Inheritance and synergism of resistance to imidacloprid in the Colorado potato beetle (Coleoptera: Chrysomelidae)

Colorado potato beetle, Leptinotarsa decemlineata (Say), adults and larvae collected from Long Island, NY, were 100.8 and 13.2 times more resistant to imidacloprid, respectively, compared with a susceptible strain. This high level of resistance appeared in only the third field season of imidacloprid use. Analysis of probit lines from F1 reciprocal crosses indicated that resistance to imidacloprid in adults was inherited autosomaly as an incompletely recessive factor. The degree of dominance of the resistance was -0.23 and -0.10, respectively, 3 and 7 d after treatment (incompletely recessive). The chi2 analysis of response ratio statistics from F1 x susceptible back crosses compared with a monogenic model suggested that more than one locus is responsible for resistance to imidacloprid. Synergism studies with piperonyl butoxide suggested that mixed-function oxidase mediated detoxification is responsible for the resistance to imidacloprid in adults. Synergism studies with S,S,S-tributyl phosphorotrithioate (DEF) indicated that esterase mediated detoxification may be an additional resistance factor. Mixed-function oxidase mediated detoxification is probably also one of the mechanisms of resistance to imidacloprid in larvae. Because the synergists used did not completely eliminate resistance in the resistant strain, there may be additional mechanisms involved. Refugia and crop rotation decrease the frequency of homozygous resistant genotypes and may be effective resistance management strategies, because of the recessive nature of the resistance.     

Baseline susceptibility to imidacloprid and cross resistance patterns in Colorado potato beetle (Coleoptera: Chrysomelidae) populations

During 1995-1998, we tested 134 geographically discrete populations of Colorado potato beetle, Leptinotarsa decemlineata (Say), from the United States, Canada, Germany, France, and Poland for susceptibility to imidacloprid. Neonates were assayed on potato-based agar diet incorporated with imidacloprid and exposed on filter paper to esfenvalerate, azinphosmethyl, and carbofuran to characterize cross-resistance. In all 4 yr, Long Island populations were the most tolerant to imidacloprid, with LC50s ranging up to 29 times higher than the most susceptible populations. Responses to imidacloprid did not change significantly on farms where populations were assayed over time, except for those from Long Island, which doubled in overall tolerance to imidacloprid since 1995. Much of this tolerance was already present before imidacloprid was used on Long Island. Correlative analysis of the populations tested over the 4 yr indicated positive cross-resistance patterns with esfenvalerate and azinphosmethyl. This response was probably caused by preexisting metabolic and excretion mechanisms selected by previous exposure. There was no significant pattern of cross-resistance with carbofuran or bensultap. Regression slopes were also significantly negatively correlated with LC50 values for imidacloprid, indicating higher heterogeneity, which could lead in further resistance development. We discuss the relative sensitivity of diet-incorporated assays with neonates compared with other bioassay studies. Based on a pooled group of susceptible populations tested in 1995, a baseline LC50 of 0.39 ppm and a discriminating concentration of 8 ppm were suggested to detect early stages of resistance in "suspect" populations. We also suggest application strategies for imidacloprid that reduce selection pressure.     

Glandular trichomes of Solanum berthaultii and resistance to the Colorado potato beetle

The defensive mechanisms of the wild potato, solanum berthaultii Hawkes, to larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say), were studied by selective removal of glandular trichomes and trichome exudates from leaflets, and by comparing performance on S. berthaultii and on the cultivated potato, S. tuberosum L., which lacks defensively active type A and B glandular trichomes. Removal of type A trichomes increased the proportion of larvae that fed on S. berthaultii. Removal of the exudate from type B trichomes increased the proportion of larvae that fed and led to a decrease in mortality. The predominant active compounds in type B exudate, i.e. fatty acid esters of sucrose, were only effective in the presence of type A trichomes. Sucrose esters did not affect larval feeding on S. tuberosum leaflets or on S. berthaultii leaf discs from which the type A trichomes had been removed. Growth of surviving larvae was not significantly affected by removing type A trichomes or type B exudate. Growth of larvae was significantly increased when S. berthaultii leaflets were presented in artificial diet which eliminated the physical barrier of the type B stalks. Growth was no different on artificial diet containing either S. berthaultii or S. tuberosum leaf material (fresh or lyophilized powder) but was poorer on these diets than on S. tuberosum leaflets. The presence of type A trichomes is a fundamental requirement for expression of S. berthaultii resistance to L1 L. decemlineata. Type B droplets containing sucrose esters increase the expression of resistance in the presence of defensively-active type A trichomes.

