Phylogenetic relationships of the tribe Toxotrypanini (Diptera: Tephritidae) based on molecular characters.

Current hypotheses of relationship among the species of the fruit fly genera Anastrepha and Toxotrypana are tested using sequence data from six DNA regions: the mitochondrial regions 16S, CAD, and COI, and the nuclear regions EF1a, PER, and PGD. DNA sequences were obtained from 146 species of Anastrepha, representing 19 of the 21 species groups as well as five of the six clades of the robusta group, and four species of Toxotrypana in addition to species of Hexachaeta, Pseudophorellia, Alujamyia, and 13 other tephritid genera used as outgroups. The results indicate that Hexachaeta is more closely related to the Molynocoelia group than to Toxotrypana and Anastrepha, and it is removed from the tribe Toxotrypanini. The group Anastrepha+Toxotrypana and the genus Toxotrypana are strongly supported as monophyletic, consistent with previous studies, but Toxotrypana arises within Anastrepha, confirming that Anastrepha as currently defined is paraphyletic. The placement of Toxotrypana within Anastrepha is clearly defined for the first time with high support, as the sister group to the cryptostrepha clade of the robusta group of Anastrepha. Within Anastrepha, the daciformis, dentata, leptozona, raveni, and striata species groups are highly supported clades. The serpentina group is recognized with lower support, and the fraterculus and pseudoparallela groups are supported with minor alterations. The robusta group is resolved as polyphyletic, but four of the six species clades within it are recovered monophyletic (one clade is not represented and another is represented by one species). The punctata and panamensis groups are resolved together in a clade. At least some species of the mucronota group are related, however this group requires further study. The benjamini, grandis, and spatulata groups appear to be polyphyletic. Relationships among the species groups are generally poorly resolved, with the following exceptions: (1) the lineage including Toxotrypana, the cryptostrepha clade, and the tripunctata group; (2) the sister group relationship of the daciformis+dentata groups; (3) a clade comprising the punctata and panamensis groups; and (4) the large clade comprising the pseudoparallela+spatulata+ramosa+grandis+serpentina+striata+fraterculus groups.

Phylogeographic Structure in Anastrepha ludens (Diptera: Tephritidae) Populations Inferred With mtDNA Sequencing.

Anastrepha ludens (Loew) (Diptera: Tephritidae), the Mexican fruit fly, is a major pest of citrus and mango. It has a wide distribution in Mexico and Central America, with infestations occurring in Texas, California, and Florida with origins believed to have been centered in northeastern Mexico. This research evaluates the utility of a sequence-based approach for two mitochondrial (COI and ND6) gene regions. We use these markers to examine genetic diversity, estimate population structure, and identify diagnostic information for A. ludens populations. We analyzed 543 individuals from 67 geographic collections and found one predominant haplotype occurring in the majority of specimens. We observed 68 haplotypes in all and see differences among haplotypes belonging to northern and southern collections. Mexico haplotypes differ by few bases possibly as a result of a recent bottleneck event. In contrast to the hypothesis suggesting northeastern Mexico as the origin of this species, we see that specimens from two southern collections show high genetic variability delineating three mitochondrial groups. These data suggest that Central America is the origin for A. ludens. We show that COI and ND6 are useful for phylogeographic studies of A. ludens.

Episodic radiations in the fly tree of life.

