Microsatellite markers reveal spatial genetic structure of Tetranychus urticae (Acari: Tetranychidae) populations along a latitudinal gradient in Europe

The genetic structure of populations of the two-spotted spider mite Tetranychus urticae was investigated along a south–north European transect spanning from southern France to The Netherlands. Mites were collected on Urtica dioica in 6 sampling zones. Microsatellite variation at 5 loci revealed considerable genetic variation with an average heterozygozity of 0.49. Significant heterozygote deficiency was found in 7 populations out of the 18 samples analyzed and one of them was completely monomorphic. Tetranychus urticae populations show some level of genetic structuring. First, genetic differentiation between localities (F _ST estimates) was significant for all comparisons. Second, the analysis of molecular variance, AMOVA, indicates that there is an effect, albeit low (9%), of the locality in accounting for allele frequency variance. Geographic distance emerges as a factor responsible for this genetic structure. The results are discussed in relation to the biological features of the species and the known patterns of migration. Related agronomical issues are addressed.     

Rapid development of 36 polymorphic microsatellite markers for Tetranychus truncatus by transferring from Tetranychus urticae

Tetranychus truncatus Ehara is a phytophagous spider mite that is now one of the most important pests of agricultural and economic crops in East and Southeast Asia. However, population genetics and other studies of T. truncatus have been impeded by the lack of microsatellite markers, which are expensive and time-consuming to identify. Previous studies indicated a high potential of cross-amplification of microsatellites in Tetranychus species, meaning that the microsatellite flanking sequences are sufficiently homologous among Tetranychus species that the primers for one species may work in another species. Here, we tested 205 primer pairs designed from the whole genome sequence of Tetranychus urticae Koch, a sister species of T. truncatus, for microsatellite markers in three populations of T. truncatus in China (N = 94). About half (102) of these primer pairs yielded the desired PCR products, 36 of which revealed polymorphism in T. truncatus. Each of the 36 markers harbored between 2 and 23 alleles, with a mean polymorphic information content of 0.589 (0.119–0.922 range). The mean observed and expected heterozygosity across loci and the three populations were 0.468 and 0.628, respectively. Of the 36 primer pairs, 22 also worked in Tetranychus piercei, but only a few of them worked in T. ludeni and T. phaselus. Cross-amplification is thus a cost-effective way to develop microsatellite markers, which can be of great value in population genetics studies.     

Isolation and characterization of microsatellite loci in Ononis repens,Leguminosae

Ten polymorphic microsatellites were isolated from Ononis repens, using a microsatellite enrichment protocol and selective hybridization with a mixed synthetic dinucleotide repeat probe. Primers for DNA amplifications using polymerase chain reaction (PCR) were designed and synthesized. The loci amplified in both O. repens and O. spinosa. All 10 loci were polymorphic in each of four populations studied. These microsatellite markers will be useful for population genetic analysis and biodiversity studies of O. repens and O. spinosa.     

Evaluation of Resistance Pattern to Fenpyroximate and Pyridaben in Tetranychus Urticae collected from Ggreenhouses and Apple Orchards using Lethal Concentration-slope Relationship

This study aimed to monitor the present and future developments of the resistance of Tetranychus urticae Koch to fenpyroximate and pyridaben, using the relationship of the LC₅₀ and slope of the concentration-mortality line in a probit model, for the provision of reliable resistance management tactics. Tetranychus urticae populations were collected from 16 commercial greenhouses, where various crops were cultivated, as well as from 10 apple orchards throughout Korea. The resistance to fenpyroximate and pyridaben of each population was estimated by calculating the median lethal concentration (LC₅₀), resistance ratio (RR) and slope of the concentration-mortality regression. Most of the greenhouse populations exhibited moderate levels of resistance, whereas the apple orchard populations showed only low levels, indicating that T. urticae populations in greenhouses were more strongly selected than those in apple orchards. Four population groups were established based on either the habitats (greenhouse and apple orchard) or acaricides (fenpyroximate and pyridaben). To test the hypothesis, “the slope is greatest at low and high levels of resistance,” the slopes were regressed as a function of the LC₅₀, and fitted to a polynomial regression. The polynomial regression model explained this relationship well for the four population groups (p < 0.05), indicating that the development of resistance toward fenpyroximate or pyridaben was consistent with the gradient. A laboratory selection study agreed with the results from both acaricide field populations. These results suggest that the gradient was a good indicator of the susceptibility of T. urticae to genetic variations, which was related to the LC₅₀. The application of these findings is also discussed in relation to the resistance management of T. urticae.     