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Résumé Les mécanismes de défense de la pomme de terre sauvage, S. berthaultii Hawkes, aux larves de Leptinotarsa decemlineata Say, ont été étudiés par ablation sélective des trichomes glandulaires et par l'élimination de leur exsudat des folioles, et par comparaison avec S. tuberosum L. qui a perdu les trichomes glandulaires défensifs A et B. L'ablation des trichomes A a augmenté la proportion de larves ayant consommé S. berthaultii. L'élimination de l'exsudat des trichomes B a augmenté la proportion de consommatrices et réduit la mortalité. Les principaux composés actifs de l'exsudat B, c'est-à-dire des esters d'acides gras de sucrose, n'étaient actifs qu'en présence de trichomes A. Les esters de sucrose n'ont pas modifié la consommation larvaire sur folioles de S. tuberosum, ou sur disques de feuilles de S. berthaultii dont les trichomes A avaient été enlevés. La croissance des larves survivantes n'a pas été modifiée significativement par l'ablation des trichomes A ou l'élimination de l'exsudat de B. La croissance des larves a été significativement augmentée quand les folioles de S. berthaultii ont été incorporés dans l'aliment artificiel après élimination de la barrière physique due aux pédoncules B. La croissance a été de même importance sur aliments artificiels contenant des feuilles (fraiches ou en poudre lyophylisée) de S. berthaultii ou de S. tuberosum, mais plus faible que sur folioles de S. tuberosum. La présence de trichomes A est indispensable à la résistance de S. berthaultii aux L, de L. decemlineata. Les gouttelettes de type B contenant des esters de sucrose augmentent l'expression de la résistance en présence d'une défense active par trichomes A.

     

The genetic base and phenotypic manifestations of Colorado potato beetle resistance to organophosphorus insecticides

A high level of resistance to organophosphorus insecticides was recorded in a local population of the Colorado potato beetle (Ufa district, Bashkortostan). Toxicological analysis demonstrated that the fraction of susceptible individuals did not exceed 0.025. DNA analysis of the polymorphism of acetylcholinesterase gene (AChE) according to the presence of mutation 980A>G in the sample of Colorado potato beetle imagoes demonstrated the absence of wild-type (sensitive, SS) homozygotes and prevalence (0.79) of the homozygotes with mutant resistant type (RR). The phenetic analysis of the variation of the integument pattern phenes demonstrated statistically significant differences between the imago phenotypes in the ratio of SS and RR genotypes.     

Combining genetic engineering and traditional breeding to provide elevated resistance in potatoes to Colorado potato beetle

The sustainable deployment of resistant crop varieties is a critical issue for the implementation of biotechnology in crop pest management. Feeding, biomass accumulation, and mortality were evaluated for susceptible, insecticide‐resistant, and Bacillus thuringiensis (Bt) Cry 3A‐selected Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera, Chrysomelidae) larvae fed on: cultivated potato, a Solanum chacoense line expressing leptine glycoalkaloids, a transformed line expressing Bt toxin, or the leptine line transformed to express Bt toxin. Larvae selected for resistance to Bt‐Cry3A performed better on Bt foliage, but not as well on the leptine foliage, compared to susceptible or insecticide‐resistant larvae. Neither leptine nor Bt toxin completely inhibited the feeding and growth of 3rd and 4th instars of all three strains of Colorado potato beetle. However, for all three strains of Colorado potato beetle on leptine + Bt foliage, feeding was almost zero, growth was zero or negative, and mortality was near 100%.     