Flies are one of four superradiations of insects (along with beetles, wasps, and moths) that account for the majority of animal life on Earth. Diptera includes species known for their ubiquity (Musca domestica house fly), their role as pests (Anopheles gambiae malaria mosquito), and their value as model organisms across the biological sciences (Drosophila melanogaster). A resolved phylogeny for flies provides a framework for genomic, developmental, and evolutionary studies by facilitating comparisons across model organisms, yet recent research has suggested that fly relationships have been obscured by multiple episodes of rapid diversification. We provide a phylogenomic estimate of fly relationships based on molecules and morphology from 149 of 157 families, including 30 kb from 14 nuclear loci and complete mitochondrial genomes combined with 371 morphological characters. Multiple analyses show support for traditional groups (Brachycera, Cyclorrhapha, and Schizophora) and corroborate contentious findings, such as the anomalous Deuterophlebiidae as the sister group to all remaining Diptera. Our findings reveal that the closest relatives of the Drosophilidae are highly modified parasites (including the wingless Braulidae) of bees and other insects. Furthermore, we use micro-RNAs to resolve a node with implications for the evolution of embryonic development in Diptera. We demonstrate that flies experienced three episodes of rapid radiation--lower Diptera (220 Ma), lower Brachycera (180 Ma), and Schizophora (65 Ma)--and a number of life history transitions to hematophagy, phytophagy, and parasitism in the history of fly evolution over 260 million y.

Genetic Diversity of Bactrocera dorsalis (Diptera: Tephritidae) on the Hawaiian Islands: Implications for an Introduction Pathway Into California.

Population genetic diversity of the oriental fruit fly, Bactrocera dorsalis (Hendel), on the Hawaiian islands of Oahu, Maui, Kauai, and Hawaii (the Big Island) was estimated using DNA sequences of the mitochondrial cytochrome c oxidase subunit I gene. In total, 932 flies representing 36 sampled sites across the four islands were sequenced for a 1,500-bp fragment of the gene named the C1500 marker. Genetic variation was low on the Hawaiian Islands with >96% of flies having just two haplotypes: C1500-Haplotype 1 (63.2%) or C1500-Haplotype 2 (33.3%). The other 33 flies (3.5%) had haplotypes similar to the two dominant haplotypes. No population structure was detected among the islands or within islands. The two haplotypes were present at similar frequencies at each sample site, suggesting that flies on the various islands can be considered one population. Comparison of the Hawaiian data set to DNA sequences of 165 flies from outbreaks in California between 2006 and 2012 indicates that a single-source introduction pathway of Hawaiian origin cannot explain many of the flies in California. Hawaii, however, could not be excluded as a maternal source for 69 flies. There was no clear geographic association for Hawaiian or non-Hawaiian haplotypes in the Bay Area or Los Angeles Basin over time. This suggests that California experienced multiple, independent introductions from different sources.

Phylogeography of Anastrepha obliqua inferred with mtDNA sequencing.

Anastrepha obliqua (Macquart) (Diptera: Tephritidae), the West Indian fruit fly, is a frugivorous pest that occasionally finds its way to commercial growing areas outside its native distribution. It inhabits areas in Mexico, Central and South America, and the Caribbean with occasional infestations having occurred in the southern tier states (California, Florida, and Texas) of the United States. This fly is associated with many plant species and is a major pest of mango and plum. We examine the genetic diversity of the West Indian fruit fly based on mitochondrial COI and ND6 DNA sequences. Our analysis of 349 individuals from 54 geographic collections from Mexico, Central America, the Caribbean, and South America detected 61 haplotypes that are structured into three phylogenetic clades. The distribution of these clades among populations is associated with geography. Six populations are identified in this analysis: Mesoamerica, Central America, Caribbean, western Mexico, Andean South America, and eastern Brazil. In addition, substantial differences exist among these genetic types that warrants further taxonomic review.

Patterns of mitochondrial haplotype diversity in the invasive pest Epiphyas postvittana (Lepidoptera: Tortricidae).

The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), is a horticultural pest of Australia and New Zealand that has more recently invaded Hawaii, Europe, and California. A 2,216-bp region of the mitochondrial genome containing the cytochrome oxidase I and II genes was sequenced from 752 individuals. Haplotype network analyses revealed a major split between a predominantly Western Australian clade and all other samples, suggestive of either a deep genetic divergence or a cryptic species. Nucleotide and haplotype diversity were highest in the country of origin, Australia, and in New Zealand populations, with evidence of haplotype sharing between New Zealand and Tasmania. Nucleotide and haplotype diversity were higher in California than within the British Isles or Hawaii. From the total of 96 haplotypes, seven were found in California, of which four were private. Within California, there have been at least two introductions; based on genetic diversity we were unable to assign a likely source for a single moth found and eradicated in Los Angeles in 2007; however, our data suggest it is unlikely that Hawaii and the British Isles are sources of the major E. postvittana population found throughout the rest of the state since 2006.

