Molecular variation of the cytochrome b DNA and protein sequences in Phytoseiulus macropilis and P. persimilis (Acari: Phytoseiidae) reflect population differentiation.

Several phytoseiid mite species are important natural enemies used in biological control strategies. In the present study, Cytb mtDNA sequences of various populations of two species, Phytoseiulus macropolis and P. persimilis, were compared to determine whether the specimens collected in Brazil could belong to P. persimilis as this latter species is reported in South America but not in Brazil. The Cytb marker was used because of its high evolution rate, assumed to capture intraspecific variation. No overlap between intra- and interspecific distances was observed but the distances were quite low for interspecific variation. This can be due to the particular biology of Phytoseiulus species and this shows the difficulty to apply a universal threshold in genetic distances to conclude about the existence of one or several species. Cytb mtDNA sequences were also considered to assess intraspecific variation. The DNA sequences of P. persimilis populations were very similar, probably because they all originated from the West Palearctic region or because of a prevalence of commercialized specimens in natura. For P. macropilis, higher genetic distances were observed and differentiation was noted according to geographic location and, to a smaller extent, pyrethroid resistance. To determine how DNA variation might impact the protein function (CytB fragment considered), the amino acid compositions of the populations studied were compared. No diagnostic mutation was observed between pyrethroid resistant and susceptible populations, whereas four mutations were identified between populations of P. macropilis separated by 1300 km (different climatic conditions). The impact of such mutations is discussed but knowledge is scarce, which makes it difficult to root testable hypotheses. The protein analysis clearly opens new perspectives in Phytoseiidae studies.

Development of microsatellite markers for the predatory mite Phytoseiulus macropilis and cross-amplification in three other species of phytoseiid mites.

Phytoseiid mites are efficient predators of mites and small pest insects. Understanding the dispersion and distribution pattern of phytoseiid mites is essential to promote the conservation of these natural enemies and support their use in biological control. Population genetic studies using molecular markers such as microsatellites have proved to be extremely informative to address questions about population structure and dispersion patterns of predatory mites. The objective of this work was to develop specific microsatellite markers for the predatory mite Phytoseiulus macropilis, aiming at improving field dispersion studies. For this purpose, the genomic DNA was extracted from the whole body of a pool of 260 adult females and used to build the genomic microsatellites-enriched library, using biotinylated probes (CT)8 and (GT)8. In total 26 pairs of primers were synthesized and screened across 30 adult females of P. macropilis for characterization. Seven loci were polymorphic, revealing from two to six alleles per locus. Cross amplifications were successfully obtained in the species Phytoseiulus persimilis, Amblyseius swirskii and Proprioseiopsis sp. The molecular markers obtained are the first developed for P. macropilis-they are effective for the detection and quantification of genetic variation, and show high transferability, thus can be used in genetic and molecular studies of this and other species of the same genus and also of close genera.

Pyrethroid resistance in Phytoseiulus macropilis (Acari: Phytoseiidae): cross-resistance, stability and effect of synergists.

Phytoseiulus macropilis Banks (Acari: Phytoseiidae) is an effective predator of Tetranychus urticae Koch (Acari: Tetranychidae). The objectives of this research were to study the stability of fenpropathrin resistance and the cross-resistance relationships with different pyrethroids, and also to evaluate the effect of synergists [piperonyl butoxide (PBO), diethyl maleate (DEM) and S,S,S-tributyl phosphorotrithioate (DEF)] on fenpropathrin resistant and susceptible strains of this predaceous mite. The stability of fenpropathrin resistance was studied under laboratory conditions, using P. macropilis populations with initial frequencies of 75 and 50% of resistant mites. The percentages of fenpropathrin resistant mites were evaluated monthly for a period of up to 12 months. A trend toward decreased resistance frequencies was observed only during the first 3-4 months. After this initial decrease, the fenpropathrin resistance was shown to be stable, maintaining constant resistance frequencies (around 30%) until the end of the evaluation period. Toxicity tests carried out using fenpropathrin resistant and susceptible strains of P. macropilis indicated strong positive cross-resistance between fenpropathrin and the pyrethroids bifenthrin and deltamethrin. Bioassays with the synergists DEM, DEF and PBO were also performed. The maximum synergism ratio (SR = LC50 without synergist/LC50 with synergist) detected for the three evaluated synergists (PBO, DEM, DEF) was 5.86 (for DEF), indicating low influence of enzyme detoxification processes in fenpropathrin resistance.

