Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance

<p>Abstract</p> <p>Background</p> <p>The New World screw-worm (NWS), <it>Cochliomyia hominivorax</it>, is one of the most important myiasis-causing flies, causing severe losses to the livestock industry. In its current geographical distribution, this species has been controlled by the application of insecticides, mainly organophosphate (OP) compounds, but a number of lineages have been identified that are resistant to such chemicals. Despite its economic importance, only limited genetic information is available for the NWS. Here, as a part of an effort to characterize the <it>C. hominivorax </it>genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology.</p> <p>Results</p> <p>Deep sequencing on the 454 platform of three normalized libraries (larval, adult male and adult female) generated a total of 548,940 reads. Eighteen candidate genes coding for three metabolic detoxification enzyme families, cytochrome P450 monooxygenases, glutathione S-transferases and carboxyl/cholinesterases were selected and gene expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Of the investigated candidates, only one gene was expressed differently between control and resistant larvae with, at least, a 10-fold down-regulation in the resistant larvae. The presence of mutations in the acetylcholinesterase (target site) and carboxylesterase E3 genes was investigated and all of the resistant flies presented E3 mutations previously associated with insecticide resistance.</p> <p>Conclusions</p> <p>Here, we provided the largest database of NWS expressed sequence tags that is an important resource, not only for further studies on the molecular basis of the OP resistance in NWS fly, but also for functional and comparative studies among Calliphoridae flies. Among our candidates, only one gene was found differentially expressed in resistant individuals, and its role on insecticide resistance should be further investigated. Furthermore, the absence of mutations in the OP target site and the high frequency of mutant carboxylesterase E3 indicate that metabolic resistance mechanisms have evolved predominantly in this species.</p

Genetic divergence in mitochondrial DNA of Anopheles nuneztovari (Diptera: Culicidae) from Brazil and Colombia

In the present study, we have examined the variability in Anopheles nuneztovari mitochondrial DNA of three populations from the Brazilian Amazon and one from western Colombia (Sitronela), using four restriction endonucleases (BclI, ClaI, HindIII, SstI). The haplotype diversity (h) was slightly elevated in all populations (0.5000 to 0.6765), whereas the nucleotide diversity (π) was lower in the Sitronela population (0.0029) and higher in populations from the Brazilian Amazon (0.0056 to 0.0098). The degree of sequence divergence (δ) estimated within the Brazilian Amazon and that in Sitronela (0.0329 to 0.0371) suggests that these geographic populations of A. nuneztovari may eventually constitute separate species. The low sequence divergence values among the three Brazilian Amazon populations (0.0012 to 0.0031) indicate that these populations are genetically similar. These results are consistent with those recently reported for allozymes of these same populations

The Phylogeographic History of the New World Screwworm Fly, Inferred by Approximate Bayesian Computation Analysis

<div><p>Insect pest phylogeography might be shaped both by biogeographic events and by human influence. Here, we conducted an approximate Bayesian computation (ABC) analysis to investigate the phylogeography of the New World screwworm fly, <i>Cochliomyia hominivorax</i>, with the aim of understanding its population history and its order and time of divergence. Our ABC analysis supports that populations spread from North to South in the Americas, in at least two different moments. The first split occurred between the North/Central American and South American populations in the end of the Last Glacial Maximum (15,300-19,000 YBP). The second split occurred between the North and South Amazonian populations in the transition between the Pleistocene and the Holocene eras (9,100-11,000 YBP). The species also experienced population expansion. Phylogenetic analysis likewise suggests this north to south colonization and Maxent models suggest an increase in the number of suitable areas in South America from the past to present. We found that the phylogeographic patterns observed in <i>C. hominivorax</i> cannot be explained only by climatic oscillations and can be connected to host population histories. Interestingly we found these patterns are very coincident with general patterns of ancient human movements in the Americas, suggesting that humans might have played a crucial role in shaping the distribution and population structure of this insect pest. This work presents the first hypothesis test regarding the processes that shaped the current phylogeographic structure of <i>C. hominivorax</i> and represents an alternate perspective on investigating the problem of insect pests.</p> </div

The Mitochondrial Control Region Of Blowflies (diptera: Calliphoridae): A Hot Spot For Mitochondrial Genome Rearrangements.

The family Calliphoridae consists of myiasis-causing flies, including species of economic, forensic, and medical importance. In this study, the complete control regions (CRs) of mitochondrial DNA from 15 calliphorid species were sequenced and structurally characterized. The CRs had a high content of adenines (A) and thymines (T) and varied in length from 854 to 2,018 bp, showing intraspecific variations in sequence and length. Two major domains were identified: the conserved domain containing conserved sequence blocks and cis-regulatory structures that may be related to the transcription and the origin of replication of mitochondrial DNA, and the variable domain, containing high sequence and length variation. Within the variable domain, duplication of the tRNA(Ile) gene, previously reported for three Chrysomya species, was identified in two more species of this genus and in two species of two other genera. The structural characterization shows the plasticity of the mitochondrial genome in dipterans. The organizational similarities of the duplicated region found in different species and the possible origin of the duplicated genes are discussed.45667-7

Genetic Structure And Demographic History Of New World Screwworm Across Its Current Geographic Range.

