Determinants of Insecticide Resistance Evolution: Comparative Analysis Among Heliothines.

It is increasingly clear that pest species vary widely in their propensities to develop insecticide resistance. This review uses a comparative approach to analyze the key pest management practices and ecological and biochemical or genetic characteristics of the target that contribute to this variation. We focus on six heliothine species, three of which, Helicoverpa armigera, Heliothis virescens, and Helicoverpa zea, have developed resistances to many pesticide classes. The three others, Helicoverpa punctigera, Helicoverpa assulta, and Helicoverpa gelotopoeon, also significant pests, have developed resistance to very few pesticide classes. We find that host range and movement between alternate hosts are key ecological traits that influence effective selection intensities for resistance. Operational issues are also critical; area-wide, cross-pesticide management practices that account for these ecological factors are key to reducing selection intensity. Without such management, treatment using broad-spectrum chemicals serves to multiply the effects of host plant preference, preadaptive detoxification ability, and high genetic diversity to create a pesticide treadmill for the three high-propensity species.Without rigorous ongoing management, such a treadmill could still develop for newer, more selective chemistries and insecticidal transgenic crops.

Heat-evolved microalgal symbionts increase coral bleaching tolerance.

Coral reefs worldwide are suffering mass mortalities from marine heat waves. With the aim of enhancing coral bleaching tolerance, we evolved 10 clonal strains of a common coral microalgal endosymbiont at elevated temperatures (31°C) for 4 years in the laboratory. All 10 heat-evolved strains had expanded their thermal tolerance in vitro following laboratory evolution. After reintroduction into coral host larvae, 3 of the 10 heat-evolved endosymbionts also increased the holobionts' bleaching tolerance. Although lower levels of secreted reactive oxygen species (ROS) accompanied thermal tolerance of the heat-evolved algae, reduced ROS secretion alone did not predict thermal tolerance in symbiosis. The more tolerant symbiosis exhibited additional higher constitutive expression of algal carbon fixation genes and coral heat tolerance genes. These findings demonstrate that coral stock with enhanced climate resilience can be developed through ex hospite laboratory evolution of their microalgal endosymbionts.

Differences in gene regulation in a tephritid model of prezygotic reproductive isolation.

The two tephritid fruit fly pests, Bactrocera tryoni and Bactrocera neohumeralis, are unusually well suited to the study of the genetics of reproductive isolating mechanisms. Sequence difference between the species is no greater than between a pair of conspecific Drosophila melanogaster populations. The two species exist in close sympatry, yet do not hybridize in the field, apparently kept separate by a strong premating isolation mechanism involving the time of day at which mating occurs. This spurred us to search for key genes for which time of day expression is regulated differently between the species. Using replicated, quantitative transcriptomes from head tissues of males of the two species, sampled in the day and night, we identified 141 transcripts whose abundance showed a significant interaction between species and time of day, indicating a difference in gene regulation. The brain transcripts showing this interaction were enriched for genes with a neurone function and 90% of these were more abundant at night than day in B. tryoni. Features of the expression patterns suggest that there may be a difference in the regulation of sleep-wake cycles between the species. In particular several genes, which in D. melanogaster are expressed in circadian pacemaker cells, are promising candidates to further explore the genetic differentiation involved in this prezygotic reproductive isolation mechanism.

Population differentiation between Australian and Chinese Helicoverpa armigera occurs in distinct blocks on the Z-chromosome.

Over the last 40 years, many types of population genetic markers have been used to assess the population structure of the pest moth species Helicoverpa armigera. While this species is highly vagile, there is evidence of inter-continental population structure. Here, we examine Z-chromosome molecular markers within and between Chinese and Australian populations. Using 1352 polymorphic sites from 40 Z-linked loci, we compared two Chinese populations of moths separated by 700 km and found virtually no population structure (n = 41 and n = 54, with <1% of variation discriminating between populations). The levels of nucleotide diversity within these populations were consistent with previous estimates from introns in Z-linked genes of Australian samples (π = 0.028 vs. 0.03). Furthermore, all loci surveyed in these Chinese populations showed a skew toward rare variants, with ten loci having a significant Tajima's D statistic, suggesting that this species could have undergone a population expansion. Eight of the 40 loci had been examined in a previous study of Australian moths, of which six revealed very little inter-continental population structure. However, the two markers associated with the Cyp303a1 locus that has previously been proposed to be a target of a selective sweep, exhibited allele structuring between countries. Using a separate dataset of 19 Australian and four Chinese moths, we scanned the molecular variation distributed across the entire Z-chromosome and found distinct blocks of differentiation that include the region containing Cyp303a1. We recommend some of these loci join those associated with insecticide resistance to form a set of genes best suited to analyzing population structure in this global pest.

Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species.

BACKGROUND: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. RESULTS: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. CONCLUSIONS: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

High nucleotide diversity and limited linkage disequilibrium in Helicoverpa armigera facilitates the detection of a selective sweep.

Insecticides impose extreme selective pressures on populations of target pests and so insecticide resistance loci of these species may provide the footprints of 'selective sweeps'. To lay the foundation for future genome-wide scans for selective sweeps and inform genome-wide association study designs, we set out to characterize some of the baseline population genomic parameters of one of the most damaging insect pests in agriculture worldwide, Helicoverpa armigera. To this end, we surveyed nine Z-linked loci in three Australian H. armigera populations. We find that estimates of π are in the higher range among other insects and linkage disequilibrium decays over short distances. One of the surveyed loci, a cytochrome P450, shows an unusual haplotype configuration with a divergent allele at high frequency that led us to investigate the possibility of an adaptive introgression around this locus.

Isomer-specific comparisons of the hydrolysis of synthetic pyrethroids and their fluorogenic analogues by esterases from the cotton bollworm Helicoverpa armigera.

The low aqueous solubility and chiral complexity of synthetic pyrethroids, together with large differences between isomers in their insecticidal potency, have hindered the development of meaningful assays of their metabolism and metabolic resistance to them. To overcome these problems, Shan and Hammock (2001) [7] therefore developed fluorogenic and more water-soluble analogues of all the individual isomers of the commonly used Type 2 pyrethroids, cypermethrin and fenvalerate. The analogues have now been used in several studies of esterase-based metabolism and metabolic resistance. Here we test the validity of these analogues by quantitatively comparing their hydrolysis by a battery of 22 heterologously expressed insect esterases with the hydrolysis of the corresponding pyrethroid isomers by these esterases in an HPLC assay recently developed by Teese et al. (2013) [14]. We find a strong, albeit not complete, correlation (r = 0.7) between rates for the two sets of substrates. The three most potent isomers tested were all relatively slowly degraded in both sets of data but three esterases previously associated with pyrethroid resistance in Helicoverpa armigera did not show higher activities for these isomers than did allelic enzymes derived from susceptible H. armigera. Given their amenability to continuous assays at low substrate concentrations in microplate format, and ready detection of product, we endorse the ongoing utility of the analogues in many metabolic studies of pyrethroids.

Metabolic enzymes associated with xenobiotic and chemosensory responses in Nasonia vitripennis.

The numbers of glutathione S-transferase, cytochrome P450 and esterase genes in the genome of the hymenopteran parasitoid Nasonia vitripennis are about twice those found in the genome of another hymenopteran, the honeybee Apis mellifera. Some of the difference is associated with clades of these families implicated in xenobiotic resistance in other insects and some is in clades implicated in hormone and pheromone metabolism. The data support the hypothesis that the eusocial behaviour of the honeybee and the concomitant homeostasis of the nest environment may obviate the need for as many gene/enzyme systems associated with xenobiotic metabolism as are found in other species, including N. vitripennis, that are thought to encounter a wider range of potentially toxic xenobiotics in their diet and habitat.

Jhe in Gryllus assimilis: cloning, sequence-activity associations and phylogeny.