Stability and fitness costs associated with etoxazole resistance in Tetranychus urticae (Acari: Tetranychidae).

The two-spotted spider mite, Tetranychus urticae Koch, is one of the most important pests on a wide range of crops worldwide. Studies on stability of resistance and possible fitness costs associated with etoxazole resistance were carried out in T. urticae to provide basic information necessary to define effective acaricide resistance management strategies for this pest. Selection for resistance to etoxazole was performed in a population of T. urticae collected from a commercial rose field, in Holambra County, in the State of São Paulo, Brazil. After five rounds of selection for resistance, the resistance ratio (R/S) at the LC50 reached 8739 fold value in comparison with a susceptible strain (Brazabrantes S Strain). The stability of etoxazole resistance was studied under laboratory conditions, using a population with initial frequency of 75 % of resistant mites. The frequencies of etoxazole resistance were evaluated monthly for a period of 13 months. The frequency of etoxazole resistance decreased from 75 to 37 % in this period. Comparison of biological traits between resistant and susceptible strains indicated the presence of fitness costs associated with etoxazole resistance. The resistant strain showed lower fecundity and a higher proportion of males in the population. The figures for net reproductive rate (R 0), intrinsic rate of natural increase (r m) and finite rate of increase (λ) were higher in the susceptible strain. The instability of etoxazole resistance is a favorable aspect for the acaricide resistance management in the spider mite.

Comparison of conventional and integrated programs for control of Tetranychus urticae (Acari: Tetranychidae).

The twospotted spider mite (TSSM), Tetranychus urticae Koch, is one of the main pests on strawberry crops in Brazil. TSSM can be difficult to control due to acaricide resistance. The objective of this work was to compare the effect of conventional and integrated strawberry production (ISP) systems on mite abundance and acaricide resistance. The control of TSSM in ISP was based on the release of Neoseiulus californicus (McGregor) or application of a selective acaricide (propargite), when TSSM monitoring indicated the timing for the release of predaceous mites (1-3 mites per leaflet on 30% leaflets) or chemical intervention (>10 mites per leaflet). Only acaricides (abamectin, fenpyroximate) were applied in the conventional system. Integrated control of TSSM were sufficient to maintain a significantly lower pest infestation level, resulting in a sixfold reduction in the frequency of acaricide applications, and consequently, a lower selection pressure for acaricide resistance. Strategies for the management of TSSM in strawberry fields are described and discussed.

Interspecific interactions involving Neoseiulus californicus (Acari: Phytoseiidae) and Agistemus brasiliensis (Acari: Stigmaeidae) as predators of Brevipalpus phoenicis (Acari: Tenuipalpidae).

Brevipalpus phoenicis (Geijskes) is associated with the transmission of Citrus leprosis which is considered the main viral disease for the Brazilian citrus production. Mites of the families Stigmaeidae and Phytoseiidae coexist in various agricultural crops, often promoting the biological control of pest mites. The aim of this work was to study the interactions of Neoseiulus californicus (McGregor) (Phytoseiidae) and Agistemus brasiliensis Matioli, Ueckermann & Oliveira (Stigmaeidae), in the presence or absence of B. phoenicis. Two experiments were carried out. In the first, a N. californicus female was placed in each leaf disc arena, with eggs of B. phoenicis and A. brasiliensis as food sources. In the second, an A. brasiliensis female was placed in each arena, with eggs of B. phoenicis and N. californicus as food sources. Adults of both predators were able to consume both types of eggs available as food sources, but they fed on considerably higher proportions of B. phoenicis than on eggs of the predator. Eggs of A. brasiliensis were not a suitable food source for N. californicus, which produced only 0.1 egg per female per day when only eggs of that species were present in the experimental unit. The results suggest that eggs of N. californicus were a suitable food source for A. brasiliensis, which oviposited 1.12 eggs per day, when only eggs of N. californicus were provided to the stigmaeid mite. The possible interactions among N. californicus, A. brasiliensis and B. phoenicis in citrus orchards are discussed.

