Maritza Reyes

Genetic architecture in codling moth populations: comparison between microsatellite and insecticide resistance markers

The codling moth, Cydia pomonella, is renowned for developing resistance to insecticides and causing significant economic damage to pome fruits worldwide. In spite of its economic importance, little is known about the patterns of movement of this pest and the effects of insecticide treatment on the population genetic structure. Here, we investigated the genetic structure of the pest in 27 orchards from France, Italy, Armenia and Chile at seven microsatellite loci and two resistance markers [biochemical activity of cytochrome P450 oxidases and proportion of knockdown resistance (kdr) alleles in the sodium channel gene]. According to the microsatellite loci, we detected isolation by distance at the supranational scale but found no evidence of geographical structure among the 24 French orchards, which were mainly structured by the intensity of the insecticide treatments. Similarly, the highest levels of metabolic resistance associated with activity of the cytochrome P450 oxidases were detected in the most treated orchards. The kdr alleles were observed in southern France and Armenia where the pyrethroid insecticides were or have been intensively sprayed. The intensity of the insecticide treatments marginally affected the allelic richness in each orchard, but not the level of inbreeding. These results suggest important and high‐distance gene flow among the codling moth populations, which were mainly structured according to the history of insecticide applications. Differences in mutation‐migration‐drift equilibrium among treated and untreated orchards also suggest that insecticide applications are the main force regulating the local dynamics of codling moth populations.

Evaluation of Azinphos-Methyl Resistance and Activity of Detoxifying Enzymes in Codling Moth (Lepidoptera: Tortricidae) from Central Chile

Abstract Regular applications of insecticides have been the main management practice against codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) in Chile. Organophosphates are the most widely used insecticides, and azinphos-methyl is an important element in spray programs. In particular, we evaluated diagnostic doses of azinphos-methyl on neonate and postdiapausing larvae from seven apple (Malus spp.) orchards. We also evaluated the activity of detoxifying enzymes, such as glutathione S-transferases (GSTs), cytochrome P450 polysubstrate monooxygenases (PSMOs), and esterases, which are likely to be involved in resistance to insecticides. Such responses were compared with an insecticide-susceptible strain that has been maintained in the laboratory for several years. Neonate larval mortality of field populations to azinphos-methyl was not significantly different from of the susceptible strain. In contrast, postdiapause larval mortality was significantly lower in the six analyzed populations than in the susceptible strain. The C. pomonella populations with reduced postdiapause mortality to azinphos-methyl also showed statistically higher GST activity. Finally, no significant differences were found in total esterase or PSMO activity between C. pomonella populations. Therefore, the observed reduction in postdiapause larval mortality to azinphos-methyl seems to be associated with an increase in GST activity.

Worldwide variability of insecticide resistance mechanisms in the codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae)

The activity of detoxifying enzymes (glutathione-S-transferases (GST), mixed-function oxidases (MFO), and esterases (EST)) and the presence of insensitive variants of target proteins (sodium channel and acetylcholinesterase) were examined in individual male and female codling moths. Twenty-nine populations from 11 countries and two laboratory strains were examined. Populations were classified as either unsprayed or sprayed. The ranges of enzyme activities across field populations varied 15-fold, 485-fold and fourfold for GST, MFO and EST, respectively. MFO was the only enzyme whose activity differed in a binomial classification of orchards based on their spray history. Few differences in enzyme activities were found due to sex among populations; and, in these cases, males had higher GST and lower MFO and EST activities than females. Activities of the three enzymatic systems across all populations were positively correlated. Populations from Greece, Argentina and Uruguay had significant percentages of moths with elevated GST and MFO activities. The co-occurrence of moths expressing both elevated MFO and low EST activities was found in conventional orchards from the Czech Republic and France. Chile was the only country where populations from treated orchards did not include a significant proportion of individuals with enhanced enzyme activity. The kdr mutation was found at significant levels in ten populations from five countries, including all French and Argentinean populations. The mutation in AChE was only detected in the Spanish population.

