microRNA profiling between Bacillus thuringiensis Cry1Ab-susceptible and -resistant European corn borer, Ostrinia nubilalis (Hübner).

Transgenic maize hybrids that express insecticidal Bacillus thuringiensis (Bt) crystalline (Cry) protein toxins effectively protect against the European corn borer, Ostrinia nubilalis, a devastating maize pest. Field monitoring and laboratory selections have detected varying levels of O. nubilalis resistance to Cry1Ab toxin. MicroRNAs (miRNAs) are short noncoding RNAs that are involved in post-transcriptional gene regulation. Their potential roles in the evolution of Bt resistance, however, remain largely unknown. Sequencing of small RNA libraries from the midgut of Cry1Ab-susceptible and resistant O. nubilalis larvae resulted in the discovery of 277 miRNAs, including 248 conserved and 29 novel. Comparative analyses of miRNA expression profiles between the laboratory strains predicted 26 and nine significantly up- and down-regulated transcripts, respectively, in the midgut of Cry1Ab resistant larvae. Amongst 15 differentially regulated miRNAs examined by quantitative real-time PCR, nine (60%) were validated as cosegregating with Cry1Ab resistance in a backcross progeny. Differentially expressed miRNAs were predicted to affect transcripts involved in cell membrane components with functions in metabolism and binding, and the putative Bt-resistance genes aminopeptidase N and cadherin. These results lay the foundation for future investigation of the potential role of miRNAs in the evolution of Bt resistance.

Quantitative trait locus mapping and functional genomics of an organophosphate resistance trait in the western corn rootworm, Diabrotica virgifera virgifera.

The western corn rootworm, Diabrotica virgifera virgifera, is an insect pest of corn and population suppression with chemical insecticides is an important management tool. Traits conferring organophosphate insecticide resistance have increased in frequency amongst D. v. virgifera populations, resulting in the reduced efficacy in many corn-growing regions of the USA. We used comparative functional genomic and quantitative trait locus (QTL) mapping approaches to investigate the genetic basis of D. v. virgifera resistance to the organophosphate methyl-parathion. RNA from adult methyl-parathion resistant and susceptible adults was hybridized to 8331 microarray probes. The results predicted that 11 transcripts were significantly up-regulated in resistant phenotypes, with the most significant (fold increases ≥ 2.43) being an α-esterase-like transcript. Differential expression was validated only for the α-esterase (ST020027A20C03), with 11- to 13-fold greater expression in methyl-parathion resistant adults (P < 0.05). Progeny with a segregating methyl-parathion resistance trait were obtained from a reciprocal backcross design. QTL analyses of high-throughput single nucleotide polymorphism genotype data predicted involvement of a single genome interval. These data suggest that a specific carboyxesterase may function in field-evolved corn rootworm resistance to organophosphates, even though direct linkage between the QTL and this locus could not be established.

Explaining the sawtooth: latitudinal periodicity in a circadian gene correlates with shifts in generation number.

Many temperate insects take advantage of longer growing seasons at lower latitudes by increasing their generation number or voltinism. In some insects, development time abruptly decreases when additional generations are fit into the season. Consequently, latitudinal 'sawtooth' clines associated with shifts in voltinism are seen for phenotypes correlated with development time, like body size. However, latitudinal variation in voltinism has not been linked to genetic variation at specific loci. Here, we show a pattern in allele frequency among voltinism ecotypes of the European corn borer moth (Ostrinia nubilalis) that is reminiscent of a sawtooth cline. We characterized 145 autosomal and sex-linked SNPs and found that period, a circadian gene that is genetically linked to a major QTL determining variation in post-diapause development time, shows cyclical variation between voltinism ecotypes. Allele frequencies at an unlinked circadian clock gene cryptochrome1 were correlated with period. These results suggest that selection on development time to 'fit' complete life cycles into a latitudinally varying growing season produces oscillations in alleles associated with voltinism, primarily through changes at loci underlying the duration of transitions between diapause and other life history phases. Correlations among clock loci suggest possible coupling between the circadian clock and the circannual rhythms for synchronizing seasonal life history. We anticipate that latitudinal oscillations in allele frequency will represent signatures of adaptation to seasonal environments in other insects and may be critical to understanding the ecological and evolutionary consequences of variable environments, including response to global climate change.