Flight take-off performance of Colorado potato beetle in relation to potato phenology

The flight take-off frequency of adult Colorado potato beetles, Leptinotarsa decemlineata (Say), from potato plants, Solanum tuberosum L. 'Red Pontiac' at the bloom stage of development was 2.2-2.5-fold that of Colorado potato beetle from plants at the vegetative stage. Tests were conducted in a flight chamber over a period of 3 h. Prefeeding Colorado potato beetles for 48 h on potato plants at the bloom or at the vegetative stage before placing them into the flight chamber resulted in the same significantly higher flight take-off frequency from potato plants at the bloom stage than from plants at the vegetative stage. These results demonstrate that the factor in potato plants in bloom that stimulates the flight take-off of the Colorado potato beetle is independent of the feeding history of the beetles and begins acting only when the beetles are in the presence of the plant. According to these results, the dispersal of adult Colorado potato beetles from potato fields in bloom to younger potato fields with plants at the vegetative stage, previously reported in the literature, is at least partly explained by the effect of plant phenology on the frequency of flight take-off. Results confirm the value of planting potato fields of similar phenology over as wide an area as possible to reduce Colorado potato beetle dispersal between fields. Results also imply that staggering the planting dates of conventional potato refuge areas near Colorado potato beetle transgenic or conventionally resistant potato fields is a sound management practice, because it promotes the movement of wild beetles over to the adjacent younger resistant crops.     

Disrupting spring colonization of Colorado potato beetle to nonrotated potato fields

Overwintering Colorado potato beetles (Leptinotarsa decemlineata (Say)) were concentrated primarily within woody borders, and mortality was lower in borders than in potato fields. After overwintering, only 15–44% of live beetles were in the potato fields. In experiments with small plots, colonization of fields from woody borders was reduced ∼60% by a trap crop, either treated with adulticide or with beetles collected daily. Such trap crops, or simply pitfall traps to prevent colonization from woody borders, could significantly reduce early-season adult numbers and subsequent larval populations. However, success is dependent on the local densities of overwintered beetles, and the prevalence of arrestment behavior in the case of trap crops.     

Evaluation of natural and engineered resistance mechanisms in potato against Colorado potato beetle in a no-choice field study

The Colorado potato beetle, Leptinotarsa decemlineata Say, is the major insect pest of potato, Solanum tuberosum L., in eastern North America and is renowned for resistance development, currently resistant to >40 insecticides worldwide. Host plant resistance may assist in delaying in resistance development to insecticides. We evaluated natural host plant resistance mechanisms (glandular trichomes and Solanum chacoense Bitter-derived resistance) and engineered resistance mechanisms (Bacillus thuringiensis [Bt] Berliner cry3A and cry1Ia1) in a no-choice cage study. Six different potato lines representing four host plant resistance mechanisms were evaluated over 2 yr. Egg masses were placed in each cage (one egg mass per plant). Almost no feeding was observed in the Bt-cry3A lines, and only minor feeding was observed in the Bt-cry1Ia1 lines in either year. On the S. chacoense-derived line, there was significantly less defoliation than on either the susceptible line or the glandular trichome line in 2003. In 2004, there was significantly higher defoliation on the S. chacoense-derived line than on the susceptible line or glandular trichome line. The defoliation of the Solanum chacoense-derived line was largely due to larvae clipping the petioles, rather than consumption of the leaves. Defoliation on the glandular trichome line did not differ significantly from the defoliation of the susceptible line, suggesting glandular trichomes may not be effective in controlling larvae and preventing defoliation. This study suggested that Bt can provide high levels of resistance, but the natural resistance mechanisms tested here are variable for control of Colorado potato beetle larvae in no-choice situations.     

