Identification of a point mutation in the ace1 gene of Therioaphis trifolli maculata and detection of insecticide resistance by a diagnostic PCR-RFLP assay.

Aphids are important agricultural pests worldwide. Their control is largely based on chemical insecticides. One species that shows important invasive abilities and host-plant-related differences is Therioaphis trifolii (Monell) (Hemiptera: Aphididae). T. trifolii maculata, also known as spotted alfalfa aphid (SAA), can be very injurious to alfalfa crops in certain regions, such as in Saudi Arabia for effective control it is essential to diagnose and monitor the resistance mechanisms in the SAA populations. In the present study, we analysed acetylcholinesterase (ace) target site insensitivity mechanisms. A 650 bp length DNA containing the putative acetylcholinesterase (ace1) precursor was obtained and compared with other Hemipteran species. The sequences of many individual aphids collected from alfalfa crops in Saudi Arabia were analysed for the presence of resistance mutations: no resistance mutations were found at the resistance mutation loci 302; however, the presence of a serine-phenylalanine substitution (S431F) was identified in one individual. The S431F substitution, has been shown to confer significant levels of both organophosphate and carbamate resistance in other aphid species, and is now found for the first time in T. trifolii. We subsequently developed a simple polymerase chain reaction-restriction fragment length polymorphism assays for the S431F mutation, using a TaqI restriction site destroyed by the S431F mutation. The novel diagnostic assay may support the implementation of Insecticide Resistance Management strategies, for the control of SAA in alfalfa crops in the Kingdom of Saudi Arabia, and other countries worldwide.

Genetic diversity and insecticide resistance during the growing season in the green peach aphid (Hemiptera: Aphididae) on primary and secondary hosts: a farm-scale study in Central Chile.

The seasonal dynamics of neutral genetic diversity and the insecticide resistance mechanisms of insect pests at the farm scale are still poorly documented. Here this was addressed in the green peach aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) in Central Chile. Samples were collected from an insecticide sprayed peach (Prunus persica L.) orchard (primary host), and a sweet-pepper (Capsicum annum var. grossum L.) field (secondary host). In addition, aphids from weeds (secondary hosts) growing among these crops were also sampled. Many unique multilocus genotypes were found on peach trees, while secondary hosts were colonized mostly by the six most common genotypes, which were predominantly sensitive to insecticides. In both fields, a small but significant genetic differentiation was found between aphids on the crops vs. their weeds. Within-season comparisons showed genetic differentiation between early and late season samples from peach, as well as for weeds in the peach orchard. The knock-down resistance (kdr) mutation was detected mostly in the heterozygote state, often associated with modified acetylcholinesterase throughout the season for both crops. This mutation was found in high frequency, mainly in the peach orchard. The super-kdr mutation was found in very low frequencies in both crops. This study provides farm-scale evidence that the aphid M. persicae can be composed of slightly different genetic groups between contiguous populations of primary and secondary hosts exhibiting different dynamics of insecticide resistance through the growing season.

Comparing the genetic structure of codling moth Cydia pomonella (L.) from Greece and France: long distance gene-flow in a sedentary pest species.

Codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) is the most important insect pest of apple production in Europe. Despite the economic importance of this pest, there is not information about the genetic structure of its population in Greece and the patterns of gene-flow which might affect the success of control programs. In this study, we analysed nine samples from apple, pear and walnut from various regions of mainland Greece using 11 microsatellite loci. Six samples from the aforementioned hosts from southern France were also examined for comparison. Bayesian clustering and genetic distance analyses separated the codling moth samples in two genetic clusters. The first cluster consisted mainly of the individuals from Greece, and the second of those from France, although admixture and miss-classified individuals were also observed. The low genetic differentiation among samples within each country was also revealed by F(ST) statistics (0.009 among Greek samples and 0.0150 among French samples compared to 0.050 global value among all samples and 0.032 the mean of the pair-wise values between the two countries). These F(ST) values suggest little structuring at large geographical scales in agreement with previous published studies. The host species and local factors (climatic conditions, topography, pest control programs) did not affect the genetic structure of codling moth populations within each country. The results are discussed in relation to human-made activities that promote gene-flow even at large geographic distances. Possible factors for the genetic differentiation between the two genetic clusters are also discussed.

