Effect of Wolbachia on insecticide susceptibility in lines of Aedes aegypti.

Two stable infections of Wolbachia pipientis, wMelPop and wMel, now established in Aedes aegypti, are being used in a biocontrol program to suppress the transmission of dengue. Any effects of Wolbachia infection on insecticide resistance of mosquitoes may undermine the success of this program. Bioassays of Ae. aegypti were conducted to test for differences in response to insecticides between Wolbachia infected (wMelPop, wMel) and uninfected lines. Insecticides screened were bifenthrin, the pyrethroid commonly used for adult knockdown, as well as larvicides: Bacillus thuringiensis var. israelensis, the organophosphate, temephos and the insect growth regulator, s-methoprene. While differences in response between lines were detected for some insecticides, no obvious or consistent effects related to presence of Wolbachia infection were observed. Spreading Wolbachia infections are, therefore, unlikely to affect the efficacy of traditional chemical control of mosquito outbreaks.

Changes in the genetic structure of Aedes aegypti (Diptera: Culicidae) populations in Queensland, Australia, across two seasons: implications for potential mosquito releases.

Diseases transmitted by mosquitoes could be controlled if vector populations were replaced with strains that have reduced vector competency. Such a strategy is being developed for control of dengue virus which is transmitted by Aedes aegypti (L.) (Diptera: Culicidae). Mosquitoes artificially infected with the bacterium, Wolbachia pipientis Hertig, are being assessed as candidates for release at the adult stage with the aim of replacement of the wild population. Wolbachia can reduce the capacity of Ae. aegypti to transmit dengue virus and has potential to be driven through the natural population via a system of cytoplasmic incompatibility. Deployment of benign mosquito strains will be influenced by population size and structure of wild-type Ae. aegypti in proposed release areas, as well as rates of gene flow among populations in the wet and dry tropical seasons. Mosquitoes from northern Queensland were screened with genetic markers to find an optimal locality for release of a benign strain of Ae. aegypti. The inland towns of Chillagoe and Charters Towers and the coastal town of Ingham had mosquito populations that were partly genetically isolated from mosquitoes in other areas across both seasons. These locations may be suitable release sites if it is important for the released strain to be restricted during initial phases of implementation. Smaller genetic differences were also evident among other regions and were consistent over two seasons (wet and dry).

Widespread pyrethroid resistance in Australian diamondback moth, Plutella xylostella (L.), is related to multiple mutations in the para sodium channel gene.

Populations of Plutella xylostella, extending over 3800 km in southern Australia, show no genetic structure as assessed by microsatellite markers; yet outbreaks of pyrethroid resistance occur sporadically in cropping areas. Since mutations in the para voltage-gated sodium channel gene have been implicated in pyrethroid resistance, we looked for DNA sequence variation at this target among Australian moths. We found two resistance mutations previously reported for this species (L1014F and T929I), as well as a novel substitution (F1020S). Of the eight possible haplotypes formed by combinations of these three biallelic polymorphisms, only four were found in Australian populations: the wild-type allele (w), the kdr mutation allele (kdr) with only L1014F, the super-kdr-like combination of L1014F and T929I (skdrl), and the crashdown allele with only F1020S (cdr). Comparison of genotype frequencies among survivors of permethrin assays with those from untreated controls identified three resistant genotypes: skdrl homozygotes, cdr homozygotes and the corresponding heterozygote, cdr/skrdl - the heterozygote being at least as resistant as either homozygote. Spatial heterogeneity of allele frequencies was conspicuous, both across the continent and among local collections, consistent with reported spatial heterogeneity of pyrethroid resistance. Further, high resistance samples were sometimes associated with high frequency of cdr, sometimes high frequency of skdrl, or sometimes with a high combined cdr+skdrl frequency. The skdrl and cdr alleles explain a high proportion of the Australia-wide resistance variation. These data add to evidence that nerve insensitivity by mutations in the para-sodium channel gene is a common pyrethroid resistance mechanism in P. xylostella.

Dynamics of the "popcorn" Wolbachia infection in outbred Aedes aegypti informs prospects for mosquito vector control.

Forty percent of the world's population is at risk of contracting dengue virus, which produces dengue fever with a potentially fatal hemorrhagic form. The wMelPop Wolbachia infection of Drosophila melanogaster reduces life span and interferes with viral transmission when introduced into the mosquito Aedes aegypti, the primary vector of dengue virus. Wolbachia has been proposed as an agent for preventing transmission of dengue virus. Population invasion by Wolbachia depends on levels of cytoplasmic incompatibility, fitness effects, and maternal transmission. Here we characterized these traits in an outbred genetic background of a potential target population of Ae. aegypti using two crossing schemes. Cytoplasmic incompatibility was strong in this background, and the maternal transmission rate of Wolbachia was high. The infection substantially reduced longevity of infected adult females, regardless of whether adults came from larvae cultured under high or low levels of nutrition or density. The infection reduced the viability of diapausing and nondiapausing eggs. Viability was particularly low when eggs were laid by older females and when diapausing eggs had been stored for a few weeks. The infection affected mosquito larval development time and adult body size under different larval nutrition levels and densities. The results were used to assess the potential for wMelPop-CLA to invade natural populations of Ae. aegypti and to develop recommendations for the maintenance of fitness in infected mosquitoes that need to compete against field insects.