Diagnosis of amitraz resistance in Brazilian populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) with larval immersion test.

Among the ectoparasites of cattle, Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae) remains a major cause of economic losses to livestock. The chemical control with acaricides is still the most efficient method available to control ticks. The aims of this study were to diagnose resistance to amitraz in 16 tick populations from the States of São Paulo (14) and Paraná (2), using the larval immersion technique (LIT), and evaluate the effect of synergists [piperonyl butoxide (PBO), diethyl maleate (DEM), triphenyl phosphate (TPP)] on amitraz resistant and susceptible strains of cattle tick. Most of the evaluated populations (68.7 %) showed to be resistant to amitraz, with resistance ratios ranging from 2.14 to 132. The results suggest that the test procedure by LIT is sensitive and adequate for detection and monitoring of amitraz resistance in cattle tick. No synergistic effect was observed for the synergists PBO, DEM and TPP, on the amitraz resistant (Poa) strain of cattle tick, indicating that increased detoxification metabolism was not involved in this resistance.

Milbemectin resistance in Tetranychus urticae (Acari: Tetranychidae): selection, stability and cross-resistance to abamectin.

Studies on artificial laboratory selections with milbemectin, stability of milbemectin resistance and possible cross-resistance with abamectin were carried out with Tetranychus urticae Koch to provide basic information for a milbemectin resistance management program. Selections for resistance and susceptibility to milbemectin were performed in a population of T. urticae, collected from a commercial chrysanthemum field in the State of São Paulo, Brazil. After six selections for resistance and five selections for susceptibility, susceptible (S) and resistant (R) strains of T. urticae to milbemectin were obtained. The resistance ratio (R/S) at the LC(50) reached 409-fold value. The stability of milbemectin resistance was also studied under laboratory conditions, using a population with initial frequency of 75% of resistant mites. The frequencies of milbemectin resistance were evaluated monthly for a period of 7 months. In order to observe possible correlation between milbemectin and abamectin resistance, the frequencies of abamectin resistance were also evaluated for that population, during the same period. The frequency of milbemectin resistance decreased from 75 to 14.5%, while the percentage of abamectin resistant mites decreased from 57 to 9.1%, in 7 months. The frequencies of milbemectin and abamectin resistance were also evaluated in 25 field populations of T. urticae, collected from several crops in the State of São Paulo. The frequencies of milbemectin resistance varied from 4.1 to 89.5%, and of abamectin, from 7.0 to 90.5%. A positive and significant correlation was observed between the frequencies of milbemectin and abamectin resistance, indicating positive cross-resistance between these acaricides. The results indicate that abamectin should be avoided for managing milbemectin resistance in T. urticae. This is the first report on milbemectin resistance in T. urticae in Brazil.

Population growth of Tetranychus urticae Koch (Acari: Tetranychidae) and predation rate of the pest mite by Neoseiulusidaeus Denmark & Muma (Acari: Phytoseiidae) in two grape cultivars

The two-spotted spider mite, Tetranychus urticae Koch, has caused significant damage to vineyards in the valley of the São Francisco River in Brazil. Neoseiulus idaeus Denmark & Muma is one of the most abundant species of predatory mites on vines in this region. This study evaluated the population growth rates of T. urticae on leaves of two grape (Vitis vinifera L.) cultivars ('Italia' and 'Superior Seedless') and jack bean [Canavalia ensiformis (L.) DC.] and, also, to estimate the predation rates of the pest mite by N. idaeus on leaves of both grape cultivars and jack bean. A higher oviposition rate of T. urticae was observed on 'Superior Seedless' than on 'Italia' grape leaves; however, there was no significant difference in the instantaneous growth rates of T. urticae for these grape cultivars. For 'Superior Seedless', the spider mite egg viability was significantly lower than on 'Italia' grape, indicating a resistance factor in this grape cultivar. The phytoseiid N. idaeus preyed higher number of T. urticae females on 'Superior Seedless' leaves than on 'Italia' grape and jack bean leaves for the densities of 10 and 20 pest mites per leaf arena (9 cm2). Evaluations of leaf trichomes in both grape cultivars indicated the occurrence of longer trichomes on the basal portion of the main veins of 'Superior Seedless' leaves. The presence of these longer trichomes may be associated with the better performance of N. idaeus on 'Superior Seedless' leaves.

