Mass-Rearing Conditions Do Not Always Reduce Genetic Diversity: The Case of the Mexican Fruit Fly, Anastrepha ludens (Diptera: Tephritidae).

The application of the sterile insect technique (SIT) requires the adaptation of insects to mass-rearing conditions. It is generally accepted that this adaptation may include a reduction in genetic diversity and an associated loss of desirable characteristics for the effective performance of sterile insects in the field. Here, we compare the genetic diversity of two mass-reared strains of the Mexican fruit fly, Anastrepha ludens, and a wild (WIL) population collected near Tapachula, Mexico, using seven DNA microsatellites as molecular genetic markers. The mass-reared strains were a bisexual laboratory strain (LAB) with approximately 130 generations under mass-rearing and a genetic sexing strain, Tapachula-7 (TA7), also under mass-rearing for 100 generations. Our results revealed an overall low level of genetic differentiation (approximately 15%) among the three strains, with the LAB and WIL populations being genetically most similar and TA7 most genetically differentiated. Although there were some differences in allele frequencies between strains, our results show that overall, the adaptation to mass-rearing conditions did not reduce genetic variability compared to the wild sample in terms of heterozygosity or allelic richness, nor did it appear to alter the level of inbreeding with respect to the wild populations. These results are contrary to the general idea that mass-rearing always results in a reduction in genetic diversity. Overall, our findings can contribute to a better understanding of the impact that adaptation to mass-rearing conditions may have on the genetic make-up of strains.

Genetic and Ecological Relationships of Anastrepha ludens (Diptera: Tephritidae) Populations in Southern Mexico.

Knowledge of the influence of evolutionary factors that promote either the differentiation or cohesion of pest insect populations is critical for the improvement of control strategies. Here, we explore the extent to which genetic differentiation occurs between populations of the Mexican fruit fly, Anastrepha ludens, in association with four plant hosts (Citrus sinensis, C. paradisi, Mangifera indica and Casimiroa edulis) in the Soconusco region of Chiapas (Mexico). Using variants from six enzymatic loci, we obtained measures of genetic diversity for three sample arrangements: (1) by sex per locality, (2) by locality and (3) by host. The extent of genetic differentiation in populations was assessed using the Analyses of Molecular Variance (AMOVA) method for each array of samples, and moderate to high levels of genetic variation were observed between the sexes, as well as among localities and host plants. A Bayesian approach was then used to assess any population structure underlying the genetic data we obtained, but this analysis showed no significant structuring due to locality or host plant. We also considered whether the observed genotypic frequencies in male and females matched those expected under a hypothesis of random mating. Here we found significant deviations from expected genotypic frequencies, suggesting that sexual selection is acting on these populations. Overall, our results indicate that sexual selection, along with the presence of some heterogeneity in environments provided by both geographical factors and availability of host plants, has influenced the evolution of pest populations in this region of Mexico. Implications for area-wide pest management strategies are discussed.

Identification and preliminary characterization of chemosensory perception-associated proteins in the melon fly Bactrocera cucurbitae using RNA-seq.

An investigation into proteins involved in chemosensory perception in the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae) is described here using a newly generated transcriptome dataset. The melon fly is a major agricultural pest, widely distributed in the Asia-Pacific region and some parts of Africa. For this study, a transcriptome dataset was generated using RNA extracted from 4-day-old adult specimens of the melon fly. The dataset was assembled and annotated via Gene Ontology (GO) analysis. Based on this and similarity searches to data from other species, a number of protein sequences putatively involved in chemosensory reception were identified and characterized in the melon fly. This included the highly conserved "Orco" along with a number of other less conserved odorant binding protein sequences. In addition, several sequences representing putative ionotropic and gustatory receptors were also identified. This study provides a foundation for future functional studies of chemosensory proteins in the melon fly and for making more detailed comparisons to other species. In the long term, this will ultimately help in the development of improved tools for pest management.

High rates of substitution of the native catfish Clarias batrachus by Clarias gariepinus in India.

The clariid catfish, Clarias batrachus commonly known as Magur, has declined drastically from natural habitats in India during the last decade. This fish is highly preferred fish by Indian consumers and has high market demand. As a result traders often substitute C. batrachus with a morphologically similar but supposedly banned exotic catfish, C. gariepinus, in India. This study uses rigorous morphological comparisons confirmed by DNA barcode analysis to examine the level of substitution of C. batracus by C. gariepinus in India. Our results indicate that up to 99% (in many cases) of the market samples sold as Magur or C. batrachus were in fact C. gariepinus.