The invasion route for an insect pest species: the tobacco aphid in the New World.

Biological invasions are rapid evolutionary events in which populations are usually subject to a founder event during introduction followed by rapid adaptation to the new environment. Molecular tools and Bayesian approaches have shown their utility in exploring different evolutionary scenarios regarding the invasion routes of introduced species. We examined the situation for the tobacco aphid, Myzus persicae nicotianae, a recently introduced aphid species in Chile. Using seven microsatellite loci and approximate Bayesian computation, we studied populations of the tobacco aphid sampled from several American and European countries, identifying the most likely source populations and tracking the route of introduction to Chile. Our population genetic data are consistent with available historical information, pointing to an introduction route of the tobacco aphid from Europe and/or from other putative populations (e.g. Asia) with subsequent introduction through North America to South America. Evidence of multiple introductions to North America from different genetic pools, with successive loss of genetic diversity from Europe towards North America and a strong bottleneck during the southward introduction to South America, was also found. Additionally, we examined the special case of a widespread multilocus genotype that was found in all American countries examined. This case provides further evidence for the existence of highly successful genotypes or 'superclones' in asexually reproducing organisms.

A small deletion in the olive fly acetylcholinesterase gene associated with high levels of organophosphate resistance.

Organophosphate resistance in the olive fly was previously shown to associate with two point mutations in the ace gene. The frequency of these mutations was monitored in Bactrocera oleae individuals of increasing resistance. In spite of the difference in resistance among the individuals, there was no correlation between mutation frequencies and resistance level, indicating that other factors may contribute to this variation. The search for additional mutations in the ace gene of highly resistant insects revealed a small deletion at the carboxyl terminal of the protein (termed Delta3Q). Significant correlation was shown between the mutation frequency and resistance level in natural populations. In addition, remaining activity of acetylcholinesterase enzyme (AChE) after dimethoate inhibition was higher in genotypes carrying the mutation. These results strongly suggest a role of Delta3Q in high levels of organophosphate (OP) resistance. Interestingly, the carboxyl terminal of AChE is normally cleaved and substituted by a glycosylphosphatidylinositol (GPI) anchor. We hypothesize that Delta3Q may improve GPI anchoring, thus increasing the amount of AChE that reaches the synaptic cleft. In this way, despite the presence of insecticide, enough enzyme would remain in the cleft for its normal role of acetylcholine hydrolysis, allowing the insect to survive. This provides a previously un-described mechanism of resistance.

Distribution of common genotypes of Myzus persicae (Hemiptera: Aphididae) in Greece, in relation to life cycle and host plant.

Microsatellite genotyping was used to identify common clones in populations of the Myzus persicae group from various hosts and regions in mainland Greece and southern Italy and to compare their distribution and occurrence on tobacco and other crops. Common clones were defined as genotypes collected at more than one time or in more than one population; and, therefore, unlikely to be participating in the annual sexual phase on peach. Sixteen common genotypes were found, accounting for 49.0% of the 482 clonal lineages examined. Eight of these genotypes were subjected, in the laboratory, to short days and found to continue parthenogenetic reproduction, i.e. they were anholocyclic. Four of the six commonest genotypes were red, and one of these accounted for 29.6% of the samples from tobacco and 29.4% of those from overwintering populations on weeds. All six commonest genotypes were found on weeds and five of them both on tobacco and on other field crops. In mainland Greece, the distribution of common clones corresponded closely with that of anholocyclic lineages reported in a previous study of life cycle variation. Common genotypes were in the minority in the commercial peach-growing areas in the north, except on weeds in winter and in tobacco seedbeds in early spring, but predominated further south, away from peach trees. This contrasts with the situation in southern Italy, reported in a previous paper, where peaches were available for the sexual phase, yet all samples from tobacco were of common genotypes.