A freeze-dried diet to test pathogens of Colorado potato beetle

The Colorado potato beetle is an important pest on potato, eggplant, and tomato. Because Colorado potato beetles develop resistance to insecticides quickly, new methods are needed for control. Bacillus thuringiensis is the only bacterium to successfully control Colorado potato beetle. Until recently, one of the drawbacks to testing bacteria against the Colorado potato beetle has been the lack of an artificial diet for screening. Previous artificial diets will only be consumed by Colorado potato beetle larvae when fresh. To improve storage, we developed a freeze-dried diet, based on a 96-well plate, suitable to feed larvae for the duration of a bioassay. Individual diet components were tested both for their effect on insect growth and on pathogen toxicity. When the preservatives, methylparaben and sorbic acid, were removed from the diet, the average weight of second instar larvae increased from 7.9 mg to greater than 9.8 mg. The preservatives inhibited the growth of two of the bacteria tested, Photorhabdus luminescens HM and Chromobacterium sp. PRAA. The removal of these preservatives also allowed for fungal growth and reduced survival from 94 to 38%. Removing diet preservatives, that inhibited the growth of Chromobacterium sp. PRAA, increased the total mortality of the larvae as well as reducing the time needed to kill 50% of the larvae. Compared to incorporation of bacteria into molten diet, the total mortality of Colorado potato beetle fed either P. luminescens HM or Chromobacterium sp. PRAA on freeze-dried diet doubled. Preparation of freeze-dried diet need not be synchronized with the insect or the pathogen. The freeze-dried diet gave consistent results as measured by low control mortality and pathogen toxicity over time.     

Use of sperm precedence to infer the overwintering cost of insecticide resistance in the Colorado potato beetle

1 Resistance to insecticides is a model system for studying adaptation. Although selection for resistance is always strong in areas and seasons where populations are exposed to insecticides, costs of resistance, which may only be expressed in the absence of insecticide use, will shape how quickly resistance will evolve.
2 We used sperm precedence to measure the shifts in resistance to imidacloprid in a natural population during winter diapause in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Because adult beetles overwinter with viable autumn sperm, but sperm from spring matings take precedence over stored sperm, we used the difference in resistance of springmated and autumn-mated overwintered females to estimate the shift in resistance during the winter. Offspring of autumn-mated females were 2.7- or 2.5-fold more resistant compared with offspring from spring-mated females in two replicate trials.
3 We also measured the resistance of late summer and spring emergent adults in fields treated and untreated with imidacloprid in the first year. Adults from the treated field were 13.7-fold more resistant and adults from the untreated field were 2.6-fold more resistant compared with the next spring's emergers.
4 These large costs of resistance observed in the field and inferred from resistance declines during diapause help to explain the observation that imidacloprid resistance has increased only slowly over the decade of widespread use against this species, and how insecticide resistance in general can cycle annually.     

Plant volatiles moderate response to aggregation pheromone in Colorado potato beetle

Abstract:  The orientation of the Colorado potato beetle, Leptinotarsa decemlineata , to a male-produced aggregation pheromone, ( S )-3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol, a three-component plant attractant blend [comprised of ( Z )-3-hexenyl acetate + (±)-linalool + methyl salicylate], and other potato volatiles (nonanal and 2-phenylethanol) were tested. All compounds were previously shown to be active in coupled gas chromatography/electroantennogram experiments. Both the three-component plant attractant blend and 2-phenylethanol were attractive to adult beetles. While male beetles oriented preferentially to both plant attractants vs. a control, females showed little preference. Combining the plant attractants with the pheromone resulted in sexually dimorphic responses similar to those seen with either plant attractant alone. Addition of nonanal abolished the sexually dimorphic response to the pheromone + 2-phenylethanol blend; the new three-component blend was attractive to both sexes. In both laboratory bioassays and field experiments, a combination of the pheromone + the three-component plant attractant was preferred over the plant attractant alone. Thus, it seems likely that combinations of pheromone + plant volatiles may be the most efficacious for field use.     

Colonization of potato fields in eastern North Carolina by Colorado potato beetle

Rotated and non-rotated commercial potato fields were sampled intensively to follow Colorado potato beetle,Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), colonization and subsequent oviposition patterns in the spring of 1990 and 1991. Maximum densities of colonizing adults ranged from 0 to 14,891/ha and maximum egg mass densities ranged from 0 to 48,451/ha. Crop rotation generally resulted in lower potato beetle populations. Regardless of crop rotation management practices, colonization of fields planted in potatoes began at field edges and progressed inward in all fields for both years. Management of potato beetles is discussed in light of these findings.     