Early detection of resistance to tebufenozide in field populations of Cydia pomonella L.: methods and mechanisms

Abstract:u2002 Four populations of codling moth Cydia pomonella L. were collected as overwintering larvae from apple orchards with different pesticide pressure (S. Michele, Roncafort, Revò and Vervò) in the Trento province (northern Italy). Mortality rate caused by a predetermined discriminating concentration of tebufenozide topically applied on overwintering larvae was evaluated. Neonate F1 progeny of the same populations were assayed for susceptibility to tebufenozide by feeding them on thinning apples treated with an appropriate discriminating dose of the insecticide. The activities of the main enzyme systems involved in the detoxification of insecticides were also evaluated in each population and related to their susceptibility to tebufenozide. The topical test detected a significant loss in susceptibility to tebufenozide in two populations, S. Michele and Roncafort, while all the overwintering larvae collected in the orchards of Revò and Vervò died when treated topically with the discriminating concentration. The apple‐dipping test performed on the neonate larvae showed a highly significant reduction in the susceptibility of the two populations of S. Michele and Roncafort. A less significant reduction in mortality rate was found in the Revò population; however, no statistical difference was found between the Vervò population and the susceptible reference. None of the four field populations significantly differed from the susceptible strain for Glutathione‐S‐transferase and esterase activity. A significantly higher frequency of individuals of the S. Michele and Roncafort populations exhibited a higher mixed function oxidase activity than the susceptible strain. The small resistance ratio values found for the two populations together with the low frequency of individuals exibiting enhanced enzymatic activity, reveals that the selection process was still at the early stage. Because of its efficiency in early detection of resistance to tebufenozide, topical application on diapausing larvae can thus be considered an appropriate, simple and robust tool for implementing resistance monitoring programmes for tebufenozide.

Resistance monitoring in codling moth: a need for standardization

BACKGROUNDnA wide range of methods has been used for monitoring resistance in Cydia pomonella L. However, the results from specific tests are not always concordant and they generate doubts over the extent to which the data represent the field situation. Therefore, the variability in the expression of resistance mechanisms and in resistance ratios between developmental stages and their reliability in a field situation were studied.nnnRESULTSnNeonate larvae showed the highest insecticide resistance, except to spinosad. Resistance ratios were from 2 to 9.5, depending on the insecticide and the strain tested, when compared with the susceptible strain. Spinosad exhibited the highest resistance ratio for diapausing larvae (4.4, 12.2 and 4.4 for Raz, Rdfb and RDelta strains, respectively). Enzymatic ratios were higher in adults than in neonates or diapausing larvae, and the highest enzymatic ratios were observed for mixed-function oxidases (7.3-36.7 for adults of resistant strains).nnnCONCLUSIONSnThe sensitivity of different instars depends on the insecticides used, and there is a differential expression of resistance mechanisms between stages. For the population considered in this study, tests on neonates provided the most consistent results to the field situation, but adult response to chlorpyrifos-ethyl and azinphos-methyl in the laboratory was also consistent with the field results.

Propolis chemical composition and honeybee resistance against Varroa destructor

Propolis is known as honeybee chemical defence against infections and parasites. Its chemical composition is variable and depends on the specificity of the local flora. However, there are no data concerning the relationship between propolis chemical composition and honeybee colony health. We tried to answer this question, studying the chemical composition of propolis of bee colonies from an apiary near Avignon, which are tolerant to Varroa destructor, comparing it with colonies from the same apiary which are non-tolerant to the mites. The results indicated that non-tolerant colonies collected more resin than the tolerant ones. The percentage of four biologically active compounds – caffeic acid and pentenyl caffeates – was higher in propolis from tolerant colonies. The results of this study pave the way to understanding the effect of propolis in individual and social immunity of the honeybees. Further studies are needed to clarify the relationship between propolis chemical composition and honeybee colony health.

Honeybee Colony Vibrational Measurements to Highlight the Brood Cycle

Insect pollination is of great importance to crop production worldwide and honey bees are amongst its chief facilitators. Because of the decline of managed colonies, the use of sensor technology is growing in popularity and it is of interest to develop new methods which can more accurately and less invasively assess honey bee colony status. Our approach is to use accelerometers to measure vibrations in order to provide information on colony activity and development. The accelerometers provide amplitude and frequency information which is recorded every three minutes and analysed for night time only. Vibrational data were validated by comparison to visual inspection data, particularly the brood development. We show a strong correlation between vibrational amplitude data and the brood cycle in the vicinity of the sensor. We have further explored the minimum data that is required, when frequency information is also included, to accurately predict the current point in the brood cycle. Such a technique should enable beekeepers to reduce the frequency with which visual inspections are required, reducing the stress this places on the colony and saving the beekeeper time.

