David S. Haymer

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 worlds 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 Illuminas 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.

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.

Y enriched and Y specific DNA sequences from the genome of the mediterranean fruit fly, Ceratitis capitata

DNA sequences that are enriched or specific to the genome of the male medfly, Ceratitis capitata, have been isolated using a differential hybridization approach. Twelve phage clones from a genomic library have been identified that consistently display more intense hybridization with a genomic DNA probe from males as opposed to one from females. Southern DNA blot analysis reveals that these recombinant clones contain at least one EcoRI fragment that is either specific to the male genome, or more highly represented in it, as compared with the female genome. These EcoRI fragments, when used as probes, all generate a similar pattern of intense multiple bands in genomic DNA of males. This suggests the presence of repetitive sequences that are at least partially homologous in these regions of the genome that are specific to or enriched in males. In situ hybridization to mitotic chromosomes confirms a Y chromosomal origin for the male specific repetitive sequences. Data on the genomic organization, representation and evolutionary conservation of these sequences that are specific to or enriched in males are presented. Studies of the genomic organization and representation of flanking sequences that are not male specific are presented as well.

Ribosomal ITS1 Polymorphisms in Ceratitis capitata and Ceratitis rosa (Diptera: Tephritidae)

Abstract Ribosomal ITS1 DNA sequences have been characterized in Ceratitis capitata (Weidemann), the Mediterranean fruit fly, and Ceratitis rosa (Karsch), the Natal fruit fly. In C. capitata the ITS1 region is 831 bp in size (on average). Relatively little variation in the makeup of the ITS1 DNA sequences was detected in individuals sampled from numerous worldwide populations of C. capitata. In C. rosa, however, a substantial difference in the size of the ITS1 region was observed in individuals from a collection made in Kenya compared with individuals from South Africa. In the C. rosa flies from South Africa, this region was ≈930 bp in size, compared with a size of 717 bp in the C. rosa Kenya flies. This difference appears to be due primarily to a single insertion or deletion event. Using polymerase chain reaction, these size polymorphisms are easily detectable and can be used for making species identification and/or population origin determinations using material from any stage of the life cycle. Despite these intra and interspecific differences, sequences from the ITS1 regions of both C. rosa collections and C. capitata can still be aligned. Quantification of the similarity of these sequences may help to resolve continuing questions over the status of species relationships in this genus.

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.

Potential for insecticide resistance in populations of Bactrocera dorsalis in Hawaii: spinosad susceptibility and molecular characterization of a gene associated with organophosphate resistance

The potential for populations to become resistant to a particular insecticide treatment regimen is a major issue for all insect pest species. In Hawaii, for example, organophosphate (OP)‐based cover sprays have been the chemical treatment most commonly applied against oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), populations since the 1950s. Moreover, bait spray treatments using spinosad were adopted as a major control tactic in the Hawaii area‐wide fruit fly pest management program beginning in the year 2000. To determine the current level of spinosad and OP tolerance of wild B. dorsalis populations, bioassays were conducted on flies collected from a range of geographic localities within the Hawaiian islands. Adult B. dorsalis flies were tested (1) for the level of susceptibility to spinosad using LC50 diagnostic criteria, and (2) for the presence of alleles of the ace gene previously shown to be associated with OP resistance. Regarding spinosad tolerance, only flies from Puna, the one area lacking prior exposure to spinosad, showed any significant difference compared to controls, and here the difference was only in terms of non‐overlap of 95% fiducial limit values. With respect to OP tolerance, specific mutations in the ace gene associated with resistance to these insecticides were found in only two populations, and in both cases, these alleles occurred at relatively low frequencies. These results suggest that at the present time, populations of B. dorsalis in Hawaii show no evidence for having acquired resistance to the insecticides widely used in control programs.

Molecular structure of yoyo, a gypsy-like retrotransposon from the Mediterranean fruit fly, Ceratitis capitata

We have isolated and characterized a new LTR-retrotransposon in the genome of the Mediterranean fruit fly (Medfly), Ceratitis capitata. This retrotransposon, which we named yoyo, appears to be a member of the gypsy/Ty3 class of elements. The yoyo element was originally discovered on the Y chromosome of the Medfly. Although the Y chromosome copy appears to be truncated, at least two other apparently complete copies of yoyo from other genomic locations have been isolated and characterized. The complete element is approximately 7.7 kb in size. In addition to fairly typical GAG and POL coding regions, the yoyo element contains a potential ENV gene. The presence of an ENV gene is a key feature distinguishing potential retroviral-like elements, such as gypsy (and possibly yoyo), from many other invertebrate retrotransposons previously described. In addition to the structural features of yoyo, evidence is provided to show that yoyo is capable of movement in the genome, including RFLPs showing variability in genomic localization of copies of yoyo between strains, and differences among individuals in the presence of yoyo at a specific site in the genome.

Codon bias in actin multigene families and effects on the reconstruction of phylogenetic relationships

Codon usage patterns and phylogenetic relationships in the actin multigene family have been analyzed for three dipteran species—Drosophila melanogaster, Bactrocera dorsalis, and Ceratitis capitata. In certain phylogenetic tree reconstructions, using synonymous distances, some gene relationships are altered due to a homogenization phenomenon. We present evidence to show that this homogenization phenomenon is due to codon usage bias. A survey of the pattern of synonymous codon preferences for I I actin genes from these three species reveals that five out of the six Drosophila actin genes show high degrees of codon bias as indicated by scaled Ξ2 values. In contrast to this, four out of the five actin genes from the other species have low codon bias values. A Monte Carlo contingency test indicates that for those Drosophila actin genes which exhibit codon bias, the patterns of codon usage are different compared to actin genes from the other species. In addition, the genes exhibiting codon bias also appear to have reduced rates of synonymous substitution. The homogenization phenomenon seen in terms of synonymous substitutions is not observed for nonsynonymous changes. Because of this homogenization phenomenon, “trees” constructed based on synonymous substitutions will be affected. These effects can be overt in the case of multigene families, but similar distortions may underlie reconstructions based on single-copy genes which exhibit codon usage bias.

Rapid Identification of the Mediterranean Fruit Fly (Diptera: Tephritidae) by Loop-Mediated Isothermal Amplification

ABSTRACT The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), ranks as one of the worlds 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.

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.