Using the rDNA Internal Transcribed Spacer 1 to Identify the Invasive Pest Rhagoletis cerasi (Diptera: Tephritidae) in North America.

The cherry-infesting fruit fly Rhagoletis cerasi Loew is a significant commercial pest in Europe that has recently invaded North America. To date, it has been trapped only in Canada and northwestern counties of New York. It has the potential to spread further and threaten production and movement of cherry commodities. Timely diagnosis of the pest will facilitate surveys and quick response to new detections. Adult morphology of the pest is distinct from other flies in North America. However, when flies are significantly damaged on traps or the immature life stages are found in fruits, molecular methods of identification are important to confirm presence and host-use records. Other than DNA sequencing of genes from flies which takes over a day to complete, there are no timely methods of molecular identification for this pest. In this study, we report the first sequence record of the internal transcribed spacer 1 (ITS1) from R. cerasi and develop two diagnostic tests for the pest based on ITS1 differences among species in North America. The tests use loop-mediated isothermal amplification (LAMP) and multiplex, conventional polymerase chain reaction (mcPCR) technologies that target the same region of the R. cerasi ITS1 sequence. Both tests performed well when tested against collections of R. cerasi from North America and Europe, generating Diagnostic Sensitivity estimates of 98.4-99.5%. Likewise, the tests had relatively high estimates of Diagnostic Specificity (97.8-100%) when tested against Rhagoletis Loew species present in North America that also use cherry as a developmental host.

Worldwide Phylogeography of Ceratitis capitata (Diptera: Tephritidae) Using Mitochondrial DNA.

The Mediterranean fruit fly, Ceratitis capitata (Weidemann), is one of the most economically important tephritid species worldwide. It has spread across six geographic regions as a result of successful invasions and continues to cause substantial losses to agricultural communities. Our study examined 1,864 flies originating from 150 localities, using mitochondrial DNA sequencing methods. We tested for population structure and revealed the genetic diversity for 1,592 specimens gathered from 144 wild fly collections from 46 countries representing the entire geographic range for this species. We also include in this study 272 Sterile Insect Technique (SIT) specimens from four SIT facilities. We recovered 202 haplotypes from the current sampling and updated previously published work to reveal a total of 231 haplotypes for this pest. These data show population structure at and below the regional level for these collections, shedding light on the current demographics for this species. We observed four common haplotypes, seen among 62% of the samples sequenced that have worldwide distribution. Three haplotypes were seen in SIT flies, with one seen as the predominant haplotype. Our work showed that two of the haplotypes were private to SIT flies, not present among wild fly collections. However, a third haplotype common among wild fly collections was also seen in one SIT facility but at a low frequency based on the current sampling. We provide guidance on the interpretation of these methods for the source estimation of current and future infestations.

Phylogenetic relationships of Ceratitis fruit flies inferred from nuclear CAD and tango/ARNT gene fragments: testing monophyly of the subgenera Ceratitis (Ceratitis) and C. (Pterandrus).

Systematic studies of Ceratitis (Tephritidae) fruit flies using molecular (i.e., COI, ND6, and period genes) and morphological (plus host-use characters) data have recently challenged the monophyly of the subgenera Ceratitis (Ceratitis) and Ceratitis (Pterandrus). In this paper, we report on the phylogenetic utility of three single-copy nuclear gene regions (two non-overlapping fragments of the carbamoylphosphate synthetase, CPS, locus of CAD, and a fragment of tango) within these taxa and investigate evolutionary relationships based on a concatenated ca. 3.4kb data set that includes the six protein encoding gene regions. Results indicate that the CAD and tango genes provide useful phylogenetic signal within the taxa and are compatible with the previously studied genes. The two subgenera, as currently classified, are not monophyletic. Our molecular phylogenetic analyses support a revised classification in which (1) the subgenus C. (Pterandrus) comprises two lineages called A and B, (2) the C. (Pterandrus) B species should be included in C. (Ceratitis), and (3) the newly defined subgenera C. (Pterandrus) (=Pterandrus section A) and C. (Ceratitis) [=C. (Ceratitis)+C. (Pterandrus) section B] are reciprocally monophyletic.

