A New qPCR Assay for the Rapid Diagnosis of Anthonomus grandis Subspecies.

Rapid and accurate identification of Anthonomus grandis subspecies is crucial for effective management and eradication. Current diagnostic methods have limitations in terms of time to diagnosis (up to seven days) and can yield ambiguous results. Here, we present the validation of a custom TaqMan SNP Genotyping Assay for the rapid and accurate identification of A. grandis grandis (boll weevil) and A. g. thurberiae (thurberia weevil) subspecies. To validate the assay, we conducted three main experiments: (1) a sensitivity test to determine the DNA concentration range at which the assay performs, (2) a non-target specificity test to ensure no amplification in non-target weevils (false positives), and (3) an accuracy test comparing the results of the new assay to previously established methods. These experiments were carried out in parallel at three independent facilities to confirm the robustness of the assay to variations in equipment and personnel. We used DNA samples from various sources, including field-collected specimens, museum specimens, and previously isolated DNA. The assay demonstrated high sensitivity (PCR success with ≥0.05 ng/µL DNA template), specificity (0.02 false positive rate), and accuracy (97.7%) in diagnosing boll weevil and thurberia weevil subspecies. The entire workflow, including DNA extraction, assay preparation, PCR run time, and data analysis, can be completed within a single workday (7-9 h) by a single technician. The deployment of this assay as a diagnostic tool could benefit boll weevil management and eradication programs by enabling same-day diagnosis of trap-captured or intercepted weevil specimens. Furthermore, it offers a more reliable method for identifying unknown specimens, contributing to the overall effectiveness of boll weevil research and control efforts.

Potential Distribution of Wild Host Plants of the Boll Weevil (Anthonomus grandis) in the United States and Mexico

The boll weevil (Anthonomus grandis Boheman) reproduces on a reported 13 species of wild host plants in North America, two in the United States and 12 in Mexico. The distributions of these plants are of economic importance to pest management and provide insight into the evolutionary history and origin of the BW. However, detailed information regarding the distributions of many of these species is lacking. In this article, we present distribution models for all of the reported significant BW host plants from Mexico and the United States using spatial distribution modelling software. Host plant distributions were divided into two groups: "eastern" and "western." In Mexico, Hampea nutricia along the Gulf Coast was the most important of the eastern group, and the wild cottons, Gossypium aridum and Gossypium thurberi were most important in the western group. Other species of Hampea, Gossypium, and Cienfuegosia rosei have relatively restricted distributions and are of apparent minimal economic importance. Cienfuegosia drummondii is the only truly wild host in the southern United States, east of New Mexico. Factors determining potential distributions were variable and indicated that species were present in five vegetation types. Ecological and economic considerations of host plant distributions are discussed, as well as threats to host plant conservation.

Exceptional larval morphology of nine species of the Anastrephamucronota species group (Diptera, Tephritidae).

Anastrepha is the most diverse and economically important genus of Tephritidae in the American tropics and subtropics. The striking morphology of the third instars of Anastrephacaballeroi Norrbom, Anastrephacrebra Stone, Anastrephahaplacantha Norrbom & Korytkowski, Anastrephakorytkowskii Norrbom, Anastrephanolazcoae Norrbom & Korytkowski, and three newly discovered and as yet formally unnamed species (Anastrepha sp. Peru-82, Anastrephasp.nr.protuberans, and Anastrepha sp. Sur-16), and the more typical morphology of Anastrephaaphelocentema Stone, are described using light and scanning electron microscopy. To contribute to a better understanding of the interspecific and intraspecific variation among species in the mucronota species group and facilitate phylogenetic studies, we integrate molecular and morphological techniques to confirm the identity and describe third instars. Larva-adult associations and the identification of described larvae were confirmed using DNA barcodes. We provide diagnostic characters to distinguish larvae among these nine species of the mucronota group and separate them from those of the 29 other Anastrepha species previously described. We introduce the vertical comb-like processes on the oral margin as a novel character, and the unusual character states, including position and shape of the preoral lobe, and dentate or fringed posterior margins of the oral ridges and accessory plates. Our comparative morphology concurs with most previously inferred phylogenetic relationships within the mucronota group.