DNA barcoding using skin exuviates can improve identification and biodiversity studies of snakes.

Snakes represent a taxonomically underdeveloped group of animals in India with a lack of experts and incomplete taxonomic descriptions being the main deterrents to advances in this area. Molecular taxonomic approaches using DNA barcoding could aid in snake identification as well as studies of biodiversity. Here a non-invasive sampling method using DNA barcoding is tested using skin exuviates. Taxonomically authenticated samples were collected and tested for validation and comparisons to unknown snake exuviate samples. This approach was also used to construct the first comprehensive study targeting the snake species from Maharashtra state in India. A total of 92 skin exuviate samples were collected and tested for this study. Of these, 81 samples were successfully DNA barcoded and compared with unknown samples for assignment of taxonomic identity. Good quality DNA was obtained irrespective of age and quality of the exuviate material, and all unknown samples were successfully identified. A total of 23 species of snakes were identified, six of which were in the list of Endangered species (Red Data Book). Intra- and inter-specific distance values were also calculated, and these were sufficient to allow discrimination among species and between species without ambiguity in most cases. Two samples were suspected to represent cryptic species based on deep K2P divergence values (>3%), and one sample could be identified to the genus level only. Eleven samples failed to amplify COI sequences, suggesting the need for alternative PCR primer pairs. This study clearly documents how snake skin exuviates can be used for DNA barcoding, estimates of diversity and population genetic structuring in a noninvasive manner.

Genetic structure of populations and conservation issues relating to an endangered catfish, Clarias batrachus, in India.

The Asian catfish, Clarias batrachus (Linnaeus, 1758), is a highly valued species endemic to India that is currently in drastic decline in most of its natural habitat. The present study was undertaken to document the genetic structure of populations of this species using mitochondrial DNA markers, specifically from the cytochrome B and D-loop regions. Specimens from eight wild populations were collected and analyzed from different regions in India. The genetic variation within and among populations was evaluated using a range of descriptive statistics. The analysis described here provides a broad and consistent view of population structure and demographic history of populations of C. batrachus. Although there was some genetic structuring consistent with regional differences, all eight populations examined here showed relatively low levels of genetic variation in terms of both haplotype and nucleotide diversities in the different analyses used. However, a number of private haplotypes were discovered, and this may provide valuable information for future selective breeding program and conservation management. The results may aid in the design and implementation of strategies for the future management of this endangered catfish C. batrachus in India.

Mitochondrial Single Nucleotide Polymorphisms in Ceratitis capitata (Diptera: Tephritidae) Can Distinguish Sterile, Released Flies from Wild Flies in Various Regions of the World.

In areas infested with pest species such as the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), many programs rely heavily on the sterile insect technique (SIT) as a form of biological control. However, when SIT treatments are used both for control of established infestations and for occasional outbreaks, for several reasons, programs are often unable to adequately quantify the success of this approach. Chief among these are difficulties associated with reliably and rapidly determining the strain of origin of males recaptured during and after the SIT program. In this study, we describe the use of a DNA-based marker that can be used to rapidly and reliably distinguish males originating from the two sterile strains that are most widely used in SIT rearing facilities from males originating from wild strains of various regions of the world. This method uses polymerase chain reaction amplification of material from individual specimens to directly analyze DNA sequence variants found within a portion of the mitochondrial ND4 subunit 4 (ND4) gene to identify single nucleotide polymorphisms (SNPs) that are diagnostic of different strains. Specifically, the SNPs described here reliably distinguish individual flies originating from the Vienna 7 and Vienna 8 strains used for sterile release from wild flies infesting various areas including Western Australia, Guatemala, and Hawaii. The availability of such markers for determination of the strain of origin of specimens, either from whole specimens or body parts (including their sperm), has great potential to improve the ability to monitor and quantify the success of any sterile release program.

Historical perspective on the synonymization of the four major pest species belonging to the Bactrocera dorsalis species complex (Diptera, Tephritidae).

