin America and the Caribbean, containing more than 300 species, many of which are undescribed. They include the most economically important pest species in the region, some that have invaded the US and other that remain serious threats to U.S. agriculture. Infestations by pest fruit fly species occur frequently in California, Florida, and Texas. The effective eradication of these infestations begins with the accurate species identification of captures. Additionally, identifying the geographic source of the pest allows for effective exclusion efforts and is important in order to reduce or eliminate further introductions and prevent establishment. For some species, however, identification to species-level using morphology is difficult to impossible, especially for the immature stages, the males of some species, and even for females that belong to species complexes. While some molecular methods have been developed, their effective diagnostic capacity is limited because sampling is inadequate or resolution among closely-related species is poor. These challenges impact accurate species identification resulting in wrong or ambiguous determinations leading to ineffective exclusionary efforts. Currently, identification of Anastrepha species is mainly accomplished using identification keys that rely on adult morphology, particularly from females. For some species, these keys are not always informative because they represent collections from incomplete sampling. They may not reflect the variation in morphology seen in species having a broad geographic distribution. Additionally, taxonomic keys that rely on larvae, pupae and egg morphology have not been developed for many species within these genera. For those that have been developed, some are ineffective because there are too few and reliable diagnostic characters present, especially at egg and early instar life stages. And while larval keys are available for several economically important species, some may be ineffective because other non-pest species share similar characters, hosts, and overlapping geographic distribution resulting in ambiguous or incorrect determinations. Using newer technologies such as Next Generation Sequencing (NGS), it is possible to develop Single Nucleotide Polymorphism (SNP) assays that provide reproducible results among different laboratories within a much shorter time period as compared to conventional methods. Adapting the SNP procedures to economically important pests should help develop a new approach to species identification and source estimation where many pests are examined using a common laboratory set up and instruments. The information presented in this suggestion therefore addresses two important goals: (1) to improve the diagnostic capacity for the Anastrepha and Toxotrypana spp. and associated fruit fly pests within these genera and (2) the implementation of new SNP technology for routine diagnosis of fruit flies. The results of this suggestion will provide USDA PPQ with an informative set of diagnostic tools used in the species identification and source estimation of captures made in the US. The suggested work is needed because methods for reliable and timely diagnosis of fruit flies are lacking for several pest species that are regularly captured in fruit producing regions of the United States.

Anastrepha and the closely related genus Toxotrypana form the largest group of fruit flies within Latin America and the Caribbean, containing more than 300 species, many of which are undescribed. They include the most economically important pest species in the region, some that have invaded the US and other that remain serious threats to U.S. agriculture. Infestations by pest fruit fly species occur frequently in California, Florida, and Texas. The effective eradication of these infestations begins with the accurate species identification of captures. Additionally, identifying the geographic source of the pest allows for effective exclusion efforts and is important in order to reduce or eliminate further introductions and prevent establishment. For some species, however, identification to species-level using morphology is difficult to impossible, especially for the immature stages, the males of some species, and even for females that belong to species complexes. While some molecular methods have been developed, their effective diagnostic capacity is limited because sampling is inadequate or resolution among closely-related species is poor. These challenges impact accurate species identification resulting in wrong or ambiguous determinations leading to ineffective exclusionary efforts. Currently, identification of Anastrepha species is mainly accomplished using identification keys that rely on adult morphology, particularly from females. For some species, these keys are not always informative because they represent collections from incomplete sampling. They may not reflect the variation in morphology seen in species having a broad geographic distribution. Additionally, taxonomic keys that rely on larvae, pupae and egg morphology have not been developed for many species within these genera. For those that have been developed, some are ineffective because there are too few and reliable diagnostic characters present, especially at egg and early instar life stages. And while larval keys are available for several economically important species, some may be ineffective because other non-pest species share similar characters, hosts, and overlapping geographic distribution resulting in ambiguous or incorrect determinations. Using newer technologies such as Next Generation Sequencing (NGS), it is possible to develop Single Nucleotide Polymorphism (SNP) assays that provide reproducible results among different laboratories within a much shorter time period as compared to conventional methods. Adapting the SNP procedures to economically important pests should help develop a new approach to species identification and source estimation where many pests are examined using a common laboratory set up and instruments. The information presented in this suggestion therefore addresses two important goals: (1) to improve the diagnostic capacity for the Anastrepha and Toxotrypana spp. and associated fruit fly pests within these genera and (2) the implementation of new SNP technology for routine diagnosis of fruit flies. The results of this suggestion will provide USDA PPQ with an informative set of diagnostic tools used in the species identification and source estimation of captures made in the US. The suggested work is needed because methods for reliable and timely diagnosis of fruit flies are lacking for several pest species that are regularly captured in fruit producing regions of the United States.