Contribution of Ldace1 gene to acetylcholinesterase activity in Colorado potato beetle

The Colorado potato beetle (CPB), Leptinotarsa decemlineata is an important economic pest of potato worldwide. Resistance to organophosphates and carbamates in CPB has been associated in some cases to point mutations in the acetylcholinesterase (AChE) gene Ldace2, an orthologue of Drosophila melanogaster Dmace2. In this paper we report cloning and sequencing of Ldace1, an orthologue of Anopheles gambiae Agace1 that was previously unknown in CPB. The Ldace1 coding enzyme contains all residues conserved in a functionally active AChE. Ldace1 is expressed at higher levels (between 2- and 11-fold) than Ldace2 in embryos, in the four larval instars and in adults. Specific interference of Ldace1 by means of dsRNA injection resulted in a reduction of AChE activity to an approximate 50% compared to control, whilst interference of Ldace2 reduced AChE activity to an approximate 85%. Analysis of zymograms of AChE activity after interference indicates that LdAChE1 is the enzyme predominantly responsible for the activity visualised. Interference of Ldace1 in CPB adults caused a significant increase in mortality (43%) as early as three days post-injection (p.i.), suggesting the essential role of Ldace1. Interference of Ldace2 also caused a significant increase in mortality (29%) compared to control, although at seven days p.i. The effect of the interference of Ldace1 on susceptibility to the organophosphate chlorpyrifos points out that LdAChE1 could be a main target for this insecticide. In the light of our results, studies associating resistance in CPB to mutations in Ldace2 should be reviewed, taking into consideration analysis of the Ldace1 gene.     

Resistance of three wild tuber-bearing potatoes to the Colorado potato beetle

The resistance of Solanum okadae Hawkes & Hjert. (PI 458367), Solanum oplocense Hawkes (PI 473368), and Solanum tarijense Hawkes (PI 414150) to the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Chrysomelidae: Chrysomelini), was studied. In replicated field trials all three accessions showed a high level of resistance to the beetle. No significant genetic variability between genotypes of the same species was found. Results from host acceptance behavior experiments, suitability for larval development tests, foliage consumption tests, and adult survival and oviposition tests supported the hypothesis that the mode of resistance differs between the three wild Solanum species. Solanum okadae and S. oplocense affected host acceptance and consumption. Because the beetle reacted differently to these two species it was hypothesized that the antifeedant chemical(s) differed in nature or quantity. S. tarijense contrasted with the other two species by affecting mostly adult colonization and oviposition.     

Quiescence in the Colorado potato beetle, Leptinotarsa decemlineata

Quiescence (death feigning) was induced in the Colorado potato beetle, Leptinotarsa decemlineata by mechanical disturbance of live and artificial plants on which beetles were walking, feeding or grooming. The response consisted of a sudden cessation of activity lasting 12 to 500 s. Recovery from this immobile state was usually preceded by either antennal or leg movement. The time taken for beetles to come out of the immobile state (i.e. recovery time) was not dependent on age or sex of beetles or on stimulus duration, but was dependent on degree of displacement and on satiation level. Repeated stimulations led to longer recovery times and to a smaller proportion of responding beetles. Stimulation within the immobile state also prolonged recovery time. The most effective frequency of mechanical disturbance for startling beetles was 30 Hz. This response is presumed to be part of the extensive defense repertoire of this beetle, but we have so far been unable to discover a natural enemy that will elicit it.     