An FAO/IAEA-sponsored coordinated research project on integrative taxonomy, involving close to 50 researchers from at least 20 countries, culminated in a significant breakthrough in the recognition that four major pest species, Bactrocera dorsalis, Bactrocera philippinensis, Bactrocera papayae and Bactrocera invadens, belong to the same biological species, Bactrocera dorsalis. The successful conclusion of this initiative is expected to significantly facilitate global agricultural trade, primarily through the lifting of quarantine restrictions that have long affected many countries, especially those in regions such as Asia and Africa that have large potential for fresh fruit and vegetable commodity exports. This work stems from two taxonomic studies: a revision in 1994 that significantly increased the number of described species in the Bactrocera dorsalis species complex; and the description in 2005 of Bactrocera invadens, then newly incursive in Africa. While taxonomically valid species, many biologists considered that these were different names for one biological species. Many disagreements confounded attempts to develop a solution for resolving this taxonomic issue, before the FAO/IAEA project commenced. Crucial to understanding the success of that initiative is an accounting of the historical events and perspectives leading up to the international, multidisciplinary collaborative efforts that successfully achieved the final synonymization. This review highlights the 21 year journey taken to achieve this outcome.

Genetic fragmentation in India's third longest river system, the Narmada.

India's third longest river, the Narmada, is studied here for the potential effects on native fish populations of river fragmentation due to various barriers including dams and a waterfall. The species we studied include a cyprinid fish, Catla catla, and a mastacembelid, Mastacembelus armatus, both of which are found in the Narmada. Our goal was to use DNA sequence information from the D-loop region of the mitochondrial DNA to explore how this fragmentation could impact the genetic structure of these fish populations. Our results clearly show that these barriers can contribute to the fragmentation of the genetic structure of these fish communities, Furthermore, these barriers enhance the effects of natural isolation by distance and the asymmetry of dispersal flows. This may be a slow process, but it can create significant isolation and result in genetic disparity. In particular, populations furthest upstream having low migration rates could be even more subject to genetic impoverishment. This study serves as a first report of its kind for a river system on the Indian subcontinent. The results of this study also emphasize the need for appropriate attention towards the creation of fish passages across the dams and weirs that could help in maintaining biodiversity.

DNA barcodes for the fishes of the Narmada, one of India's longest rivers.

This study describes the species diversity of fishes of the Narmada River in India. A total of 820 fish specimens were collected from 17 sampling locations across the whole river basin. Fish were taxonomically classified into one of 90 possible species based on morphological characters, and then DNA barcoding was employed using COI gene sequences as a supplemental identification method. A total of 314 different COI sequences were generated, and specimens were confirmed to belong to 85 species representing 63 genera, 34 families and 10 orders. Findings of this study include the identification of five putative cryptic or sibling species and 43 species not previously known from the Narmada River basin. Five species are endemic to India and three are introduced species that had not been previously reported to occur in the Narmada River. Conversely, 43 species previously reported to occur in the Narmada were not found. Genetic diversity and distance values were generated for all of the species within genera, families and orders using Kimura's 2 parameter distance model followed by the construction of a Neighbor Joining tree. High resolution clusters generated in NJ trees aided the groupings of species corresponding to their genera and families which are in confirmation to the values generated by Automatic Barcode Gap Discovery bioinformatics platform. This aided to decide a threshold value for the discrimination of species boundary from the Narmada River. This study provides an important validation of the use of DNA barcode sequences for monitoring species diversity and changes within complex ecosystems such as the Narmada River.

Using DNA chips for identification of tephritid pest species.

BACKGROUND: The ability correctly to identify species in a rapid and reliable manner is critical in many situations. For insects in particular, the primary tools for such identification rely on adult-stage morphological characters. For a number of reasons, however, there is a clear need for alternatives. This paper reports on the development of a new method employing DNA biochip technology for the identification of pest species within the family Tephritidae. RESULTS: The DNA biochip developed and tested here quickly and efficiently identifies and discriminates between several tephritid species, except for some that are members of a complex of closely related taxa and that may in fact not represent distinct biological species. The use of these chips offers a number of potential advantages over current methods. Results can be obtained in less than 5 h using material from any stage of the life cycle and with greater sensitivity than other methods currently available. CONCLUSIONS: This technology provides a novel tool for the rapid and reliable identification of several major pest species that may be intercepted in imported fruits or other commodities. The existing chips can also easily be expanded to incorporate additional markers and species as needed.

High levels of genetic diversity in Penaeus monodon populations from the east coast of India.