The phylogeographical history of the pest fly screwworm, Cochliomyia hominivorax (Coquerel), was studied using partial mitochondrial DNA sequences of the control region, Cytochrome c oxidase (CO) subunit I and CO subunit II from 361 individuals collected across its current geographic range. Analyses showed marked genetic differentiation on a macrogeographic scale. The genetic diversity in the species is structured into four main regional groups, corresponding to Cuba, the Dominican Republic, and the North and South Amazon region. Results indicated that the distribution of screwworm genetic diversity was mainly shaped by historical events, i.e., colonization of Caribbean islands, vicariance in the Amazon region and population expansion. Demographic history analyses revealed that the population expansion started approximately 20-25,000 yr ago and recently increased exponentially. We hypothesized that the initial period of expansion was probably associated with environmental amelioration in the late Pleistocene and the exponential increase with resource availability in recent times. The population expansion is probably responsible for the low divergence and the lack of genetic and geographic correlation in the South Amazon region but did not erase the genetic structure pattern on a continental scale. The screwworm is one of the most damaging livestock pests in South and Central America, and the pattern of genetic variability distribution reported here suggests that the Caribbean area and the North and South Amazon regions could be considered as independent units for future pest control programs.48280-9

Incongruent Nuclear and Mitochondrial Genetic Structure of New World Screwworm Fly Populations Due to Positive Selection of Mutations Associated with Dimethyl- and Diethyl-Organophosphates Resistance

<div><p>Livestock production is an important economic activity in Brazil, which has been suffering significant losses due to the impact of parasites. The New World screwworm (NWS) fly, <i>Cochliomyia hominivorax</i>, is an ectoparasite and one of the most important myiasis-causing flies endemic to the Americas. The geographic distribution of NWS has been reduced after the implementation of the Sterile Insect Technique (SIT), being eradicated in North America and part of Central America. In South America, <i>C</i>. <i>hominivorax</i> is controlled by chemical insecticides, although indiscriminate use can cause selection of resistant individuals. Previous studies have associated the Gly137Asp and Trp251Leu mutations in the active site of carboxylesterase E3 to resistance of diethyl and dimethyl-organophosphates insecticides, respectively. Here, we have sequenced a fragment of the carboxylesterase E3 gene (<i>ChαE7</i>), comprising part of intron iII, exon eIII, intron iIII and part of exon eIV, and three mitochondrial gene sequences (CR, COI and COII), of NWS flies from 21 locations in South America. These markers were used for population structure analyses and the <i>ChαE7</i> gene was also investigated to gain insight into the selective pressures that have shaped its evolution. Analysis of molecular variance (AMOVA) and pairwise FST analysis indicated an increased genetic structure between locations in the <i>ChαE7</i> compared to the concatenated mitochondrial genes. Discriminant analysis of principal components (DAPC) and spatial analysis of molecular variance (SAMOVA) indicated different degrees of genetic structure for all markers, in agreement with the AMOVA results, but with low correlation to geographic data. The NWS fly is considered a panmitic species based on mitochondrial data, while it is structured into three groups considering the <i>ChαE7</i> gene. A negative association between the two mutations related to organophosphate resistance and Fay & Wu’s H significant negative values for the exons, suggest that these mutations evolved under positive selection.</p></div

Carboxylesterase E3 <i>ChαE7</i> schematic view.

<p>Comparison of exon and intron lengths and position between (A) <i>L</i>. <i>cuprina</i> and (B) <i>C</i>. <i>hominivorax</i>. Exons and introns were named according to <i>L</i>. <i>cuprina</i> (e = exon; I = intron) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128441#pone.0128441.ref027" target="_blank">27</a>]. The position of primers used for gene region characterization (7F0a, RN2, 7FIn1 and 7R1aN) and for posterior populational analyses (7FIn2, RN2 and 7R3a) are indicated by blue and red arrows, respectively. RN2 is indicated by a green arrow (used for both characterization and population analyses).</p

Hardy-Weinberg equilibrium tests for the two polymorphic codon positions related to the determination of susceptibility and resistance.

<p>Statistically significant values are in bold. Ho = observed heterozygosity; He = expected heterozygosity; s.d. = standard deviation.</p><p>Hardy-Weinberg equilibrium tests for the two polymorphic codon positions related to the determination of susceptibility and resistance.</p

Structure analyses results for esterase data (K = 3).

<p>(A) SAMOVA F indices. F_CT: differentiation between groups; F_SC: differentiation between sampling locations within groups; F_ST: differentiation between sites between groups. (B) Pie charts of the posterior probability to be from a group estimated by DAPC (i.e. Red, Yellow and Green) and SAMOVA grouping (i.e. I, II and III). SAMOVA groups (I, II and III), considered for posterior analyses, are defined by the grey, purple and blue contours.</p

Frequencies of polymorphic nucleotides related to the determination of susceptibility and resistance in the codons 137 and 251 of the <i>ChαE7</i> sequence.

<p>The structure in groups I, II and III (K = 3) was analyzed. Codons were named for the encoded amino acid, <i>ChαE7</i> sequence position (amino acid number) and the nucleotide substitution of that codon (Gly137-G, Asp137-A, Trp251-G, Leu251-T and Ser251-C).</p