James A. Ottea

Genetics, Synergists, and Age Affect Insecticide Sensitivity of the Honey Bee, Apis mellifera

The number of honey bee colonies in the United States has declined to half of its peak level in the 1940s, and colonies lost over the winter have reached levels that are becoming economically unstable. While the causes of these losses are numerous and the interaction between them is very complex, the role of insecticides has garnered much attention. As a result, there is a need to better understand the risk of insecticides to bees, leading to more studies on both toxicity and exposure. While much research has been conducted on insecticides and bees, there have been very limited studies to elucidate the role that bee genotype and age has on the toxicity of these insecticides. The goal of this study was to determine if there are differences in insecticide sensitivity between honey bees of different genetic backgrounds (Carniolan, Italian, and Russian stocks) and assess if insecticide sensitivity varies with age. We found that Italian bees were the most sensitive of these stocks to insecticides, but variation was largely dependent on the class of insecticide tested. There were almost no differences in organophosphate bioassays between honey bee stocks (<1-fold), moderate differences in pyrethroid bioassays (1.5 to 3-fold), and dramatic differences in neonicotinoid bioassays (3.4 to 33.3-fold). Synergism bioassays with piperonyl butoxide, amitraz, and coumaphos showed increased phenothrin sensitivity in all stocks and also demonstrated further physiological differences between stocks. In addition, as bees aged, the sensitivity to phenothrin significantly decreased, but the sensitivity to naled significantly increased. These results demonstrate the variation arising from the genetic background and physiological transitions in honey bees as they age. This information can be used to determine risk assessment, as well as establishing baseline data for future comparisons to explain the variation in toxicity differences for honey bees reported in the literature.

Selection, egg viability, and fecundity of the sugarcane borer (Lepidoptera: Crambidae) with tebufenozide.

Abstract Two separate attempts to select the sugarcane borer, Diatraea saccharalis (F.), for resistance to tebufenozide were unsuccessful. Both selected colonies were lost after the fourth generation due to a lack of oviposition. Differences were not detected in fecundity or percent egg viability for 5-d-old third instars exposed to concentrations (EC5, EC15, and EC30) of tebufenozide for 7 d. Decreases (P ≤ 0.01) in mean female pupal weights were detected in larvae exposed to EC15 and EC30 concentrations. An ovicidal impact using serial dilutions of tebufenozide (10, 100, and 200 ppm) also was detected. Percent viability was reduced from 98% for untreated eggs to 61% for eggs dipped in 10 ppm and below 6% for eggs dipped in ≥100 ppm. Eggs treated with 200 ppm did not hatch. Though some embryonic development was observed on eggs treated with the high concentrations (100 and 200 ppm), sclerotization of head capsule was not apparent. The ovicidal property of tebufenozide may enhance its effectiveness in controlling populations of the D. saccharalis on an area-wide basis. Fecundity and egg viability were affected in later generations of selection; however, separate studies assessing individuals that were exposed to sublethal concentration (EC5, EC15, and EC30) of tebufenozide as third instars for 7 d in one generation did not detect differences.

Systemic effects of thiamethoxam and chlorantraniliprole seed treatments on adult Lissorhoptrus oryzophilus (Coleoptera: Curculionidae) in rice.

BACKGROUND Feeding assays using adult rice water weevils and foliage of plants treated as seeds with chlorantraniliprole and thiamethoxam at different rates were conducted to evaluate the systemic adulticidal and feeding effects. Dose-mortality relationships were determined for thiamethoxam seed treatments by combining leaf area lost due to feeding and insecticide residues analyzed by LC/MS/MS. Changes in adulticidal activity of thiamethoxam were also investigated by contrasting adult mortalities at the 5-6-leaf and tillering stages of rice. RESULTS Adult weevil mortalities and leaf consumption rates on foliage were affected in thiamethoxam but not in chlorantraniliprole treatments when rice was at the 6-7-leaf stage. The LD(50) for weevils feeding on thiamethoxam-treated rice at the 2-3-leaf stage was 447 pg insecticide weevil(-1) (95% CL: 25-830 pg weevil(-1)) but was lower (142 pg weevil(-1); 95% CL: 102-180 pg weevil(-1)) in experiments with 3-4-leaf-stage plants. Mortalities on leaves from 5-6-leaf-stage plants were consistently higher than on leaves from tillering plants. Thiamethoxam residues measured by ELISA increased with seed treatment rate and differed between plant stages. CONCLUSION The LD(50) values developed in this study are the first values for leaf-feeding insects on foliage of plants treated as seeds with thiamethoxam. The attrition of adulticidal activity of thiamethoxam in foliage of older plants may help to explain the reduced effectiveness of seed treatments against rice water larvae that is seen at later stages of rice growth in field studies. The differential activity of these two seed treatments on adults suggests that adult mortality contributes to the field efficacy of thiamethoxam but not to that of chlorantraniliprole.