Quality production of the shrimp Penaeus monodon in hatchery operations depends heavily on the evaluation of genetic diversity and population structure of brood stocks. Mitochondrial DNA (mtDNA) sequences have been widely used to study genetic variability and relationships in many crustacean groups, and these same markers may be incorporated into evaluation studies of shrimp broods and populations. For this purpose we looked at variation in mitochondrial D-loop sequences as an indicator of genetic diversity in shrimp populations from a region of India that represents the main sources of new material for brood stocks. In our study of these populations the overall mean genetic diversity was 0.191. The highest level of genetic diversity (0.357) was observed in the Kakinada population, whereas the lowest diversity (0.0171) was observed in the Nellore population. The results also indicate that overall, the populations along the Andhra Pradesh coast are genetically diverse despite the fact that there is considerable gene flow between them. From the results, it is evident that east cost of India shows high genetic diversity among P. monodon broods and no evidence of loss of diversity due to excessive inbreeding. The fact that the genetic variability of these populations has been maintained, despite ten years of dependence on these broods, shows that at the present time there is no indication of over exploitation.

Discovery of genes related to insecticide resistance in Bactrocera dorsalis by functional genomic analysis of a de novo assembled transcriptome.

Insecticide resistance has recently become a critical concern for control of many insect pest species. Genome sequencing and global quantization of gene expression through analysis of the transcriptome can provide useful information relevant to this challenging problem. The oriental fruit fly, Bactrocera dorsalis, is one of the world's most destructive agricultural pests, and recently it has been used as a target for studies of genetic mechanisms related to insecticide resistance. However, prior to this study, the molecular data available for this species was largely limited to genes identified through homology. To provide a broader pool of gene sequences of potential interest with regard to insecticide resistance, this study uses whole transcriptome analysis developed through de novo assembly of short reads generated by next-generation sequencing (NGS). The transcriptome of B. dorsalis was initially constructed using Illumina's Solexa sequencing technology. Qualified reads were assembled into contigs and potential splicing variants (isotigs). A total of 29,067 isotigs have putative homologues in the non-redundant (nr) protein database from NCBI, and 11,073 of these correspond to distinct D. melanogaster proteins in the RefSeq database. Approximately 5,546 isotigs contain coding sequences that are at least 80% complete and appear to represent B. dorsalis genes. We observed a strong correlation between the completeness of the assembled sequences and the expression intensity of the transcripts. The assembled sequences were also used to identify large numbers of genes potentially belonging to families related to insecticide resistance. A total of 90 P450-, 42 GST-and 37 COE-related genes, representing three major enzyme families involved in insecticide metabolism and resistance, were identified. In addition, 36 isotigs were discovered to contain target site sequences related to four classes of resistance genes. Identified sequence motifs were also analyzed to characterize putative polypeptide translational products and associate them with specific genes and protein functions.

Monitoring resistance to spinosad in the melon fly (Bactrocera cucurbitae) in Hawaii and Taiwan.

Spinosad is a natural insecticide with desirable qualities, and it is widely used as an alternative to organophosphates for control of pests such as the melon fly, Bactrocera cucurbitae (Coquillett). To monitor the potential for development of resistance, information about the current levels of tolerance to spinosad in melon fly populations were established in this study. Spinosad tolerance bioassays were conducted using both topical applications and feeding methods on flies from field populations with extensive exposure to spinosad as well as from collections with little or no prior exposure. Increased levels of resistance were observed in flies from the field populations. Also, higher dosages were generally required to achieve specific levels of mortality using topical applications compared to the feeding method, but these levels were all lower than those used for many organophosphate-based food lures. Our information is important for maintaining effective programs for melon fly management using spinosad.

Molecular taxonomic identification of Dacus and Ceratitis species from Sub-Saharan Africa using mitochondrial haplotypes.

Several different taxa within the genera Dacus and Ceratitis (Diptera: Tephritidae) are important agricultural pests in Sub-Saharan Africa. Although the status of many of these taxa as distinct species and their phylogenetic relationships is unclear, it is clear that these pests use a wide range of host plants and are highly invasive. The great potential for economic damage inflicted by these pests requires the ability to make accurate and reliable taxonomic identification of specimens. However, many limitations and uncertainties are encountered when these species are examined using traditional approaches based on morphological identification techniques. We describe here the amplification and analysis of DNA sequences from the mitochondrial cytochrome oxidase II (COII)-tRNA(lys)-tRNA(ASP) genes from individuals of various Dacus and Ceratitis species and populations from Sub-Saharan Africa. The variation detected in the DNA sequences of these individuals is used both for clarification of their taxonomic status and the analysis of phylogenetic relationships of these taxa.