Life-history of predatory mites Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae) on four spider mite species as prey, with special reference to Tetranychus evansi (Acari: Tetranychidae)

The commercially available strains of Phytoseiulus persimilis Athias-Henriot, the biological control agent of Tetranychus urticae Koch, perform poorly in the Western Mediterranean, probably because they are not well adapted to local climatic conditions. For that reason, efforts are being focused on the development of a biological control programme using native phytoseiid mites. Four species of red spider mites can be found in vegetable crops in eastern Spain: T. urticae, Tetranychus turkestani Ugarov and Nikolski, Tetranychus ludeni Zacher and the recently introduced Tetranychus evansi Baker and Pritchard. To evaluate their potential role as biological control agents, the present study evaluates the life-history of local populations of Neoseiulus californicus (McGregor) and P. persimilis when fed on T. urticae, T. turkestani, T. evansi, and T. ludeni in the laboratory. Results indicate that N. californicus and P. persimilis are able to feed and complete their development on the four tested red spider mite species. The predators may exhibit a particularly high capacity for population increase when fed on T. urticae, T. turkestani, and T. ludeni, thus may be able to provide effective control of these species in the field. When fed T. evansi, however, predator performance was poor; significant increase in development and preoviposition times, and a reduction in oviposition period and fecundity were recorded. The resultant low capacity for population growth suggests poor ability of the two tested predators to suppress T. evansi populations on commercial crops. It is unlikely therefore that P. persimilis and N. californicus, now being widely used to control T. urticae in greenhouse crops in Central Europe, will be able to halt any spread of T. evansi to greenhouse crops in temperate areas.     

Establishment of an acaricide-susceptible Tetranychus urticae strain and its species confirmation based on morphological and molecular characters

Acquisition of a reference Tetranychus strain that is completely susceptible to acaricides and retains an identical genetic background to acaricide-resistant strains is an essential step in elucidating mechanisms of resistance. To establish a strain completely susceptible to various acaricides, we collected Tetranychus mite populations from several regions in South Korea, including both remote and heavily cultivated regions. We tested their suitability as a susceptible reference strain by determining baseline susceptibility to six acaricides and by determining species identity as Tetranychus urticae. The UD strain, originally collected from a remote island region, was found to be most susceptible to all five major acaricides tested and was confirmed to as T. urticae on the basis of both morphological and molecular evidence. Moreover, molecular phylogenetic analysis revealed that the UD strain is an ancestral strain of other prevalently collected green-type strains. Taken together, we propose that the UD strain can be used as a susceptible reference strain for T. urticae resistance studies as it provides baseline susceptibility to acaricides and possesses a common genetic background with most other acaricide-resistant strains.     

Does host adaptation of Tetranychus urticae populations in clementine orchards with a Festuca arundinacea cover contribute to a better natural regulation of this pest mite?