Population dynamics of phytophagous and predaceous mites on coffee in Brazil, with emphasis on Brevipalpus phoenicis (Acari: Tenuipalpidae).

The objective of this work was to study the population dynamics of Brevipalpus phoenicis (Geijskes) and predaceous mites (Phytoseiidae and Stigmaeidae), as well as the interactions among these mite species, in a coffee plantation in the State of São Paulo, Brazil. Tydeids were also evaluated because of the high frequency of these mites on coffee plants. Samples of leaves, branches and fruits were taken fortnightly, from April 2001 to June 2003, from plants randomly chosen in the coffee plantation. B. phoenicis mites were found on leaves in higher number during the drier periods of the year. Among the predaceous mites, Euseius citrifolius Denmark & Muma and Euseius concordis (Chant) were the most frequent species on the surface of leaves. During the evaluations, 72,534 domatia were cut and opened to remove the mites, from 6,360 leaves examined. Zetzellia malvinae Matioli, Ueckermann & Oliveira was the most frequent predator species found inside domatia. Significant correlations were observed between population dynamics of, among others, E. concordis and B. phoenicis, Z. malvinae and B. phoenicis, and Z. malvinae and E. concordis. Significant correlations were also verified between the number of domatia and the population densities of B. phoenicis, E. concordis, Lorryia sp. and Z. malvinae. Interactions between predator-prey and predator-predator on coffee plants are discussed. The influence of the meteorological factors temperature and precipitation on the most frequent mite species is also discussed.

Management of Tetranychus urticae (Acari: Tetranychidae) in strawberry fields with Neoseiulus californicus (Acari: Phytoseiidae) and acaricides.

The purpose of this study was to evaluate the performance of Neoseiulus californicus (McGregor) for the control of Tetranychus urticae Koch in commercial strawberry fields, under greenhouse conditions, in association or not with the use of acaricides. The N. californicus strain used in this study was tolerant or resistant to several pesticides. Three experiments were carried out in the State of São Paulo, Brazil. For the first experiment, the initial infestation of T. urticae was 87.1 active stages per leaflet. Two applications of propargite were made on the first and the 14th day of the experiment. Approximately 2 h after each propargite application, N. californicus was released at a rate of 3.0 and 1.9 adult mites per plant, respectively, for each application. The population of T. urticae decreased from 87.1 to 2.8 mites per leaflet in the first three weeks. After this period, the population of T. urticae was maintained at low levels (

Monooxygenase activity in methidathion resistant and susceptible populations of Amblyseius womersleyi (Acari: Phytoseiidae).

The purpose of this study was to evaluate the cytochrome P450-dependent monooxygenase activities in methidathion resistant and susceptible strains of Amblyseius womersleyi Schicha. Artificial laboratory selections for resistance and susceptibility to methidathion were performed in an organophosphate resistant strain of A. womersleyi (Kanaya strain). Selections for susceptibility were also performed in a susceptible strain of this predaceous mite (Ishigaki Strain). After the selection process, the LC(50) of methidathion for the selected strains of A. womersleyi were 816 mg/l (Kanaya R), 4.61 mg/l (Kanaya S) and 1.59 mg/l (Ishigaki S). The monooxygenase activities were determined biochemically by the O-deethylation of 7-ethoxycoumarin (7-EC). The monooxygenase activity in adult females of Kanaya R strain (51.1 pmol/30 min/mg protein) was 3.60- and 5.42-fold higher than the activity observed for Kanaya S and Ishigaki S strains, respectively. Significant correlation between monooxygenase activity and LC(50) (mg/l) of methidathion was observed analyzing 16 populations of A. womersleyi with different susceptibilities to methidathion. Monooxygenase activity was also evaluated in different life stages (egg, larva, protonymph, deutonymph and adult) of A. womersleyi. The lowest activity was observed for the larval stage, which presented the highest susceptibility to methidathion. Protonymph, deutonymph and adult presented the highest monooxygenase activities. These stages were the most tolerant to methidathion. Monooxygenase activities of the Kanaya R strain were higher than of the Kanaya S strain in all developmental stages. The present study can be helpful for the implementation of a program involving release of insecticide-resistant populations of A. womersleyi in the field. The monooxygenase activity determination is easier and quicker than the estimation of LC(50), requiring fewer mites.