Genetic variation among Helicoverpa armigera populations as assessed by microsatellites: a cautionary tale about accurate allele scoring.

The existence of genetic differences among Australian populations of the pest moth Helicoverpa armigera based on microsatellite markers is contentious. To resolve this issue, we analyzed microsatellite variation in moth samples from multiple locations simultaneously in two laboratories that have previously reported contrasting patterns. Alleles and allele numbers detected in the laboratories differed, as did the genetic differences found between the samples. The automated scoring system used in one of the laboratories combined with non-denaturing polyacrylamide gels led to inaccurate identification of alleles and high FST values between the populations. However, H. armigera in Australia is probably not structured geographically, with high gene flow between populations. This influences management of H. armigera and the development of area-wide control options, as populations need to be considered as one panmictic unit. The results also highlight potential problems of automated scoring systems when these are not checked carefully.

Genetic structure of Aedes aegypti in Australia and Vietnam revealed by microsatellite and exon primed intron crossing markers suggests feasibility of local control options.

The distribution of Aedes aegypti (L.) in Australia is currently restricted to northern Queensland, but it has been more extensive in the past. In this study, we evaluate the genetic structure of Ae. aegypti populations in Australia and Vietnam and consider genetic differentiation between mosquitoes from these areas and those from a population in Thailand. Six microsatellites and two exon primed intron crossing markers were used to assess isolation by distance across all populations and also within the Australian sample. Investigations of founder effects, amount of molecular variation between and within regions and comparison of F(ST) values among Australian and Vietnamese populations were made to assess the scale of movement ofAe. aegypti. Genetic control methods are under development for mosquito vector populations including the dengue vector Ae. aegypti. The success of these control methods will depend on the population structure of the target species including population size and rates of movement among populations. Releases of modified mosquitoes could target local populations that show a high degree of isolation from surrounding populations, potentially allowing new variants to become established in one region with eventual dispersal to other regions.

Assessing the susceptibility of cruciferous Lepidoptera to Cry1Ba2 and Cry1Ca4 for future transgenic cruciferous vegetables.

Advances in transgenic plants expressing Bacillus thuringiensis (Bt) insecticidal gene(s) offer a promising alternative to traditional insecticides for control of lepidopteran pests on important cruciferous vegetable crops such as cabbage and cauliflower. A public-private partnership, the Collaboration on Insect Management for Brassicas in Asia and Africa (CIMBAA), was formed in 2005 with the goal of developing dual-gene Bt cauliflower and cabbage, initially for India, to replace the use of broad spectrum, traditional insecticides. As a first step in this effort, the major lepidopteran pests of cruciferous vegetable crops [Plutella xylostella (L.), Pieris rapae (L.), Pieris brassicae (L.), Crocidolomia binotalis (L.), Hellula undalis (F.), Diacrisia obliqua Walker, Spodoptera litura F., and Helicoverpa armigera (Hübner)] were collected over a large geographic area (India, Indonesia, Taiwan, China, Australia, and the United States) and tested against purified Cry1Ba2 and Cry1Ca4 toxins, the toxins proposed to be expressed in the CIMBAA plants. Our results demonstrate that Cry1Ba2 and Cry1Ca4 were effective against the primary target of the CIMBAA plants, P. xylostella, regardless of geographic location, and had LC50 values <1.3 ppm. Furthermore, one or both toxins were effective against the other major pest Lepidoptera, except for S. litura or H. armigera which were less susceptible. No cross-resistance has been found between Cry1Ba2 and Cry1Ca4, suggesting cry1Ba2+cry1Ca4 cauliflower and cabbage could be an effective and sustainable tool to control, P. xylostella, the key lepidopteran pest on cruciferous vegetable crops, as well as most other Lepidoptera. As the CIMBAA plants are being developed, further tests are needed to determine whether they will express these proteins at sufficient levels to control all the Lepidoptera. Sustainable use of the dual-gene plants also is discussed.

The effects of local selection versus dispersal on insecticide resistance patterns: longitudinal evidence from diamondback moth (Plutella xylostella (Lepidoptera: Plutellidae)) in Australia evolving resistance to pyrethroids.