The 458 amino acid sequence of a mature JHE protein from the cricket Gryllus assimilis was identified after isolating the partial cDNA sequence encoding this protein from a fat body and midgut cDNA library. This hemimetabolan JHE sequence shows over 40% amino acid similarity to the known JHE sequences of several holometabolous insects. It also includes previously determined peptide sequences for G. assimilis JHE as well as two other motifs associated with JHE enzymes in holometabolous insects. The predicted molecular weight of the protein agrees with that of the JHE previously purified from G. assimilis. Partial genomic sequence encoding the Jhe contains two large (1330 and 2918bp) introns. No coding DNA sequence variation was observed over a 1293bp region between selected lines differing six to eight-fold in hemolymph JHE activity. However, a 19bp indel was found in one of the introns; the insertion was strongly associated with elevated hemolymph activity, both in the selected lines and in the F(2) progeny of crosses between them. Phylogenetic analyses localised the G. assimilis JHE to a clade containing dipteran and coleopteran JHEs, with lepidopteran JHEs occurring in a separate clade.

Hydrolysis of individual isomers of fluorogenic pyrethroid analogs by mutant carboxylesterases from Lucilia cuprina.

We previously showed that wild-type E3 carboxylesterase of Lucilia cuprina has high activity against Type 1 pyrethroids but much less for the bulkier, alpha-cyano containing Type 2 pyrethroids. Both Types have at least two optical centres and, at least for the Type 1 compounds, we found that wild-type E3 strongly prefers the less insecticidal configurations of the acyl group. However, substitutions to smaller residues at two sites in the acyl pocket of the enzyme substantially increased overall activity, particularly for the more insecticidal isomers. Here we extend these analyses to Type 2 pyrethroids by using fluorogenic analogs of all the diastereomers of cypermethrin and fenvalerate. Wild-type E3 hydrolysed some of these appreciably, but, again, not those corresponding to the most insecticidal isomers. Mutations in the leaving group pocket or oxyanion hole were again generally neutral or deleterious. However, the two sets of mutants in the acyl pocket again improved activity for the more insecticidal acyl group arrangements as well as for the more insecticidal configuration of the cyano moiety on the leaving group. The activities of the best mutant enzyme against the analogs of the most insecticidal isomers of cypermethrin and fenvalerate were more than ten and a hundred fold higher, respectively, than those of wild-type. The implications for resistance development are discussed.

Molecular population genetics of the alpha-esterase5 gene locus in original and colonized populations of Drosophila buzzatii and its sibling Drosophila koepferae.

Several studies have suggested that esterase-2 (EST-2) may be the target of natural selection in the cactophilic fly Drosophila buzzatii. In this work, we analyzed nucleotide variation in a fragment of alpha-esterase5 (alphaE5), the gene encoding EST-2, in original (Argentinian) and colonized (Australian) populations of D. buzzatii and in its sibling D. koepferae. Estimates of nucleotide heterozygosity in D. buzzatii were similar in Australia and Argentina, although we detected a loss of singletons in colonized populations, suggesting a moderate founder effect. Interspecific comparisons revealed that D. buzzatii was more polymorphic for nonsynonymous variation, whereas D. koepferae was more variable for synonymous and noncoding sites. The two major chromosomal arrangements (2st and 2j) in D. buzzatii displayed similar levels of nucleotide variation, whereas 2jz3 was monomorphic. The sequenced region allowed the discrimination of a greater number of EST-2 protein variants in the Australian sample than in the Argentinean sample. In D. koepferae, nucleotide variation in alphaE5 does not depart from neutral expectations, although tests of population structure were significant for silent variation. In contrast, D. buzzatii has probably undergone a recent population expansion in its South American range. In addition, the McDonald and Kreitman test revealed an excess of nonsynonymous polymorphism in both original and colonized populations of this species.

A deficit of detoxification enzymes: pesticide sensitivity and environmental response in the honeybee.