Rapid identification of the Mediterranean fruit fly (Diptera: Tephritidae) by loop-mediated isothermal amplification.

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), ranks as one of the world's most destructive agricultural pests. This pest is also widespread and highly invasive; thus, it is a high priority for pest detection and quarantine programs. Although Mediterranean fruit fly adult and third-instar larvae can usually be identified and distinguished from other species by morphological keys, it is often difficult or impossible to identify or distinguish this species from other tephritids by using material from other stages of development. In such situations, use of a molecular technique known as loop-mediated isothermal amplification (LAMP) would be valuable as a rapid and robust alternative species diagnostic tool. This method uses isothermal conditions and requires only relatively inexpensive equipment. In this study we have developed a simple and rapid procedure that combines a Chelex-based DNA extraction procedure with LAMP to rapidly detect the presence of Mediterranean fruit fly DNA and discriminate it from other species, by using material from different stages of development. Amounts of DNA as little as that recovered from a single egg were shown to be adequate for the analysis, and LAMP itself required only 45 min to complete.

Alterations of the acetylcholinesterase enzyme in the oriental fruit fly Bactrocera dorsalis are correlated with resistance to the organophosphate insecticide fenitrothion.

Alterations of the structure and activity of the enzyme acetylcholinesterase (AChE) leading to resistance to organophosphate insecticides have been examined in the oriental fruit fly, Bactrocera dorsalis (Hendel), an economic pest of great economic importance in the Asia-Pacific region. We used affinity chromatography to purify AChE isoenzymes from heads of insects from lines showing the phenotypes of resistance and sensitivity to insecticide treatments. The AChE enzyme from a strain selected for resistance to the insecticide fenitrothion shows substantially lower catalytic efficiency for various substrates and 124-, 373- and 5810-fold less sensitivity to inhibition by paraoxon, eserine and fenitroxon, respectively, compared to that of the fenitrothion susceptible line. Using peptide mass fingerprinting, we also show how specific changes in the structure of the AChE enzymes in these lines relate to the resistant and sensitive alleles of the AChE (ace) gene characterized previously in this species (described in Hsu, J.-C., Haymer, D.S., Wu, W.-J., Feng, H.-T., 2006. Mutations in the acetylcholinesterase gene of Bactrocera dorsalis associated with resistance to organophosphorus insecticides. Insect Biochem. Mol. Biol. 36, 396-402). Polyclonal antibodies specific to the purified isoenzymes and real-time PCR were also used to show that both the amount of the isoenzyme present and the expression levels of the ace genes were not significantly different between the R and S lines, indicating that quantitative changes in gene expression were not significantly contributing to the resistance phenotype. Overall, our results support a direct causal relationship between the mutations previously identified in the ace gene of this species and qualitative alterations of the structure and function of the AChE enzyme as the basis for the resistance phenotype. Our results also provide a basis for further comparisons of insecticide resistance phenomena seen in closely related species, such as Bactrocera oleae, as well as in a wide range of more distantly related insect species.

Mutations in the acetylcholinesterase gene of Bactrocera dorsalis associated with resistance to organophosphorus insecticides.

Mutations in the gene encoding the enzyme acetylcholinesterase (AChE) of the oriental fruit fly, Bactrocera dorsalis, associated with resistance to an organophosphorus insecticide have been characterized. Three point mutations producing nonsynonymous changes in the predicted amino acid sequence of the product of the B. dorsalis ace gene in resistant vs. susceptible flies have been identified. One of these changes is unique to B. dorsalis while the other two occur at sites that are identical to mutations previously described for another Bactrocera species. Although the precise role of the third mutation is not clearly established, the independent origin of two identical alterations in these two species strongly supports the idea proposed previously that molecular changes associated with insecticide resistance in key genes and enzymes such as AChE are largely constrained to a limited number of sites. The results obtained here also suggest that the widespread use of organophosphorus insecticides will likely lead to a predictable acquisition of resistance in wild populations of B. dorsalis as well as other pest species. For surveys of B. dorsalis populations that may develop resistance, diagnostic tests using PCR-RFLP based methods for detecting the presence of all three mutations in individual flies are described.