Orientation of Colorado potato beetle to natural and synthetic blends of volatiles emitted by potato plants

1 Behavioural responses of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), to volatiles emitted from solanaceous host plants (potato and tomato), a non‐host legume (soybean), and 13 synthetic blends or three individual chemicals emitted by potato plants were investigated in laboratory bioassays. 2 Both male and female CPB were attracted to volatiles emitted by mechanically damaged potato foliage, but not to mechanically damaged tomato foliage; CPB offered a choice between the two damaged solanaceous plants did not show a preference. 3 Among 16 odourous blends or individual chemical components of potato plant emissions tested, six blends were attractive, two were repellent, and eight elicited no preference in laboratory bioassays. Volatile blends containing relatively high amounts of the green leaf volatiles (E)‐2‐hexen‐1‐ol and (Z)‐3‐hexen‐1‐ol, or the sesquiterpene β‐ caryophyllene, were unattractive or repellent. Minimal blends attractive to CPB were comprised of (Z)‐3‐hexenyl acetate (±)‐linalool and methyl salicylate: the combination of all three chemicals elicited sexually dimorphic attraction of males; two component blends comprised of (Z)‐3‐hexenyl acetate and either (±)‐linalool and methyl salicylate attracted both sexes. Individual compounds were inactive. No significant difference was noted between two attractive blends, or an attractive synthetic blend vs. mechanically damaged potato foliage. 4 These results show that CPB are attracted to blends of specific chemicals emitted by their host plants and provide a basis for the use of plant attractants as a component of integrated management of pestiferous populations.     

A QTL that confers resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]) in tetraploid potato populations segregating for leptine

Genetic resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]) from Solanum chacoense has been incorporated in the tetraploid potato selection, ND4382-19, which is highly resistant and contains moderate level of foliar leptines. We recently reported using ND4382-19 progeny, population ND5873 (ND4382-19 × Chipeta), to map two genes that segregated as complementary epistatic genes that allow accumulation of leptinidine (Lep) and acetyl-leptinidine (AL) on chromosomes 2 and 8, respectively. We describe here the characterization of a second half-sib population NDG116 (ND4382-19 × N142-72). In this population, solasodine from parent N142-72, which has Solanum berthaultii in its background, was predominant over solanidine-based alkaloids. Concentrations of solanidine, leptinidine, and acetyl-leptinidine were 15-, 5-, and 14-fold lower than in the ND5873 population. Nevertheless, Lep and AL mapped to the same locations on chromosomes 2 and 8 of parent ND4382-19, respectively. The two populations were evaluated for resistance to Leptinotarsa in field assays, and by detached leaf assay for population NDG116. In both families, QTL analysis identified a major QTL from ND4382-19 on the distal end of chromosome 2, close to the Lep locus. The contribution of this QTL to resistance ranged from 11 to 34% for ND5873 at four field sites. Contribution to resistance from the linkage group that contains the gene AL for the accumulation of leptine was not detected. In family NDG116, the same chromosome 2 QTL was detected for field and detached leaf assays, explaining 26 and 12% of the variance for defoliation and larval development, respectively. These data may indicate another resistance mechanism besides leptine in the Leptinotarsa resistance observed in these populations.     

Photosynthetic responses of potato to Colorado potato beetle injury and differences in injury between adult males and females

Establishing rates of injury to plants and the physiological impact of this injury provides essential data in the development of economic injury levels, but variation of sex effects is not often considered. Here, we examined injury by the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), larvae and adult males and females on potato, Solanum tuberosum L. (Solanaceae). Specifically, we looked for adult sex differences between males and females in injury rates (= leaf consumption rates), and examined the impact of all types of injury (larval, adult male, and adult female) on gas exchange parameters of remaining potato leaf tissue. Experiments were conducted in the field and in growth chambers on Frito‐Lay proprietary and Pike chipping‐potato varieties at pre‐blooming and blooming stages. We found no change in photosynthetic rates on remaining (uninjured) leaf tissue infested with male, female, or fourth‐stage larva of Colorado potato beetle. However, when the midrib was cut in trials with male beetles, the remaining tissue above the injury exhibited photosynthetic rate reductions as a result of stomatal limitations. These findings are consistent with the pattern that we and other researchers have observed with gross tissue removal by various insects on other plant species. Adult females consumed more tissue than males, and temperature was positively correlated with feeding rates for both sexes. Sex‐related differences in feeding rate are most important to studies quantifying consumption rates for economically important species because of its potential impact on resulting economic injury level calculations.