The honeybee genome has substantially fewer protein coding genes ( approximately 11 000 genes) than Drosophila melanogaster ( approximately 13 500) and Anopheles gambiae ( approximately 14 000). Some of the most marked differences occur in three superfamilies encoding xenobiotic detoxifying enzymes. Specifically there are only about half as many glutathione-S-transferases (GSTs), cytochrome P450 monooxygenases (P450s) and carboxyl/cholinesterases (CCEs) in the honeybee. This includes 10-fold or greater shortfalls in the numbers of Delta and Epsilon GSTs and CYP4 P450s, members of which clades have been recurrently associated with insecticide resistance in other species. These shortfalls may contribute to the sensitivity of the honeybee to insecticides. On the other hand there are some recent radiations in CYP6, CYP9 and certain CCE clades in A. mellifera that could be associated with the evolution of the hormonal and chemosensory processes underpinning its highly organized eusociality.

Amplification of DNA from preserved specimens shows blowflies were preadapted for the rapid evolution of insecticide resistance.

Mutations of esterase 3 confer two forms of organophosphate resistance on contemporary Australasian Lucilia cuprina. One form, called diazinon resistance, is slightly more effective against commonly used insecticides and is now more prevalent than the other form, called malathion resistance. We report here that the single amino acid replacement associated with diazinon resistance and two replacements associated with malathion resistance also occur in esterase 3 in the sibling species Lucilia sericata, suggesting convergent evolution around a finite set of resistance options. We also find parallels between the species in the geographic distributions of the polymorphisms: In both cases, the diazinon-resistance change is absent or rare outside Australasia where insecticide pressure is lower, whereas the changes associated with malathion resistance are widespread. Furthermore, PCR analysis of pinned specimens of Australasian L. cuprina collected before the release of organophosphate insecticides reveals no cases of the diazinon-resistance change but several cases of those associated with malathion resistance. Thus, the early outbreak of resistance in this species can be explained by the preexistence of mutant alleles encoding malathion resistance. The pinned specimen analysis also shows much higher genetic diversity at the locus before organophosphate use, suggesting that the subsequent sweep of diazinon resistance in Australasia has compromised the scope for the locus to respond further to the ongoing challenge of the insecticides.

Enzymatic bioremediation: from enzyme discovery to applications.

1. Enzymatic bioremediation is potentially a rapid method of removing environmental pesticide residues. Applications include the treatment of residues resulting from agricultural production and processing industries, such as the treatment of irrigation waters, surface-contaminated fruit and vegetables and spent dip liquors. 2. A specific application for some organophosphate-degrading enzymes involves detoxification of nerve agent stockpiles. Effective and affordable remediation requires highly specialized enzymes, so protein engineering techniques are being used to improve properties of various source enzymes to enhance catalytic rates, stability and substrate range. 3. Trials with an optimized organophosphate-degrading enzyme have shown the feasibility of such technology in various applications. 4. The enzymes developed for environmental remediation for specific pesticide classes also have applications as antidotes for high-dose pesticide poisonings and as prophylaxis for people at risk of high pesticide doses.

Hydrolysis of organophosphorus insecticides by in vitro modified carboxylesterase E3 from Lucilia cuprina.

Resistance of the blowfly, Lucilia cuprina, to organophosphorus (OP) insecticides is due to mutations in LcalphaE7, the gene encoding carboxylesterase E3, that enhance the enzyme's ability to hydrolyse insecticides. Two mutations occur naturally, G137D in the oxyanion hole of the esterase, and W251L in the acyl binding pocket. Previous in vitro mutagenesis and expression of these modifications to the cloned gene have confirmed their functional significance. G137D enhances hydrolysis of diethyl and dimethyl phosphates by 55- and 33-fold, respectively. W251L increases dimethyl phosphate hydrolysis similarly, but only 10-fold for the diethyl homolog; unlike G137D however, it also retains ability to hydrolyse carboxylesters in the leaving group of malathion (malathion carboxylesterase, MCE), conferring strong resistance to this compound. In the present work, we substituted these and nearby amino acids by others expected to affect the efficiency of the enzyme. Changing G137 to glutamate or histidine was less effective than aspartate in improving OP hydrolase activity and like G137D, it diminished MCE activity, primarily through increases in Km. Various substitutions of W251 to other smaller residues had a broadly similar effect to W251L on OP hydrolase and MCE activities, but at least two were quantitatively better in kinetic parameters relating to malathion resistance. One, W251G, which occurs naturally in a malathion resistant hymenopterous parasitoid, improved MCE activity more than 20-fold. Mutations at other sites near the bottom of the catalytic cleft generally diminished OP hydrolase and MCE activities but one, F309L, also yielded some improvements in OP hydrolase activities. The results are discussed in relation to likely steric effects on enzyme-substrate interactions and future evolution of this gene.