Genetic variation among Mediterranean populations of Sesamia nonagrioides (Lepidoptera: Noctuidae) as revealed by RFLP mtDNA analysis.

Restriction fragment length polymorphism analysis of two segments of mitochondrial DNA (COI and 16S rRNA) was used to examine genetic variation in Sesamia nonagrioides (Lefèbvre) populations from the Mediterranean basin. Four populations were collected from central and southern Greece, and five from northern latitudes: Greece, Italy, France and Spain. No variation was observed in COI, while 16S rRNA segment proved highly polymorphic and 28 different haplotypes were found. Lower intra-population polymorphism was found in the northern populations than in southern ones. Although no significant isolation by distance was found, the UPGMA tree based on Nei's raw number of nucleotide differences separated the populations into two major groups, i.e. one with the northern (40.6 degrees N-43.4 degrees N) and the other with the southern populations (37.3 degrees N-39.2 degrees N). Analysis of molecular variance revealed that most of the variation was between the two major groups (Phi(CT)=0.559), and all pairwise comparisons between the northern and southern populations resulted in high and significant F(ST) values (overall F(ST)=0.604). The high F(ST) values and the strong spatial genetic structure indicate that long-distance migration may be a rare event. The populations do not seem to have experienced a strong historical bottleneck. The occurrence of a few widespread haplotypes and the genetic similarity of the northern populations could be attributed to a historical expansion of certain haplotypes from the south towards to the northern borders of the species' distribution area.

Morphological discrimination of Aphis gossypii (Hemiptera: Aphididae) populations feeding on Compositae.

Aphis gossypii Glover is a polyphagous aphid pest with a worldwide distribution. However, there is evidence that on a global scale the name A. gossypii is being applied to a number of forms with different life cycles and/or host-plant associations. Morphometric variation of A. gossypii samples from crops and non-cultivated plants in many parts of the world was examined, to determine whether this variation is correlated with the hosts from which the aphids originated. Samples of A. gossypii were collected from Cucurbitaceae and Malvaceae in Europe, and from Compositae in various parts of the world. Morphometric data for 13 parameters measured from 97 clonal lineages (728 specimens) and 27 field-collected samples (313 specimens) were analysed by a series of canonical variates analyses, using the field sample/clonal lineage as grouping factor. Clonal lineages were reared on a common host in controlled conditions to standardize the effect of host and environment on morphology. The analyses provided a clear morphometric separation of the aphids originating from Compositae and those collected on Cucurbitaceae and Malvaceae, regardless of the geographical origin of the aphids and the host plant on which they were reared. This indicates that within A. gossypii there are two widely distributed host races or subspecies with different plant family associations. The taxonomic implications are discussed.

Genetic variation of Marchalina hellenica (Hemiptera: Margarodidae) sampled from different hosts and localities in Greece.

Random amplified polymorphic DNA (RAPD) analysis was applied to 120 individuals of Marchalina hellenica (Gennadius) representing six populations collected in northern, central and southern mainland Greece. One population was sampled on one species of fir tree and the others on two species of pine trees. Four random decamer primers were used to evaluate genetic variation among the populations examined. The results revealed intra- and interpopulation polymorphism both related to host type and region of origin. Phylogenetic analysis based on genetic distances estimated by the RAPD frequencies revealed an important genetic differentiation in samples collected on fir trees in southern Greece and to a lesser extent in samples from pine trees in central and northern Greece. Furthermore, considerable subdivision and restricted gene flow among the populations examined were observed. The results are discussed in relation to the biology and geographical distribution of M. hellenica in Greece.

Performance of Myzus persicae (Hemiptera: Aphididae) clones on different host-plants and their host preference.