The rapid loss of biodiversity due to human activities has adversely affected ecosystem functions and services around the world, and is a pressing concern to all nations. The importance of better understanding and protecting biodiversity cannot be overstated. The United States and China occupy large areas of North America and eastern Asia at similar temperate latitudes. While both are rich in biodiversity, China harbors more species. Unfortunately, China is also losing its diversity at a higher rate. . The two regions offer many opportunities for comparative studies of biodiversity at all levels and in different dimensions, from the origin, evolution, and distribution of species to the community assembly and ecological functions. Recent increases in research funding from the Chinese government have greatly enhanced the research capacity in China and led to an enhancement of biodiversity studies and training of a younger generation of investigators in China. This provides the right timing for large-scale collaboration between researchers in the US and China in support of innovative and integrative research to address questions about biodiversity that are unique to these geographic regions which can greatly benefit the two countries and other geographic regions. To promote more collaboration on biodiversity research between the US and China, we proposed to hold two 3-day US-China collaborative workshops, one in the US and the other in China, to bring biologists from China and the US together to foster new research partnerships via presentations, discussions, and field visits to research sites. Intellectual merits: The workshops will integrate keynote talks from invited speakers, mini-talks of all participants in topical groups, discussion periods and three field trips. The invited talks will present the state of the science in biodiversity studies, successful examples of collaborations, discuss frontier issues on US-China biodiversity, and innovative approaches and new opportunities for collaborative studies. The mini-talks in topical groups will communicate expertise and interest of the participants and facilitate seeking appropriate partners by participants. The workshops will target leading scientists, including some who have received NSF dimensions awards as invited speakers to share their cutting-edge research, and recruit junior PIs, graduate students, and postdoctoral researchers from diverse fields including evolutionary biology, ecology, systematics, and bioinformatics, as participants from both countries. The group discussion and field trips to the to coastal, Piedmont, and mountain research sites will foster broad interactions seeking new research directions and inspiring new collaboration. Broader Impacts: The workshops will expose the younger generation of biologists to cutting-edge research on biodiversity and will engage participants from across diverse disciplines and institutions. A wiki website will be constructed to post the talks and relevant information. With respect to the North American meeting, through local news report, NESCent and NC Natural History Museum, the workshop event can outreach to local communities and increase the awareness of biodiversity research and education.

Building directly on infrastructure developed through the NSF-funded Planetary Biodiversity Inventories award to the American Museum of Natural History for the study of plant bugs (Miridae) and database initiatives at the New York Botanical Garden and the University of California, Riverside, we propose the following: 1. Form a collaboration of 6 lead institutions to direct the project and to encompass expertise in the major subgroups of Hemiptera, the taxa that parasitize them, and the plants that many of them utilize as hosts, in order to create a tri-trophic evolutionary- ecological database for the North American biota. Recruit data from 30?40 additional collections as a way of capturing the broadest cross section of relevant data. 2. Create a specimen database for the approximately 11,000 species of Hemiptera (aphids, scales, hoppers, true bugs) for North America. The AMNH currently has ~67,000 georeferenced specimen records for Heteroptera from North America, including Mexico, many of those with associated host data. Some other collections already have a significant number of digital specimen records in one or more hemipteran groups. Project goals therefore include the recruitment of these previously captured data as well as the capture new information, with the ultimate goal of assembling a database approaching 1 million records. 3. Capture and/or incorporate data for the parasitoids of Hemiptera with emphasis on the hymenopteran family groups Aphelinidae, Encyrtidae, Trichogrammatidae (Hymenoptera, Chalcidoidea) and Platygastroidea. 4. Collaborate with the New York Botanical Garden and additional herbaria with strong collections from North America, to facilitate the capture of additional relevant plant data, and to therefore great expand our ability to provide collection-based information on insect-plant relationships. 5. Emphasize the incorporation of taxa of economic importance, including species of agricultural significance as well as potential invasive species from outside North America. 6. Utilize the web-based database application developed with PBI funding (and also used for the AMNH Bee Databasing Project recently funded by the NSF) to capture the majority of the insect-specimen data. The simplicity of use of this database would simplify training of data-entry personnel, while direct writing of data to a single server would allow us to perform georeferencing and monitor data quality on a centralized basis. All specimens would be uniquely identified through the use of machine readable labels that incorporate your museum acronym. A copy of the data captured through this application would then be repatriated to each institution contributing data under our proposed approach. 7. Focus data capture on specimens of maximum value, as for example those with host information in the case of phytophagous species and those that capture range and temporal extensions.