Effects of Chlorantraniliprole and Thiamethoxam Rice Seed Treatments on Egg Numbers and First Instar Survival of Lissorhoptrus oryzophilus (Coleoptera: Curculionidae)

ABSTRACT Effects of treatment of rice seeds with an anthranilic diamide, chlorantraniliprole, and a neonicotinoid, thiamethoxam, on egg laying and first instar survival in rice water weevil, Lissorhoptrus oryzophilus Kuschel, were examined under greenhouse conditions. Exposure of adult weevils to rice (6–7 leaf stage) grown from seeds treated with chlorantraniliprole and thiamethoxam resulted in reduction in numbers of eggs and first instars. The low egg numbers by adults exposed to chlorantraniliprole-treated plants was confirmed as a sublethal effect on adults: adult survival was not impacted after 4 d of feeding on foliage from chlorantraniliprole-treated plants but the number of eggs laid by these weevils was reduced when released on untreated plants. Furthermore, a comparison of first instar emergence from chlorantraniliprole-treated plants and from untreated plants infested with weevils previously exposed to this chemical suggested that chlorantraniliprole was also reducing egg or first instar survival. In contrast, adults that fed on foliage from thiamethoxam-treated plants showed increased mortality. Possible sublethal effects of thiamethoxam on the number of eggs laid by adults were investigated by infesting untreated plants with weevils that survived exposure to thiamethoxam via foliar feeding (7 µg active ingredient / seed). Prior exposure to thiamethoxam through adult feeding reduced egg numbers. However, potential larvicidal or ovicidal effects of thiamethoxam seed treatments could not be detected in this study because of low first instar emergence from both thiamethoxam-treated plants and from untreated plants infested with weevils previously exposed to this chemical. These experiments revealed that the two seed treatments accomplish weevil control in different ways.

Susceptibility of Diatraea saccharalis (Lepidoptera: Crambidae) to Tebufenozide

Abstract Dosage-mortality baselines were determined for first and newly molted third instars of the sugarcane borer, Diatraea saccharalis (F.), with tebufenozide incorporated into an artificial diet. Using death as an endpoint for these assays, LC50 and LC90 values were estimated to be 0.17 and 0.41 ppm for the first and 0.87 and 3.67 ppm for third instars, respectively. Developmental effects also were observed and were defined to include mortality as well as physical impairment and retarded or prolonged development with absence of feeding. Concentrations causing developmental effects in treated larvae (EC50 and EC90) were 5.8- and 3.8-fold lower than the LC50 and LC90, respectively. The critical time of exposure to tebufenozide was determined for developmentally synchronous third instars to be less than 12 h postecdysis. Mortality through adult emergence significantly decreased from 92% (treatment at 0 h postecdysis) to 30% (treatment at 12 h postecdysis), when exposed to tebufenozide at the EC50. For surviving larvae, length of development time until pupation was significantly increased and female pupal weight significantly decreased when larvae were treated at 0 h postecdysis into the third instar. In addition, to modifying the traditional ways of assessing mortality 0–3 d postapplication, this study points out the value of using other approaches to pesticide assessment, especially where insect growth regulators are involved.

Activity of chlorantraniliprole and thiamethoxam seed treatments on life stages of the rice water weevil as affected by the distribution of insecticides in rice plants

BACKGROUND The systemic insecticides chlorantraniliprole (CAP) and thiamethoxam (TMX), applied to rice as seed treatments, may affect multiple life stages of the rice water weevil, Lissorhoptrus oryzophilus. Effects of CAP and TMX on adult survival, egg-laying and first- and late-instar survivals were determined by infesting plants treated as seeds with different rates of insecticides. The biological activity was related to insecticidal concentrations in leaves, shoots and roots. RESULTS CAP did not affect adult survival but decreased egg numbers and reduced the survival of the first and late instars. The greatest reduction in weevil population occurred in late instars feeding on roots. In contrast, TMX reduced adult survival and egg and larval numbers. The high biological activity of CAP on root-feeding stages was consistent with the accumulation of CAP in roots, whereas in TMX-treated plants the high activity on adults correlated with high concentrations of TMX in leaves and stems. CONCLUSIONS The differential activity of insecticides on adults suggests poor inherent potency of CAP as an adulticide and/or its limited systemicity in foliage. The distribution of insecticide in specific plant parts can be attributed to the different physicochemical properties of CAP and TMX. The field implications of this research on management of L. oryzophilus are discussed.

Multiple mechanisms for enhancement of glutathione S-transferase activities in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Abstract Glutathione S-transferase activities toward 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were measured from fat body homogenates of chemically-treated and untreated Spodoptera frugiperda larvae. In assays with untreated insects, four GST activities were separable on the basis of substrate preference and pH optima: two active in DCNB metabolism and two with activity toward CDNB. Both GST activities toward DCNB increased following injection of 8-methoxypsoralen (8-MOP) but were unaffected by pentamethylbenzene (PMB). Induction by 8-MOP was inhibited in a dose-dependent manner by co-administration of PMB, suggesting that PMB acts as an antagonist of 8-MOP-mediated induction. In contrast, activities toward CDNB increased following PMB, but not 8-MOP injection, and co-administration of 8-MOP blocked PMB-mediated enhancement of one GST/CDNB activity, but had no effect on the other. These results confirm previous reports of multiple forms of GSTs in insects and suggest that expression of GST activities in this insect is controlled by multiple mechanisms.