Developmental expression and gene/enzyme identifications in the alpha esterase gene cluster of Drosophila melanogaster.

Here we show how the 10 genes of the alpha esterase cluster of Drosophila melanogaster have diverged substantially in their expression profiles. Together with previously described sequence divergence this suggests substantial functional diversification. By peptide mass fingerprinting and in vitro gene expression we have also shown that two of the genes encode the isozymes EST9 (formerly ESTC) and EST23. EST9 is the major 'alpha staining' esterase in zymograms of gut tissues in feeding stages while orthologues of EST23 confer resistance to organophosphorus insecticides in other higher Diptera. The results for EST9 and EST23 concur with previous suggestions that the products of the alpha esterase cluster function in digestion and detoxification of xenobiotic esters. However, many of the other genes in the cluster show developmental or tissue-specific expression that seems inconsistent with such roles. Furthermore, there is generally poor correspondence between the mRNA expression patterns of the remaining eight genes and isozymes previously characterized by standard techniques of electrophoresis and staining, suggesting that the alpha cluster might only account for a small minority of the esterase isozyme profile.

Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution.

Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences.

Isolation and characterization of a Mycobacterium strain that metabolizes the insecticide endosulfan.

AIM: The aim of this study was to isolate and characterize a bacterium capable of metabolizing endosulfan. METHODS AND RESULTS: A endosulfan-degrading bacterium (strain ESD) was isolated from soil inoculum after repeated culture with the insecticide as the sole source of sulfur. Analysis of its 16S rRNA gene sequence, and morphological and physiological characteristics revealed it to be a new fast-growing Mycobacterium, closely related to other Mycobacterium species with xenobiotic-degrading capabilities. Degradation of endosulfan by strain ESD involved both oxidative and sulfur-separation reactions. Strain ESD did not degrade endosulfan when sulfite, sulphate or methionine were present in the medium along with the insecticide. Partial degradation occurred when the culture was grown, with endosulfan, in the presence of MOPS (3-(N-morpholino)propane sulphonic acid), DMSO (dimethyl sulfoxide), cysteine or sulphonane and complete degradation occurred in the presence of gutathione. When both beta-endosulfan and low levels of sulphate were provided as the only sources of sulfur, biphasic exponential growth was observed with endosulfan metabolism being restricted to the latter phase of exponential growth. CONCLUSIONS: This study isolated a Mycobacterium strain (strain ESD) capable of metabolizing endosulfan by both oxidative and sulfur-separation reactions. The endosulfan-degrading reactions are a result of the sulfur-starvation response of this bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: This describes the isolation of a Mycobacterium strain capable of degrading the insecticide endosulfan. This bacterium is a valuable source of enzymes for use in enzymatic bioremediation of endosulfan residues.

Development of a simple and sensitive fluorimetric method for isolation of coumaphos-hydrolysing bacteria.

AIMS: To develop a simple, rapid and sensitive fluorimetric assay to detect, isolate and characterize a soil bacterium capable of degrading the organophosphorus pesticide, coumaphos. METHODS AND RESULTS: A high throughput microtitre plate-based method was used to quantify coumaphos hydrolysis by the bacterium. The fluorescent hydrolysis product of coumaphos, chlorferon, was detected at levels as low as 10 nmol l(-1). Incorporation of coumaphos into agar plates allowed the rapid detection of coumaphos-hydrolysing bacteria when exposed to an excitation wavelength of approximately 340 nm. The coumaphos-hydrolysing enzyme could be visualized when bacterial cell extracts were separated on SDS-PAGE, incubated with coumaphos and exposed to an excitation source as above. CONCLUSIONS: This method is 100-fold more sensitive than the currently used spectrophotometric method for coumaphos. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a unique and versatile tool to screen for bacteria possessing phosphotriesterase activity.