Pathway analysis of Ceratitis capitata (Diptera: Tephritidae) using mitochondrial DNA.

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method described in a previous publication to document worldwide population structure of Mediterranean fruit flies and analyze probable sources of fly introductions is re-evaluated. Analysis of representatives of the RFLP-based markers using the ND5-tRNA-ND4 mitochondrial locus through DNA sequencing shows that the original markers produced haplotypes that are homoplasic and exclude valuable information. DNA sequences also identified errors in data generated with the protocol because of failure of the XbaI enzyme to efficiently cut PCR product. In all, the PCR-RFLP method is able to document haplotypes present in medfly populations outside of Sub-Saharan Africa and is a cost-effective approach to pathway analysis. New primer pairs for analysis of this locus are reported that reduce the number of PCR steps required to analyze the RFLP marker and the time of each PCR run. For comparison of the PCR-RFLP method, the utility of a DNA sequence-based approach to Mediterranean fruit fly pathway analysis is reported using the ND5-ND4 locus and the proposed "barcode region" of the COI gene. Both gene sequences are more informative than the PCR-RFLP method and document high genetic variation within Sub-Saharan Africa. Application of these methods as tools and their interpretation is discussed.

The Genetic Diversity of Bactrocera dorsalis (Diptera: Tephritidae) in China and Neighboring Countries: A Review From Published Studies.

For more than a decade, various research groups have tracked the population genetics of the oriental fruit fly, Bactrocera dorsalis (Hendel) in China and neighboring countries using mitochondrial cytochrome c oxidase subunit I (COI) DNA. Although most research has reported high levels of mtDNA variation, to date no efforts have been made to integrate and compare the results from these studies simultaneously. Here, we show that: 1) despite the fact that a large portion of the sampling effort has focused on the Yunnan province beginning in 2005, each subsequent study recovers only a small number of previously sampled haplotypes; 2) new haplotypes of B. dorsalis remain to be found, a projection of new haplotypes versus the number of individuals sampled suggest that sampling the species mtDNA diversity is far from reaching an asymptote; 3) it is unlikely that the observed genetic variation is the result of NUMTs (nuclear mitochondrial DNA), as most differences between haplotypes are silent substitutions; and 4) although all studies employed the 3' end of COI, the length of COI fragment sequenced differs among studies, making comparisons challenging. Therefore, we offer these results with the caveat that mtDNA diversity might be underestimated in China.

Range-wide population genomics of the Mexican fruit fly: Toward development of pathway analysis tools.

Recurrently invading pests provide unique challenges for pest management, but also present opportunities to utilize genomics to understand invasion dynamics and inform regulatory management through pathway analysis. In the southern United States, the Mexican fruit fly Anastrepha ludens is such a pest, and its incursions into Texas and California represent major threats to the agricultural systems of those regions. We developed a draft genome assembly for A. ludens, conducted range-wide population genomics using restriction site-associated DNA sequencing, and then developed and demonstrated a panel of highly differentiated diagnostic SNPs for source determination of intercepted flies in this system. Using 2,081 genomewide SNPs, we identified four populations across the range of A. ludens, corresponding to western Mexico, eastern Mexico/Texas, Guatemala/Belize/Honduras, and Costa Rica/Panama, with some intergradation present between clusters, particularly in Central America. From this population genomics framework, we developed a diagnostic panel of 28 highly differentiated SNPs that were able to recreate the genomewide population structure in this species. We demonstrated this panel on a set of test specimens, including specimens intercepted as part of regular trapping surveillance in Texas and California, and we were able to predict populations of origin for these specimens. This methodology presents a highly applied use of genomic techniques and can be implemented in any group of recurrently invading pests.