Ácaros de cafeeiro (Coffea spp.) no Estado de São Paulo, Brasil: Parte II. Prostigmata / Mites of coffee plants (Coffea spp.) in State of São Paulo: Part II. Prostigmata

Este trabalho teve como objetivo fazer um levantamento das espécies de ácaros pertencentes à subordem Prostigmata em Coffea spp. em diferentes localidades do Estado de São Paulo. Folhas e ramos de cafeeiros (Coffea spp.) foram coletados em 24 municípios do Estado de São Paulo e em diferentes épocas do ano. Todos os ácaros encontrados foram montados em lâminas de microscopia. A identificação dos ácaros foi feita até o nível específico quando possível. Foram identificadas 79 espécies de ácaros pertencentes às famílias Anystidae, Bdellidae, Cheyletidae, Cunaxidae, Diptilomiopidae, Eriophyidae, Eupalopsellidae, Eupodidae, Iolinidae, Meyerellidae, Nanorchestidae, Paratydeidae, Raphignathidae, Stigmaeidae, Tarsonemidae, Tenuipalpidae, Tetranychidae, Trombidiidae, Tuckerellidae e Tydeidae.

The objective of this study was to carry out a survey of mite species belonging to the suborder Prostigmata on Coffea spp. in different localities of the State of São Paulo. Samples of coffee (Coffea spp.) leaves and branches were collected in 24 municipalities of the State of São Paulo, in different periods of the year. All mites collected were mounted on microscope slides. Whenever possible, mites were identified to species level. Seventy-nine species were identified, belonging to the families Anystidae, Bdellidae, Cheyletidae, Cunaxidae, Diptilomiopidae, Eriophyidae, Eupalopsellidae, Eupodidae, Iolinidae, Meyerellidae, Nanorchestidae, Paratydeidae, Raphignathidae, Stigmaeidae, Tarsonemidae, Tenuipalpidae, Tetranychidae, Trombidiidae, Tuckerellidae e Tydeidae.

Ácaros associados ao cafeeiro (Coffea spp. ) no estado de São Paulo, Brasil: Parte I: Mesostigmata / Mites associated with coffee plants (Coffea spp. ) in the state of São Paulo, Brazil: Part I: Mesostigmata

Este trabalho teve como objetivo fazer um levantamento das espécies de ácaros pertencentes à ordem Mesostigmata em Coffea spp. em diferentes localidades do estado de São Paulo. Folhas e ramos de cafeeiros (Coffea spp.) foram coletados em 27 municípios do estado de São Paulo e em diferentes épocas do ano. Todos os ácaros encontrados foram montados em lâminas de microscopia, em meio de Hoyer. A identificação dos ácaros foi feita até o nível específico quando possível. Foram identificadas 39 espécies de ácaros pertencentes às famílias Ameroseiidae, Ascidae, Laelapidae, Macrochelidae, Parasitidae, Phytoseiidae e a um grupo que ainda está sem uma família definida, Africoseius sp.

The objective of this study was to carry out a survey of mite species belonging to the order Mesostigmata on Coffea spp. in different localities of the State of São Paulo. Samples of coffee (Coffea spp.) leaves and branches were collected in 27 municipalities of the State of São Paulo, in different periods of the year. All collected mites were mounted in Hoyer's medium on microscope slides. The identification of mites was at species level when possible. Thirty-nine mite species were identified, belonging to the families Ameroseiidae, Ascidae, Laelapidae, Macrochelidae, Parasitidae, Phytoseiidae and to a group for which the family is still not defined, Africoseius sp.