Role of a γ-aminobutryic acid (GABA) receptor mutation in the evolution and spread of Diabrotica virgifera virgifera resistance to cyclodiene insecticides.

The western corn rootworm, Diabrotica virgifera virgifera, is a damaging pest of cultivated corn that was controlled by applications of cyclodiene insecticides from the late 1940s until resistance evolved ∼10 years later. Range expansion from the western plains into eastern USA coincides with resistance development. An alanine to serine amino acid substitution within the Rdl subunit of the gamma-aminobutyric acid (GABA) receptor confers resistance to cyclodiene insecticides in many species. We found that the non-synonymous single nucleotide polymorphism (SNP) G/T at the GABA receptor cDNA position 838 (G/T(838)) of D. v. virgifera resulted in the alanine to serine change, and the codominant SNP allele T(838) was genetically linked to survival of beetles in aldrin bioassays. A phenotypic gradient of decreasing susceptibility from west to east was correlated with higher frequencies of the resistance-conferring T(838) allele in the eastern-most populations. This pattern exists in opposition to perceived selective pressures since the more eastern and most resistant populations probably experienced reduced exposure. The reasons for the observed distribution are uncertain, but historical records of the range expansion combined with the distribution of susceptible and resistant phenotypes and genotypes provide an opportunity to better understand factors affecting the species' range expansion.

The spatial genetic differentiation of the legume pod borer, Maruca vitrata F. (Lepidoptera: Crambidae) populations in West Africa.

The legume pod borer, Maruca vitrata, is an endemic insect pest that causes significant yield loss to the cowpea crop in West Africa. The application of population genetic tools is important in the management of insect pests but such data on M. vitrata is lacking. We applied a set of six microsatellite markers to assess the population structure of M. vitrata collected at five sites from Burkina Faso, Niger and Nigeria. Observed polymorphisms ranged from one (marker 3393) to eight (marker 32008) alleles per locus. Observed and expected heterozygosities ranged from 0.0 to 0.8 and 0.0 to 0.6, respectively. Three of the loci in samples from Nigeria and Burkina Faso deviated significantly from Hardy-Weinberg Equilibrium (HWE), whereas no loci deviated significantly in samples from Niger. Analysis of molecular variance (AMOVA) indicated that 67.3% level of the genetic variation was within individuals compared to 17.3% among populations. A global estimate of F ST=0.1 (ENA corrected F ST=0.1) was significant (P⩽0.05) and corroborated by pairwise F ST values that were significant among all possible comparisons. A significant correlation was predicted between genetic divergence and geographic distance between subpopulations (R2=0.6, P=0.04), and cluster analysis by the program STRUCTURE predicted that co-ancestry of genotypes were indicative of three distinct populations. The spatial genetic variance among M. vitrata in West Africa may be due to limited gene flow, south-north seasonal movement pattern or other reproductive barriers. This information is important for the cultural, chemical and biological control strategies for managing M. vitrata.

Comparison of the transcriptional profiles of head and body lice.