Genetic structure and diversity in Brazilian populations of Anastrepha obliqua (Diptera: Tephritidae).

Anastrepha obliqua (Macquart), the West Indian fruit fly, is one of the most economically important pest species in the Neotropical region. It infests an extensive range of host plants that include over 60 species. The geographic range of A. obliqua is from northern Mexico to southern Brazil and includes the Caribbean Islands. Previous molecular studies have revealed significant genetic structure among populations. We used sequences from a fragment of the mitochondrial protein-coding gene cytochrome c oxidase I to estimate structure and genetic diversity of A. obliqua populations from Brazil. We analyzed a total of 153 specimens from the Amazon Forest, Atlantic Forest, Cerrado, and Caatinga biomes. Our study revealed weak genetic structure among the A. obliqua Brazilian populations sampled. Collections from the Amazon Forest had similar haplotype diversity compared to previously reported estimates for collections from the Caribbean and both populations are also closely related to each other, thus challenging the hypothesis that A. obliqua originated in the Caribbean and then moved to other regions of the Americas. Therefore, further evidence is necessary to draw a definite conclusion about the putative center of origin for A. obliqua. Additionally, we suggest a putative historical migration from the west to the east for the A. obliqua Brazilian populations, which could explain the high genetic diversity for this fly in the Amazon Forest and low genetic diversity in the other Brazilian biomes.

Identifying Anastrepha (Diptera; Tephritidae) Species Using DNA Barcodes.

Molecular identification of fruit flies in the genus Anastrepha (Diptera; Tephritidae) is important to support plant pest exclusion, suppression, and outbreak eradication. Morphological methods of identification of this economically important genus are often not sufficient to identify species when detected as immature life stages. DNA barcoding a segment of the mitochondrial cytochrome oxidase I gene has been proposed as a method to identify pests in the genus. The identification process for these fruit flies, however, has not been explained in prior DNA barcode studies. DNA barcode methods assume that available DNA sequence records are biologically meaningful. These records, however, can be limited to the most common species or lack population-level measurements of diversity for pests. In such cases, the available data used as a reference are insufficient for completing an accurate identification. Using 539 DNA sequence records from 74 species of Anastrepha, we demonstrate that our barcoding data can distinguish four plant pests: Anastrepha grandis (Macquart) (Diptera; Tephritidae), Anastrepha ludens (Loew), Anastrepha serpentina (Wiedemann), and Anastrepha striata Schiner. This is based on genetic distances of barcode records for the pests and expert evaluation of species and population representation in the data set. DNA barcoding of the cytochrome oxidase I gene alone cannot reliably diagnose the pests Anastrepha fraterculus (Wiedemann), Anastrepha obliqua (Macquart), and Anastrepha suspensa (Loew).

A New Diagnostic Resource for Ceratitis capitata Strain Identification Based on QTL Mapping.

The Mediterranean fruit fly Ceratitis capitata (Wiedemann) is a destructive agricultural pest and the subject of exclusion efforts in many countries. Suppression and eradication of invasive populations to prevent its establishment is facilitated by the release of sterile males using the sterile insect technique (SIT). In SIT release areas, it is critical to accurately discriminate between released sterile males and wild individuals to detect extremely rare invasive individuals in areas inundated with millions of sterile male flies. Current methods for discrimination exist but are not always definitive, and a more reliable method is necessary. To address this, we developed a genotyping assay that can be used to discriminate between sterile males from the SIT strain and wild individuals. This was achieved by identifying single nucleotide polymorphisms (SNPs) linked to the maintained traits that facilitate male-only releases, white pupae (wp) and temperature-sensitive lethal (tsl), via QTL mapping. This resulted in the identification of one SNP that was in near-perfect linkage disequilibrium between genotype at this locus and the pupal color phenotype. Medfly from many SIT colonies and wild individuals from across its geographic range were genotyped for this locus, and results show its consistency in identifying SIT flies. In addition, linkage and QTL mapping of wp and tsl have larger impacts as they can serve as foundational tools to identify the genetic basis of traits that facilitate the separation of males from female flies, which can be used to develop SIT programs in related species.