The performance of eighteen clones of Myzus persicae (Sulzer) on pepper and tobacco plants at 20 degrees C and L16:D8 and the choice of young adult apterae between tobacco and pepper leaf-discs were examined. The clones were collected from weeds and peach in two tobacco-growing regions: Katerini, northern Greece and Karditsa, central Greece (only from weeds) and from Lehonia, central eastern Greece where tobacco is not cultivated. All clones did well on both hosts. However, the analysis of data revealed a significant effect of "region / host plant origin" on aphid performance. The mean values of adult weight, intrinsic rate of increase and fecundity of the clones collected in Lehonia and reared on tobacco were significantly lower than the observed values for clones from Katerini and Karditsa. Aphids from Lehonia had significantly higher mean values for developmental time on tobacco than clones from the other regions whereas the opposite was observed when aphids were reared on pepper. Aphids collected in Lehonia performed better on pepper than those originating from the tobacco-growing regions. A choice test revealed differences among the clones originating from different regions. Fifty three percent and 43% of aphids from weeds and peach from Lehonia, respectively, chose pepper. By comparison 41.5% and 40.0% of aphids from peach and weeds from Katerini, respectively and 49.5% of aphids from Karditsa preferred tobacco. The results are discussed in relation to host specialization in M. persicae.

Co-existence of different host-adapted forms of the Myzus persicae group (Hemiptera: Aphididae) in southern Italy.

Multivariate morphometric analysis (method of canonical variates) was used to compare 38 parthenogenetic lineages and three field collections of aphids of the Myzus persicae (Sulzer) group sampled on peach and tobacco in the Caserta region of southern Italy. Comparisons were also made with the morphology of lineages collected on peach in Lehonia, in central eastern Greece away from tobacco-growing regions, and on tobacco in Naphplion, in southern Greece. The lineages were measured after parthenogenetic rearing for one to three generations on the same host (potato) under constant conditions. As in previous work, the multivariate morphometric approach separated the aphids from peach from those feeding on tobacco. The life cycle category of the lineages was also examined. Almost all the lineages from peach in Caserta were holocyclic, yet the lineages from tobacco in the same region were unable to produce sexual morphs. The results suggest that tobacco-adapted and non-tobacco-adapted forms co-exist in the same region in southern Italy, not only because they colonize different host plants, but also because they have different life cycles. This is in complete contrast to the situation in peach-growing areas of northern Greece, and shows that the ecology and population structure of M. persicae is different in neighbouring counties of the Mediterranean area, even where climatic conditions and cultivated crops are similar.

Biological characteristics of the mirids Macrolophus costalis and Macrolophus pygmaeus preying on the tobacco form of Myzus persicae (Hemiptera: Aphididae).

Biological and population parameters of the predatory bugs Macrolophus costalis Fieber and Macrolophus pygmaeus Rambur preying on the tobacco aphid Myzus persicae (Sulzer) were examined. Tobacco was used as host plant and all experiments were carried out at 23 degrees C and L16:D8. In M. costalis, the developmental time for eggs and the total duration of nymphal instars was 13.1 and 22.2 days and in M. pygmaeus 13.0 and 20.2 days, respectively. Both species completed their nymphal development feeding only on plant juices although they required significantly more time to attain adulthood than when feeding on prey. The longevity of M. costalis females and the preoviposition period were 49.9 and 5.6 days and those of M. pygmaeus were 50.3 and 6.3 days, respectively. Adult males showed a higher longevity than females in both species. The intrinsic rate of increase and the mean total number of eggs laid were 0.0644 and 121 in M. costalis and 0.0615 and 104 in M. pygmaeus, respectively. Mean total aphid consumption by nymphs of M. costalis was 61 aphids, about twice that of M. pygmaeus (37 aphids). Total aphid consumption by a male and female pair of M. costalis and M. pygmaeus until the death of the female was 244 and 285 aphids, respectively. The results of the study are discussed in relation to the impact of the two predatory bugs on tobacco aphid populations.

Discrimination of the closely related biocontrol agents Macrolophus melanotoma (Hemiptera: Miridae) and M. pygmaeus using mitochondrial DNA analysis.