Head and body lice are both blood-feeding parasites of humans although only the body louse is a potent disease vector. In spite of numerous morphological and life history differences, head and body lice have recently been hypothesized to be ecotypes of the same species. We took a comparative genomics approach to measure nucleotide diversity by comparing expressed sequence tag data sets from head and body lice. A total of 10 771 body louse and 10 770 head louse transcripts were predicted from a combined assembly of Roche 454 and Illumina sequenced cDNAs from whole body tissues collected at all life stages and during pesticide exposure and bacterial infection treatments. Illumina reads mapped to the 10 775 draft body louse gene models from the whole genome assembly predicted nine presence/absence differences, but PCR confirmation resulted in a single gene difference. Read per million base pair estimates indicated that 14 genes showed significant differential expression between head and body lice under our treatment conditions. One novel microRNA was predicted in both lice species and 99% of the 544 transcripts from Candidatus riesia indicate that they share the same endosymbiont. Overall, few differences exist, which supports the hypothesis that these two organisms are ecotypes of the same species.

A novel class of miniature inverted repeat transposable elements (MITEs) that contain hitchhiking (GTCY)(n) microsatellites.

The movement of miniature inverted repeat transposable elements (MITEs) modifies genome structure and function. We describe the microsatellite-associated interspersed nuclear element 2 (MINE-2), that integrates at consensus WTTTT target sites, creates dinucleotide TT target site duplications (TSDs), and forms predicted MITE-like secondary structures; a 5' subterminal inverted repeat (SIR; AGGGTTCCGTAG) that is partially complementary to a 5' inverted repeat (IR; ACGAAGCCCT) and 3'-SIRs (TTACGGAACCCT). A (GTCY)(n) microsatellite is hitchhiking downstream of conserved 5'MINE-2 secondary structures, causing flanking sequence similarity amongst mobile microsatellite loci. Transfection of insect cell lines indicates that MITE-like secondary structures are sufficient to mediate genome integration, and provides insight into the transposition mechanism used by MINE-2s.

Mining an Ostrinia nubilalis midgut expressed sequence tag (EST) library for candidate genes and single nucleotide polymorphisms (SNPs).

Genes expressed in lepidopteran midgut tissues are involved in digestion and Bacillus thuringiensis (Bt) toxin resistance traits. Five hundred and thirty five unique transcripts were annotated from 1745 high quality O. nubilalis larval midgut expressed sequence tags (ESTs). Full-length cDNA sequence of 12 putative serine proteinase genes and 3 partial O. nubilalis aminopeptidase N protein genes, apn1, apn3, and apn4, were obtained, and genes may have roles in plant feeding and Bt toxin resistance traits of Ostrinia larvae. The EST library was not normalized and insert frequencies reflect transcript levels under the initial treatment conditions and redundancy of inserts from highly expressed transcripts allowed prediction of putative single nucleotide polymorphisms (SNPs). Ten di-, tri- or tetranucleotide repeat unit microsatellite loci were identified, and minisatellite repeats were observed within the C-termini of two encoded serine proteinases. Molecular markers showed polymorphism at 28 SNP loci and one microsatellite locus, and Mendelian inheritance indicated that markers were applicable to genome mapping applications. This O. nubilalis larval midgut EST collection is a resource for gene discovery, expression information, and allelic variation for use in genetic marker development.

Sequence variation in trypsin- and chymotrypsin-like cDNAs from the midgut of Ostrinia nubilalis: methods for allelic differentiation of candidate Bacillus thuringiensis resistance genes.

Midgut expressed alkaline serine proteases of Lepidoptera function in conversion of Bacillus thuringiensis (Bt) protoxin to active toxin, and reduced level of transcript T23 is associated with Ostrinia nubilalis resistance to Dipel Bt formulations. Three groups of trypsin- (OnT25, OnT23, and OnT3) and two chymotrypsin-like (OnC1 and OnC2) cDNAs were isolated from O. nubilalis midgut tissue. Intraspecific groupings are based on cDNA similarity and peptide phylogeny. Derived serine proteases showed a catalytic triad (His, Asp, and Ser; except transcript OnT23a), three substrate specificity-determining residues, and three paired disulphide bonds. RT-PCR indicated all transcripts are expressed in the midgut. Mendelian-inherited genomic markers for loci OnT23, OnT3 and OnC1 will be useful for association of alleles with bioassayed Bt toxin resistance phenotypes.