Tetranychus urticae Koch (Acari: Tetranychidae) is a key pest of clementine mandarins, Citrus clementina Tanaka (Rutaceae), in Spain. This mite is highly polyphagous and can be easily found in clementine orchards, both in the trees and in the associated flora. In a previous study we found that the use of a cover of Festuca arundinacea Schreber (Poaceae) offered a better regulation of T. urticae populations than either bare soil or the traditional wild cover, which included a mix of weed species. We hypothesized that the selection of two host races of T. urticae, specialized in F. arundinacea and C. clementina, could partly explain the results obtained (bottom‐up regulation). Reciprocal transplant experiments show that sympatric deme × host combinations had higher mean fitness values than the allopatric combinations in clementine, but not in F. arundinacea, for most of the fitness parameters evaluated in our study. Because local adaptation implies mean deme fitness to be systematically higher for the sympatric deme × habitat combinations than for the allopatric ones, these results can be taken as indicative of occurrence of local adaptation in T. urticae. Molecular genetic analyses with microsatellite markers support this conclusion and indicate that local adaptation of T. urticae found in our system may indeed contribute to a better natural regulation of this mite.     

Development and characterization of ten novel microsatellite markers from olive ridley sea turtle (Lepidochelys olivacea)

Olive ridleys, one of the widely distributed marine turtle species has undergone declines in recent years due to multiple anthropogenic factors warranting conservation efforts for which assessment of genetic variability in existing populations become critical. Here we describe development of ten new microsatellite markers from a short sequence repeat-enriched partial genomic DNA library, which are found to be highly informative for genetic studies. Eight of these markers when tested on 83 olive ridley turtles revealed high allelic diversity (4–27 alleles per marker), and high observed and expected heterozygosity estimates that ranged from 0.29 to 0.82 and 0.62 to 0.94, respectively. Two microsatellites were monomorphic in the tested olive ridley samples, but were found to be informative/polymorphic when tested on related marine turtle species. More importantly, nine of the new markers showed robust cross-species amplifications in three related species Dermochelys coriacea, Chelonia mydas and Eretmochelys imbricata. Thus, this study describes ten new microsatellite markers and also demonstrates their potential as efficient genetic markers in studies related to parentage analysis, population structure, phylogeography and species relationships of olive ridleys and other marine turtle species.     

Effects of feeding frequency and sugar concentration on behavior and longevity of the adult aphid parasitoid: Aphidius ervi (Haliday) (Hymenoptera: Braconidae)

The effectiveness of the predatory mite, Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseidae), as a suppressive agent of the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), was evaluated on greenhouse ivy geraniums at predator:prey release ratios of 1:60, 1:20, and 1:4. Releases at each predator:prey ratio were made at moderate and high T. urticae densities to determine if initial pest population size influenced the suppressive ability of the predator. At ratios of 1:4 and 1:20, P. persimilis significantly reduced T. urticae populations 1 week after release and kept them at low levels thereafter. Plant damage also was significantly reduced at these densities. After 4 weeks, the P. persimilis that were released at a ratio of 1:4 consistently reduced T. urticae populations from densities as high as 30 T. urticae per leaf to fewer than 0.6 per leaf. We found no interaction between release ratio and T. urticae density, indicating that predator effectiveness remains constant, at least within the range of T. urticae densities used. Our work demonstrates the potential of P. persimilis to provide effective control of T. urticae on a greenhouse-grown floricultural crop at a moderately low predator:prey ratio (1:20) and over a range of initial pest densities. However, we recommend that P. persimilis be released at a ratio of 1:4 for greatest reliability and successful control of T. urticae on ivy geraniums.     

Isolation and characterization of microsatellite loci in Geum urbanum (Rosaceae) and their transferability within the genus Geum

Thirteen novel polymorphic microsatellite loci are presented for Geum urbanum (Rosaceae). The microsatellites will be useful tools to analyse the influence of landscape structure and land‐use intensity in agricultural landscapes on genetic diversity within and among populations of Geum urbanum. Transferability was tested in 19 other Geum species and two Waldsteinia species. In most species polymerase chain reaction (PCR) products of the expected range were obtained, therefore the markers reported here appear to be applicable across the whole genus.     