Organophosphate Resistance and Its Main Mechanism in Populations of Codling Moth (Lepidoptera: Tortricidae) from Central Chile

ABSTRACT n The codling moth, Cydia pomonella (L.), is the key pest of apple production worldwide. Insecticide resistance has been reported in all producing countries, based on five different mechanisms. Codling moth in Chile has resistance to azinphos-methyl and tebufenozide in post-diapausing larvae. However, there are no studies about the susceptibility of these populations to insecticides from other chemical groups. Therefore, the efficacy of azinphos-methyl, chlorpyrifos-ethyl, esfenvalerate, methoxyfenozide, tebufenozide, and thiacloprid on neonate and post-diapausing larvae from six field populations was investigated, and identified resistance mechanisms in this species were evaluated. Neonate larvae were susceptible to all insecticides studied, but post-diapausing larvae from four populations were resistant to chlorpyrifos, one of them was also resistant to azinphos-methyl, and another one was resistant to tebufenozide. The acetylcholinesterase insensitivity mutation was not detected, and the sodium channel knockdown resistance mutation was present in a low frequency in one population. Detoxifying enzymatic activity of glutathione S-transferases, esterases, and cytochrome P-450 monooxygenases in adults differed among populations, but chlorpyrifos resistance was associated only with a decreased esterase activity as shown by a significant negative correlation between chlorpyrifos mortality and esterase activity.

Cytochrome P450 monooxygenase activity levels in phytoplasma-infected and uninfected Amplicephalus curtulus (Hemiptera: Cicadellidae): possible implications of phytoplasma infections

The process of pass-through and multiplication of phytoplasma in host-insect tissues could cause some pathogenic effects in insect vectors and may increase the susceptibility to some insecticides. We propose that ‘Candidatus phytoplasma ulmi-infected Amplicephalus curtulus had reduced cytochrome P450 (P450s) activity compared with uninfected leafhoppers. P450s activity and phytoplasmas were quantified in adult A. curtulus at 25, 35 and 45xa0days after the access acquisition period (AAP) in the head-thorax and abdomen sections. Real-time PCR analysis showed that 67 and 78xa0% of insect samples were positive to phytoplasma at 35 and 45xa0days after the AAP, respectively. None of the samples tested positive to phytoplasma at 25xa0days after the AAP. P450s activity did not change at 35xa0days of incubation, but 45xa0days after the AAP, the enzymatic activity remained 112xa0% higher in phytoplasma-infected than in noninfected leafhoppers. P450s activity in the abdomen and head-thorax sections in phytoplasma-infected leafhoppers was 28 and 81xa0% more than in uninfected leafhoppers, respectively. Females had a higher concentration of phytoplasma than males, with 38xa0% more in the abdomen than in the head-thorax section. These results indicate that infection with ‘Ca. Phytoplasma ulmi alters A. curtulus P450s activity because of phytoplasma invasion in the host, and it is recognized as probably being an exogenous agent for a specific time period in the life of the insect vector.

Infection of ‘Candidatus Phytoplasma ulmi’ reduces the protein content and alters the activity of detoxifying enzymes in Amplicephalus curtulus

It has been reported that insecticide‐detoxifying enzymes such as glutathione S‐transferases (GST) and esterases are affected by microbial infections in hemipteran insect vectors. The total protein content, and GST and α‐ and β‐esterase activities were quantified in ‘Candidatus Phytoplasma ulmi’‐infected and uninfected adults of Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae) at 25, 35, and 45 days after the acquisition access period (AAP) in the head‐thorax and abdomen sections. The total protein content was lower in phytoplasma‐infected leafhoppers 25, 35, and 45 days after the AAP. Thirty‐five days after the AAP, the GST and β‐esterase activities had increased (26 and 69%, respectively) compared to the control. However, 45 days after the AAP, the phytoplasma‐infected leafhoppers displayed lower GST (87%) and β‐esterase (253%) activities than the uninfected individuals. On the other hand, the α‐esterase activity proved to be unaffected by the phytoplasma infection. Forty‐five days after the AAP, females had a higher phytoplasma titer (46%) in their head‐thorax than in their abdomen sections, whereas males showed a higher titer in their abdomens (75%). In addition, the GST and β‐esterase activities in the abdomen were affected negatively by 96–98% as a result of the increasing ‘Ca. Phytoplasma ulmi’ titer. These results indicate that an infection of ‘Ca. Phytoplasma ulmi’ alters the metabolic activities of A. curtulus.