New species and host plants of Anastrepha (Diptera: Tephritidae) primarily from Suriname and Par, Brazil.

Seventeen new species of Anastrepha, primarily from Suriname, French Guiana and Par, Brazil, are described and illustrated: A. aithogaster Norrbom from Brazil (Par), French Guiana, and Suriname; A. aliesae Norrbom from Suriname; A. brownsbergiensis Norrbom from Suriname; A. crassaculeus Norrbom Rodriguez Clavijo from Colombia (Magdalena, Norte de Santander) and Suriname; A. curvivenis Norrbom from Brazil (Amazonas), Ecuador (Zamora-Chinchipe), Peru (San Martn), and Suriname; A. fuscoalata Norrbom from Brazil (Par), French Guiana, and Suriname; A. gangadini Norrbom from Suriname; A. juxtalanceola Norrbom from Brazil (Par) and Suriname; A. microstrepha Norrbom from Brazil (Bahia) and Suriname; A. mitaraka Norrbom from French Guiana; A. neptis Norrbom from Brazil (Par), Ecuador (Orellana), Peru (Loreto) and Suriname; A. sobrina Norrbom from Brazil (Par), French Guiana, and Suriname; A. surinamensis Norrbom from Suriname; A. tenebrosa Norrbom from Brazil (Par) and Peru (Loreto); A. triangularis Norrbom from Suriname; A. wachiperi Norrbom from French Guiana and Peru (Cusco); and A. wittiensis Norrbom from Suriname. The following host plant records are reported: A. aithogaster from fruit of Parahancornia fasciculata (Poir.) Benoist (Apocynaceae); A. aliesae from fruit of Passiflora coccinea Aubl. and P. glandulosa Cav. (Passifloraceae); A. crassaculeus from fruit of an undetermined species of Pouteria (Sapotaceae); A. fuscoalata from fruit of Trymatococcus oligandrus (Benoist) Lanj. (Moraceae); A. sobrina from fruit of Eugenia lambertiana DC. (Myrtaceae); and A. wittiensis from fruit of Manilkara bidentata (A. DC.) A. Chev. (Sapotaceae).

Population genomics and phylogeography of the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), in the United States, northern Mexico, and Argentina.

The boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), is an important pest of commercial cotton across the Americas. In the United States, eradication of this species is complicated by re-infestations of areas where eradication has been previously successful and by the existence of morphologically similar variants that can confound identification efforts. To date, no study has applied a high-throughput sequencing approach to better understand the population genetic structure of the boll weevil. Furthermore, only a single study has investigated genetic relationships between populations in North and South America. We used double digest restriction site-associated DNA sequencing (ddRADseq) to resolve the population genomic structure of the boll weevil in the southern United States, northern Mexico, and Argentina. Additionally, we assembled the first complete mitochondrial genome for this species and generated a preliminary whole genome assembly, both of which were used to improve the identification of informative loci. Downstream analyses revealed two main lineages-one consisting of populations found geographically west of the Sierra Madre Occidental mountain range and the second consisting of populations found to the east-were revealed, and both were sub-structured. Population geographic structure was consistent with the isolation by distance model, indicating that geogrpahic distance is likely a primary mechanism driving divergence in this species. Boll weevil populations from Argentina were found to be more closely related to the eastern lineage, suggesting a recent colonization of South America by the eastern lineage, but additional sampling across Mexico, Central America and South America is needed to further clarify their origin. Finally, we uncovered an instance of population turnover or replacement, highlighting the temporal instability of population structure.

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.

Genetic Variation in Anastrepha obliqua (Diptera: Tephritidae) in a Highly Diverse Tropical Environment in the Mexican State of Veracruz.