The fine-scale genetic structure of the two-spotted spider mite in a commercial greenhouse

The fine-scale genetic structure of Tetranychus urticae Koch was studied to estimate local gene flow within a rose tree habitat in a commercial greenhouse using seven microsatellite markers. Two beds of rose trees with different population densities were selected and 18 consecutive quadrats of 1.2 m length were sequentially established in each bed. Heterozygote deficiency was positive within quadrats, which was most likely a result of the Wahlund effect because the mites usually form small breeding colonies. Low population density and frequent inbreeding could also accelerate genetic differentiation among the breeding colonies. A short-range (2.4–3.6 m) positive autocorrelation and clear genetic cline among quadrat populations was detected within a bed. This suggests that gene flow was limited to a short range even if population density was substantially increased. Therefore, large-scale dispersal such as aerial dispersal contributed very little to gene flow in the greenhouse.     

Pre-adult development of Phytoseiulus persimilis on diets of Tetranychus urticae and Tetranychus lintearius: implications for the biological control of Ulex europaeus

Predation by the phytoseiid mite, Phytoseiulus persimilis, is considered a major threat to the effectiveness of biological control of gorse, Ulex europaeus, using Tetranychus lintearius. To assess this threat and to determine if the impact of P. persimilis on T. lintearius populations is comparable to its impact on T. urticae populations, its development and predator : prey generation time ratios were assessed. The pre-adult mortality and development time of two populations of P. persimilis fed on two diets, T. urticae and T. lintearius, were determined at two temperatures, 14 and 24°C. There were no significant differences in either mortality or development time between the two populations of P. persimilis at these temperatures. There is therefore no evidence that the two tested populations of P. persimilis are behaving as different strains. Similarly, diet had no significant effect on either mortality or development time at these temperatures. At 14°C the mortality of P. persimilis was significantly higher and development was significantly longer than at 24°C. Using pre-adult development as a surrogate for generation times, predator : prey generation time ratios were calculated between P. persimilis and both T. urticae and T. lintearius using data from this and other studies. The predator : prey generation time ratios between P. persimilis and T. lintearius were lower than those between P. persimilis and T. urticae. These results indicate that the impact of P. persimilis on T. lintearius populations is likely to be comparable to its impact on T. urticae populations. This provides further evidence that predation by P. persimilis is having a deleterious effect on T. lintearius populations and therefore reducing its effectiveness as a biological control agent for gorse.     

Characterization of nine microsatellite loci in the sea star Astropecten aranciacus and cross-species amplification for related taxa

So far, only few microsatellite markers have been developed and extensively tested for echinoderms. To study the population genetic structure of the sea star Astropecten aranciacus, we developed primers for nine polymorphic microsatellite loci and tested them on two populations from Faro in Portugal (N = 25) and from La Herradura in Spain (N = 20). Within populations, allele numbers varied from four to 20, while expected and observed heterozygosities ranged from 0.593 to 0.936 and from 0.222 to 0.900, respectively. Additional cross‐species amplifications were polymorphic at some loci, indicating their potential usefulness for related taxa.     

The G126S substitution in mitochondrially encoded cytochrome b does not confer bifenazate resistance in the spider mite Tetranychus urticae

Several genetic variants of the cd1- and ef-helices of the Q_o site of mitochondrial cytochrome b have been associated with bifenazate resistance in the spider mite Tetranychus urticae, an important crop pest around the world. Maternal inheritance of bifenazate resistance has provided strong evidence for the involvement of many of these mutations alone or in combination. A number of populations highly resistant to bifenazate were uncovered that carried the G126S substitution in combination with other target-site mutations. This G126S mutation has therefore been investigated in several studies in the context of resistance evolution and the development of diagnostic markers. However, experimental data that link bifenazate resistance with the presence of the G126S mutation without additional cd1- and ef-helices mutations, remain very limited. Here, we genotyped 38 T. urticae field populations for cytochrome b and uncovered nine field populations with a fixed or segregating G126S substitution without other target-site mutations in the conserved cd1- and ef-helices of the cytochrome b Q_o pocket. Toxicity bioassays showed that all nine field populations were very susceptible to bifenazate, providing strong evidence that G126S alone does not confer bifenazate resistance. These findings also implicate that previous T. urticae populations with G126S found to be low to moderately resistant to bifenazate, evolved alternative mechanisms of resistance, and more importantly, that this mutation cannot be used as a molecular diagnostic for bifenazate resistance.