The separation of the closely related predatory species Macrolophus melanotoma Costa (= M. caliginosus Wagner) and Macrolophus pygmaeus (Rambur) based exclusively on the different colour pattern of the first antennal segment (white central band in M. melanotoma and entirely black in M. pygmaeus) is rather precarious and their taxonomic status is still in doubt. In the present study their taxonomic status was evaluated by DNA confirmatory analysis and hybridization experiments between M. pygmaeus and a Macrolophus taxon, resembling M. melanotoma, with a first antennal segment entirely black or with a white central band collected from Dittrichia viscosa (L.) W. Greuter (Asteraceae) in southern Greece. Adult females from Dittrichia plants hybridized with males of M. pygmaeus and vice versa did not produce viable eggs. The Macrolophus species from Dittrichia irrespective of the first antennal segment coloration differed from M. pygmaeusin digestive patterns generated by AseI, XbaI, and MseI on 16S rRNA and in RAPD profiles produced by the primers OPA-18 and OPA-20. These results demonstrate that on Dittrichia plants there is a distinct dimorphic taxon, M. melanotoma, as it is the only species of the genus Macrolophus bearing a first antennal segment with a central white band. Given the limitation of the coloration pattern, the mtDNA genetic markers are the appropriate method for the identification of M. melanotomaand M. pygmaeus.

Variation in the life cycle and morphology of the tobacco host-race of Myzus persicae (Hemiptera: Aphididae) in relation to its geographical distribution.

Morphological variation and life cycle category were examined in 121 clones of Myzus persicae (Sulzer). The clones were collected from tobacco from three localities in Greece (Xanthi, Nea Efessos and Naphplion), one in Germany (Rheinstetten), one in France (Bergerac) and one in Spain (Madrid). Before morphometrics, all aphids were laboratory-reared on potato. The morphological variation was investigated using both canonical variates analysis and a novel non-parametric classification tree method. The life cycle category was examined by rearing the clones for three generations under short day conditions. In Nea Efessos a relative high proportion of clones was found to overwinter as eggs on the primary host. In the other regions all collected clones were non-holocyclic. Intermediate genotypes were found in all regions at percentages ranging from 4.0 to 24.0%. Androcyclic clones were found only in Xanthi, Greece (4.0%) and Rheinstetten, Germany (16.7%). The canonical variates analysis and the tree classification method revealed important intrapopulation polymorphisms in clones from Bergerac, Nea Efessos and Madrid. Both methods separated the populations originating from Greece from those collected elsewhere in western Europe. The observed morphological variation was probably due to genetic differences, since all clones were reared in a common environment. The results are discussed in relation to factors responsible for genetic divergence in M. persicae populations.

Life cycle variation of Myzus persicae (Hemiptera: Aphididae) in Greece.

During the years 1995-1999 the life cycle category of 2797 clones of Myzus persicae (Sulzer) was examined. The clones originated from primary and secondary hosts from different localities of North and Central Greece and the island of Crete in the south. Four different overwintering life cycle strategies were found that have also been described for M. persicae and other heteroecious species previously. A geographical variation was found in the proportion of holocyclic clones from tobacco and other secondary hosts associated with the abundance of the primary host in the sampling regions. In Central Macedonia, around the main peach-growing regions, the proportion of holocyclic clones was mostly above 50% and in some cases reached 100%. In localities of East Macedonia, holocyclic clones were also frequent. On the other hand, further south or in north-eastern Greece, where peach is not common, the proportion of holocyclic clones varied between 0 and 33%. Fifty seven percent of examined anholocyclic clones produced males under short day conditions, suggesting that androcyclic clones in Greece represent an important factor of genetic variability. Intermediate clones were sampled from all host-plants but at low frequencies (3.6% of total examined clones and 6.9% of non-holocyclic ones). Moreover, a regional variation was found in different colour forms feeding on tobacco plants. Red clones were predominant in regions where aphids overwinter parthenogenetically on weeds or winter crops. However, almost all clones from the primary host were green. The ecological aspects of life cycle variation are discussed.