There has been considerable interest in understanding biological, ecological, historical, and evolutionary processes that contribute to the diversification of species and populations among tephritid fruit flies. Only a limited number of studies have examined the genetic diversity and population biology of species belonging to the genus Anastrepha considering fine-scale differentiations associated to locality as well as hosts over an entire fruiting season. To expand our understanding of population structure and genetic diversity in one of the critical Anastrepha fruit flies populations in a highly diverse tropical environment we analyzed Anastrepha obliqua (Macquart) (Diptera: Tephritidae) in the Mexican state of Veracruz from five host fruit species and 52 geographic collections using sequence data from mtDNA and microsatellite markers from nuclear DNA. Indeed, we examined the population structure of this pest in a micro-geographic region and report on relationships and historical processes for individuals collected within a small portion of the geographic range of its distribution. Analyses of 1055 bp mtDNA sequences from CO1and ND1genes across 400 individuals detected 34 haplotypes. Haplotype and nucleotide diversity was low, with 53% of the individuals exhibiting a single haplotype (OBV1). Host association and fine-scale differentiation at 17 microsatellite markers across 719 individuals from 32 of the 52 geographic collections reveal fragmented A. obliqua populations. These findings have important implications for the implementation of the Sterile Insect Technique (SIT) and other pest management programs used to control this pestiferous fruit fly.

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.

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.

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.

Molecular diagnosis of populational variants of Anthonomus grandis (Coleoptera: Curculionidae) in North America.

The utility of the cytochrome oxidase I (COI) DNA sequence used for DNA barcoding and a Sequence Characterized Amplified Region for diagnosing boll weevil, Anthonomus grandis Boheman, variants was evaluated. Maximum likelihood analysis of COI DNA sequences from 154 weevils collected from the United States and Mexico supports previous evidence for limited gene flow between weevil populations on wild cotton and commercial cotton in northern Mexico and southern United States. The wild cotton populations represent a variant of the species called the thurberia weevil, which is not regarded as a significant pest. The 31 boll weevil COI haplotypes observed in the study form two distinct haplogroups (A and B) that are supported by five fixed nucleotide differences and a phylogenetic analysis. Although wild and commercial cotton populations are closely associated with specific haplogroups, there is not a fixed difference between the thurberia weevil variant and other populations. The Sequence Characterized Amplified Region marker generated a larger number of inconclusive results than the COI gene but also supported evidence of shared genotypes between wild and commercial cotton weevil populations. These methods provide additional markers that can assist in the identification of pest weevil populations but not definitively diagnose samples.

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.

Microsatellite markers reveal population structure and low gene flow among collections of Bactrocera cucurbitae (Diptera: Tephritidae) in Asia.

The melon fruit fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae), is widespread agricultural pest, and it is known to have the potential to establish invasive populations in various tropical and subtropical areas. Despite the economic risk associated with a putative stable presence of this fly, the population genetics of this pest have remained relatively unexplored in Asia, the main area for distribution of this pest. The goals for this study were to employ nuclear markers to examine geographic collections for population genetic structure and quantify the extent of gene flow within these Southeast Asian and Chinese populations. To achieve these goals, we used 12 polymorphic microsatellite markers. A low level of genetic diversity was found among collections from China and higher levels were seen in Southeast Asia collections. Three genetically distinct groups, Southeast Asia, southwest China, and southeast China, were recovered by Bayesian model-based clustering methods, the phylogenetic reconstruction and the principal coordinate analysis. The Mantel test clearly shows geographical distance contributed in the genetic structuring of B. cucurbitae's populations. No recent bottlenecks for any of the populations examined. The results of clustering, migration analyses, and Mantel test, strongly suggest that the regional structure observed may be due to geographical factors such as mountains, rivers, and islands. We found a high rate of migration in some sites from the southwest China region (cluster 1) and the southeast China region (cluster 2), suggesting that China-Guangdong-Guangzhou (GZ) may be the center of melon fruit fly in the southeast China region.