When strong directional selection acts on a trait, the spatial distribution of phenotypes may reflect effects of selection, as well as the spread of favoured genotypes by gene flow. Here we investigate the relative impact of these factors by assessing resistance to synthetic pyrethroids in a 12-year study of diamondback moth, Plutella xylostella, from southern Australia. We estimated resistance levels in populations from brassicaceous weeds, canola, forage crops and vegetables. Differences in resistance among local populations sampled repeatedly were stable over several years. Levels were lowest in samples from weeds and highest in vegetables. Resistance in canola samples increased over time as insecticide use increased. There was no evidence that selection in one area influenced resistance in adjacent areas. Microsatellite variation from 13 populations showed a low level of genetic variation among populations, with an AMOVA indicating that population only accounted for 0.25% of the molecular variation. This compared to an estimate of 13.8% of variation accounted for by the resistance trait. Results suggest that local selection rather than gene flow of resistance alleles dictated variation in resistance across populations. Therefore, regional resistance management strategies may not limit resistance evolution.

Microsatellites reveal a lack of structure in Australian populations of the diamondback moth, Plutella xylostella (L.).

The diamondback moth, Plutella xylostella, is renowned for developing resistance to insecticides and causing significant economic damage to Brassica vegetable crops throughout the world. Yet despite its economic importance, little is known about the population structure and movement patterns of this pest both at local and regional scales. In Australia, the movement patterns and insecticide resistance status of P. xylostella infesting canola, vegetables, forage brassicas and weeds have fundamental implications for the management of this pest. Here we use six polymorphic microsatellite loci to investigate population structure and gene flow in Australian populations of P. xylostella. Samples of P. xylostella from New Zealand, Malaysia, Indonesia and Kenya were also scored at these loci. We found no evidence of population structure within Australia, with most populations having low inbreeding coefficients and in Hardy-Weinberg equilibrium. In addition, a sample from the North Island of New Zealand was indistinguishable from the Australian samples. However, large genetic differences were found between the Australia/New Zealand samples and samples from Kenya, Malaysia and Indonesia. There was no relationship between genetic distance and geographic distance among Australian and New Zealand samples. Two of the loci were found to have null alleles, the frequency of which was increased in the populations outside the Australia/New Zealand region. We discuss these results with reference to insecticide resistance management strategies for P. xylostella in Australia.

Evaluation of fixed sample-size plans for Plutella xylostella (Lepidoptera: Plutellidae) on broccoli crops in Australia.

Fixed sample-size plans for monitoring Plutella xylostella (L.) (Lepidoptera: Plutellidae) on broccoli and other Brassica vegetable crops are popular in Australia for their simplicity and ease of application. But the sample sizes used are often small, approximately 10-25 plants per crop, and it may be that they fail to provide sufficient information upon which to base pest control decisions. We tested the performance of seven fixed sample-size plans (10, 15, 20, 30, 35, 40, and 45 plants) by resampling a large data set on P. xylostella in commercial broccoli crops. For each sample size, enumerative and presence-absence plans were assessed. The precision of the plans was assessed in terms of the ratio of the standard error to the mean; and at least 45 and 35 samples were necessary for the enumerative and presence-absence plans, respectively, to attain the generally accepted benchmark of < or = 0.3. Sample sizes of 10-20 were highly imprecise. We also assessed the consequences of classifications based on action thresholds (ATs) of 0.2 and 0.8 larvae per plant for the enumerative case, and 0.15 and 0.45 proportion of plants of infested for the presence-absence case. Operating characteristic curves and investigations of the frequency of correct decisions suggest improvements in the performance of plans with increased sample size. In both the enumerative and presence-absence cases, the proportion of incorrect decisions was much higher for the lower of the two ATs assessed, and type II errors (i.e., failure to suggest pest control upon the AT is exceeded) generally accounted for the majority of this error. Type II errors are the most significant from a producer's standpoint. Further consideration is necessary to determine what is an acceptable type II error rate.

A dynamic binomial sequential sampling plan for Plutella xylostella (Lepidoptera: Plutellidae) on broccoli and cauliflower in Australia.

Binomial sequential sampling plans have been used widely for monitoring invertebrate pest populations. Such plans are typically based upon a single action threshold (AT), which represents the level of infestation that the grower is prepared to accept before using a control measure. For many cropping systems this acceptable infestation level is likely to vary, being dependent on factors such as the growth stage of the crop and the value or demands of the destination market (e.g., local or high-quality export). We developed and validated a computer-assisted plan that uses a dynamic AT. The plan has been developed for monitoring diamondback moth, Plutella xylostella (L.) on broccoli (Brassica oleracea variety botrytis L.) and cauliflower (Brassica oleracea variety botrytis L.), but the concepts and methodologies could be readily applied to other systems.