Production of sexual morphs by apterous virginoparae of Myzus persicae (Hemiptera: Aphididae) in relation to pre- and postnatal exposure to short day conditions.

The production of males and females by apterae of a holocyclic clone of Myzus persicae (Sulzer) was studied in the laboratory by transferring apterous virginoparae from long day to short day conditions at different stages of their pre- and post-natal development. Prenatal exposure was also examined in nine additional clones coming from different regions of Greece. By increasing the number of short day cycles at 17 degrees C, the aphids switched to male production earlier and produced more males. The highest mean number of male progeny was observed in apterae that were transferred to short day conditions 13-14 days prenatally. Apterae that were transferred to short day conditions after the fourth nymphal instar produced only females. Apterous virginoparae were produced by aphids that were transferred to short day conditions 0-1 days prenatally or postnatally. Aphids, given short days prenatally or in the first instar, produced alate female progeny that were all gynoparae. The later the instar aphids were transferred to short day conditions the fewer the gynoparae produced among their female progeny. A reduction in the total number of progeny and embryos per ovariole was observed as aphids were transferred to short day conditions in earlier stages of their development. The increase in male production, which follows the increase in the duration of long night exposure, was also observed in the additional eight out of nine examined clones.

A modified method for the detection of differentially expressed mRNAs without using radioactivity.

We present here a modification of the original differential display approach using a single oligo(dT) primer for the reverse transcription reaction (instead of the various oligo(dT)NM primers that subdivide the pool of mRNAs) and a combination of 25-mer or 26-mer arbitrary primers together with 30-mer anchored primers for the PCR reaction. The PCR products are, then, efficiently separated in a non-denaturing polyacrylamide gel and the bands are visualized after staining with silver nitrate. The model for the development of our differential display approach was seven clones of an insect species: the aphid Myzus pesicae (Sulzer) (Homoptera: Aphididae). We believe that our modified differential display technique, with the efficient resolution of the DNA bands in a non-denaturing gel and staining with silver can be applied as an alternative non-radioactive detection of differentially expressed messages in various cell populations. In addition, the method could be used as a supplementary tool to other techniques for examining inter- and intraspecific genetic variation in aphids.

Host-correlated morphological variation of Myzus persicae (Hemiptera: Aphididae) populations in Greece.

Morphological variation in nine characters of 157 clones of Myzus persicae (Sulzer) was examined by multivariate analysis. The clones were collected from peach, Prunus persica, the primary host and the secondary hosts tobacco, Nicotiana tabacum, cabbage, Brassica oleracea, sugarbeet, Beta vulgaris and pepper Capsicum annuum. The 156 clones originated from various regions of Greece, both in the north, where a large part of the population has an annual bisexual generation on peach, and in more southerly regions, where populations are predominantly unisexual. One clone was collected from tobacco in Caserta, Italy. All clones were laboratory-reared on potato. Canonical variate analysis, hierarchical cluster analysis and a non-parametric classification tree method both revealed morphological differences associated with the host-plant on which they were collected. The scores of the first two canonical variates separated the tobacco-feeding clones from those originating from other secondary host-plants. However, in tobacco-growing areas the tobacco-feeding form predominated in spring populations on peach, and was sometimes found on other secondary hosts. In addition, using cluster analysis, the clones from tobacco which were sampled in the most southeasterly region showed a relatively large phenotypic distance from those collected further north and west. Moreover, clonal phenotypes were affected both by host plant and by long-term parthenogenetic rearing. However, in spite of these effects, the tobacco form was generally distinguishable from aphids originating from other hosts, indicating that the difference must have a genetic basis. In separate analyses of the clones originating from secondary hosts no association was found between morphology and either life cycle category or colour. Discriminant analysis showed that 89% of 1723 specimens could be correctly classified into the two groups.