Association of Esterases with Insecticide Resistance in Culex quinquefasciatus (Diptera: Culicidae)

ABSTRACT The southern house mosquito, Culex quinquefasciatus Say, is a competent vector of human disease and an important target of mosquito abatement programs. However, these management programs have been compromised by development of insecticide resistance. In the current study, susceptibilities to naled and resmethrin, two adulticides used in mosquito abatement, were monitored using a topical and contact bioassay, respectively, in five field- collected populations of C. quinquefasciatus (MARC, HOOD1, HOOD2, MINLOVE, and THIB). Frequencies of resistance, measured as survival after treatment with discriminating concentrations (i.e., sufficient to kill>90% of a reference susceptible strain) were high (88.0–96.8%) in all field collections treated with naled, but were variable (3.3–94.2%) with resmethrin. In addition, esterase activities in mosquitoes from these collections were quantified using &agr;-naphthyl acetate and ranged from 1.08 to 3.39 µmol &agr;-naphthol produced min-1 mg prot-1. Heightened activities were associated with decreased insecticide susceptibility in HOOD1, THB3, and MINLOVE but not HOOD2. Esterases were visualized using native Polyacrylamide gel electrophoresis, and intra- and interstrain differences in banding patterns were detected. In addition, esterases from MINLOVE mosquitoes were more numerous and intensely staining when compared with those from a laboratory-susceptible strain. Finally, naled synergized the toxicity of resmethrin in populations with decreased insecticide susceptibility and increased esterase activity by 2.5– (MINLOVE) to three-fold (THIB). Results from this study will allow management strategies for populations of C. quinquefasciatus to be optimized, and provide a foundation for further studies exploring use of esterase inhibitors as synergists of pyrethroid toxicity.

Pteridine levels and head weights are correlated with age and colony task in the honey bee, Apis mellifera

Background. The age of an insect strongly influences many aspects of behavior and reproduction. The interaction of age and behavior is epitomized in the temporal polyethism of honey bees in which young adult bees perform nurse and maintenance duties within the colony, while older bees forage for nectar and pollen. Task transition is dynamic and driven by colony needs. However, an abundance of precocious foragers or overage nurses may have detrimental effects on the colony. Additionally, honey bee age affects insecticide sensitivity. Therefore, determining the age of a set of individual honey bees would be an important measurement of colony health. Pteridines are purine-based pigment molecules found in many insect body parts. Pteridine levels correlate well with age, and wild caught insects may be accurately aged by measuring pteridine levels. The relationship between pteridines and age varies with a number of internal and external factors among many species. Thus far, no studies have investigated the relationship of pteridines with age in honey bees. Methods. We established single-cohort colonies to obtain age-matched nurse and forager bees. Bees of known ages were also sampled from colonies with normal demographics. Nurses and foragers were collected every 3–5 days for up to 42 days. Heads were removed and weighed before pteridines were purified and analyzed using previously established fluorometric methods. Results. Our analysis showed that pteridine levels significantly increased with age in a linear manner in both single cohort colonies and colonies with normal demography. Pteridine levels were higher in foragers than nurses of the same age in bees from single cohort colonies. Head weight significantly increased with age until approximately 28-days of age and then declined for both nurse and forager bees in single cohort colonies. A similar pattern of head weight in bees from colonies with normal demography was observed but head weight was highest in 8-day old nurse bees and there was no relationship of head weight with age of foragers. Discussion. Although the relationship between pteridine levels and age was significant, variation in the data yielded a +4-day range in age estimation. This allows an unambiguous method to determine whether a bee may be a young nurse or old forager in colonies with altered demographics as in the case of single cohort colonies. Pteridine levels in bees do not correlate with age as well as in other insects. However, most studies used insects reared under tightly controlled laboratory conditions, while we used free-living bees. The dynamics of head weight change with age is likely to be due to growth and atrophy of the hypopharyngeal glands. Taken together, these methods represent a useful tool for assessing the age of an insect. Future studies utilizing these methods will provide a more holistic view of colony health.

Characterization and transcriptional analyses of cDNAs encoding three trypsin‐ and chymotrypsin‐like proteinases in Cry1Ab‐susceptible and Cry1Ab‐resistant strains of sugarcane borer, Diatraea saccharalis

Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is responsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab‐susceptible (Cry1Ab‐SS) and Cry1Ab‐resistant (Cry1Ab‐RR) strains. Full‐length cDNAs encoding three trypsin‐ and three chymotrypsin‐like proteinases were sequenced from Cry1Ab‐SS and Cry1Ab‐RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, including signal peptide, conserved His‐Asp‐Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab‐SS and Cry1Ab‐RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab‐SS and Cry1Ab‐RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab‐SS and Cry1Ab‐RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly associated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.