A Real-Time PCR Assay for the Separation of Autographa gamma (Noctuidae: Plusiinae) From Morphologically Similar Species in North America.

The silver Y moth, Autographa gamma L. (Noctuidae: Plusiinae), is a pest of major economic importance in its native range of Europe, Asia, and North Africa. Although not present in North America, larvae of A. gamma are commonly intercepted in commodity shipments at U.S. ports, and adult surveys are conducted each year in more than 20 states. Because of the similarity of A. gamma to several native North American species that are attracted to the same pheromone lure, morphological identification of adults is difficult and requires dissection. In 2010, a specimen of Autographa californica (Speyer, 1875) (Lepidoptera: Noctuidae) from Pennsylvania was incorrectly identified as A. gamma, signaling the need for an alternative method of rapid identification. Here we detail a real-time PCR assay capable of identifying A. gamma specimens in approximately 45 min using extracted DNA. The assay uses a hydrolysis probe that targets a species-specific segment of the CO1 DNA barcode region, while a control probe targets a conserved region of 18S rDNA. The assay was tested with two independent runs of 452 specimens of Plusiinae representing 23 different species. The assay provided unambiguous data 99.7% of the time and did not result in any false positives; these data were used to develop a rule set for interpreting the real-time PCR results. In addition, the same diagnostic probe was tested in bulk sample simulations using real-time PCR and droplet digital PCR where A. gamma could be detected in concentrations as low as 1:1,000,000 (gamma:californica). These experiments provide baseline data for developing a bulk sample assay.

A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World.

The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult-adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae.

Molecular phylogenetics of the genus Ceratitis (Diptera: Tephritidae).

The Afrotropical fruit fly genus Ceratitis MacLeay is an economically important group that comprises over 89 species, subdivided into six subgenera. Cladistic analyses of morphological and host use characters have produced several phylogenetic hypotheses for the genus. Only monophyly of the subgenera Pardalaspis and Ceratitis (sensu stricto) and polyphyly of the subgenus Ceratalaspis are common to all of these phylogenies. In this study, the hypotheses developed from morphological and host use characters are tested using gene trees produced from DNA sequence data of two mitochondrial genes (cytochrome oxidase I and NADH-dehydrogenase subunit 6) and a nuclear gene (period). Comparison of gene trees indicates the following relationships: the subgenus Pardalaspis is monophyletic, subsection A of the subgenus Pterandrus is monophyletic, the subgenus Pterandrus may be either paraphyletic or polyphyletic, the subgenus Ceratalaspis is polyphyletic, and the subgenus Ceratitis s. s. might not be monophyletic. In addition, the genera Ceratitis and Trirhithrum do not form reciprocally monophyletic clades in the gene trees. Although the data statistically reject monophyly for Trirhithrum under the Shimodaira-Hasegawa test, they do not reject monophyly of Ceratitis.

Mutations in raised Drosophila melanogaster affect experience-dependent aspects of sexual behavior in both sexes.

Many aspects of the reproductive behavior of Drosophila melanogaster are modified dramatically by experience and age. Males' courtship of immature males and fertilized females decreases over time. Females' receptivity to copulation, and the behaviors that females perform and elicit, are affected by their age and sexual experience. We show that mutations in a raised stock affect all of these age- and experience-dependent aspects of male and female sexual behavior. Experience has no effect on raised males' courtship of immature males and has opposite effects on raised and wild-type males' courtship of fertilized females. In comparison to controls, raised females become sexually mature at an earlier age, and sexually mature raised virgin females copulate more quickly. Following mating, raised females elicit more courtship and remate faster and more frequently than control females.