     

Host plant-mediated interactions between Tetranychus urticae and Eotetranychus carpini borealis (Acari: Tetranychidae)

Competitive interactions between Tetranychus urticae and Eotetranychus carpini borealis were investigated under laboratory and greenhouse conditions. When placed first to colonize red raspberry leaves, T. urticae caused extinction of E. carpini borealis populations. E. carpini borealis had detrimental effects on T. urticae but did not cause T. urticae population extinction. The area used by E. carpini borealis for egg deposition decreased with an increase in the number of T. urticae eggs. Red raspberry showed an induced response to T. urticae and E. carpini borealis feeding after two weeks of infestation. Population growth of each species was reduced on plants that were previously infested by conspecifics but inducible response by one species had no effects on the other species.     

Identification and characterization of nuclear microsatellite loci in Abies alba Mill.

Eleven polymorphic nuclear microsatellite markers for Abies alba Mill. were developed from an enriched genomic library. An average of 5.2 alleles per locus and a mean expected heterozygosity of 0.532 were found in a sample of 24 Abies alba individuals from different populations within Europe. These loci can be used in studies of genetic diversity for parentage analysis and for estimation of gene flow in silver fir populations. Moreover, successful amplifications were obtained for eight other Mediterranean Abies species, suggesting that these loci may be useful for similar applications in other fir species.     

Isolation and variability of polymorphic microsatellite loci in Aedes aegypti,the vector of dengue viruses

We isolated five microsatellite sequences from an enriched‐(CAA)_n library of 5000 recombinant clones in Aedes aegypti. Two polymorphic microsatellites from our library and four from other sequence databases were tested: we investigated their polymorphism and Mendelian inheritance in mosquito populations. Our results indicate that trinucleotide repeat markers could be used to differentiate Ae. aegypti populations, making them valuable tools for the study of population genetic structure.     

Isolation and characterization of highly polymorphic microsatellite markers in Hypochaeris radicata (Asteraceae)

We developed five highly polymorphic dinucleotide microsatellite loci for the grassland species Hypochaeris radicata (Asteraceae). Polymorphism of these markers was examined in six populations in the Netherlands. All loci were polymorphic in all populations. The number of alleles per locus varied between 18 and 43. Expected heterozygosity was between 0.86 and 0.91. Cross‐species amplification was tested in six Hypochaeris species and was successful for three different loci in four species. These microsatellites are a useful tool in population genetic, dispersal and metapopulation studies or in testing levels of inbreeding.     

Evolutionary adaptation to host plants in a laboratory population of the phytophagous mite Tetranychus urticae Koch

Summary For evolutionary expansion of host range to occur in an herbivore population, genetic variation in ability to survive on and/or accept new hosts must be present. To determine whether a population of the phytophagous mite Tetranychus urticae contained such variation, I established lines from the population on two hosts on which mites initially showed both high juvenile mortality and low acceptance, tomato and broccoli. In less than ten generations, mites from the line kept on each host showed both lower mortality and greater acceptance on it than mites from a control line kept on lima bean, a favorable host for T. urticae. Host acceptance was measured by the proportion of mites attempting to disperse from leaves of the host. The line kept on tomato but not the one kept on broccoli also increased in development rate on its host. These results and those of a similar previous experiment on cucumber indicate that T. urticae populations can adapt to a diversity of initially unfavorable hosts. T. urticae populations therefore should be able to respond to temporal and spatial variation in host availability by adapting to the most abundant hosts.