The DOMON domain protein LmKnk contributes to correct chitin content, pore canal formation and lipid deposition in the cuticle of Locusta migratoria during moulting.

Insects prevent uncontrolled penetration of water and xenobiotics by producing an impermeable cuticle. The major component of the cuticle is chitin that adopts a crystalline structure thereby contributing to cuticle stability. Our understanding of the contribution of chitin to the cuticle barrier function is limited. Here, we studied the role of the DOMON domain protein Knickkopf (LmKnk) that is involved in chitin organization and cuticle permeability in the migratory locust Locusta migratoria. We show that LmKnk localizes to the chitin layer in the newly produced cuticle. Injection of double-stranded RNA targeting LmKnk (dsLmKnk) in locust nymphs caused failure of moulting to the next stage. Histological experiments revealed that apolysis, i.e., the detachment of the old cuticle from the body surface, was normal; however, the newly synthesized cuticle was thinner than the cuticle of the control insects. Indeed, chitin content dropped after suppression of LmKnk expression. As seen by transmission electron microscopy, crystalline chitin organization was lost in dsLmKnk-treated insects. In addition, the structure of pore canals, which are lipid transporting routes in the cuticle, was abnormal. Consistently, their content was reduced and, probably by consequence, lipid deposition on the cuticle was decreased after injection of dsLmKnk. Suppression of LmKnk transcript levels rendered L. migratoria more susceptible to each of four selected insecticides including malathion, chlorpyrifos, carbaryl and deltamethrin. Overall, our data show that LmKnk is needed for correct chitin amounts and organization, and their changes ultimately affect cuticular permeability in L. migratoria.

V-ATPase subunit a is required for survival and midgut development of Locusta migratoria.

The vacuolar-type H+ -ATPase (V-ATPase) is an ATP-dependent proton pump, which regulates various cellular processes. To date, most functional studies on V-ATPases of insects have focused on subunits of the V1 complex, and there is little information on the VO genes. In this study, two cDNA sequences of LmV-ATPase a were identified in Locusta migratoria. RT-qPCR analysis revealed that LmV-ATPase a1 and LmV-ATPase a2 are differentially expressed in various tissues and developmental stages. Injection of dsRNA for the common region of LmV-ATPase a1 and LmV-ATPase a2 into third-instar nymphs resulted in a significant suppression of LmV-ATPase a. The injected nymphs ceased feeding, lost body weight and finally died at a mortality of 98.6%. Furthermore, aberrations of midgut epithelial cells, the accumulation of electron-lucent vesicles in the cytoplasm, and a partially damaged brush border were observed in dsLmV-ATPase a-injected nymphs using transmission electron microscopy. Especially, the mRNA level of wingles, and notch genes were dramatically down-regulated in the dsLmV-ATPase a-injected nymphs. Taken together, our results suggest that LmV-ATPase a is required for survival and midgut development of L. migratoria. Hence, this gene could be a good target for RNAi-based control against locusts.

[The value of serum dehydroepiandrosterone sulfate in differential diagnosis of Cushing's syndrome].

Objective: To evaluate the changes and diagnostic value of serum dehydroepiandrosterone sulfate (DHEAS) in Cushing's syndrome (CS) with different etiologies. Methods: The study retrospectively recruited patients diagnosed as CS in Drum Tower Hospital affiliated to Nanjing University Medical School between January 2012 and June 2019, including 36 patients (8 males, 28 females, with an average age of 44 years) with Cushing disease (CD) and 64 patients (6 males, 58 females, with an average age of 39 years) with adrenal CS (ACS). Meanwhile, 97 patients diagnosed as nonfunctional adrenal adenoma (NFA) were also included as controls. Clinical characteristics, laboratory data, adrenocorticotropic hormone (ACTH), serum DHEAS level and sex-and age-adjusted DHEAS ratio of the three groups were collected. The sensitivity and specificity of DHEAS and its ratio in differential etiology diagnosis of CS were compared using receiver operating characteristic (ROC) curve analysis. Results: Compared to NFA group, ACS patients had lower DHEAS levels [0.39 (0.39, 0.63) µmol/L vs 2.96 (1.92, 4.60) µmol/L, P<0.01] and lower DHEAS ratio [0.58 (0.27, 0.98) vs 3.95 (3.08, 6.83), P<0.01]. DHEAS [6.49 (4.32, 11.63) µmol/L] and DHEAS ratio [9.17 (4.49, 15.41)] in CD patients were significantly higher compared to those in NFA and ACS patients (all P<0.01). There were 53 ACS patients (82.8%) with suppressed ACTH level (<2.2 pmol/L) and 11 patients (17.2%) with normal/high ACTH level (≥2.2 pmol/L). The level of 24 hour urine free cortisol in normal/high ACTH level group was lower than the suppressed ACTH group [(1 299±511) nmol/24 h vs (1 972±876) nmol/24 h, P=0.04]. No significant differences were found in the DHEAS and DHEAS ratio between the two groups. ROC analysis showed that the area under the curve of serum DHEAS and DHEAS ratio in diagnosing ACS from CD was 0.997 and 0.990, respectively. The optimal cut-off values for DHEAS and its ratio were 2.06 µmol/L and 2.10, respectively. The diagnostic sensitivity and specificity of DHEAS were 97.5% and 100%, and those of DHEAS ratio were 95.0% and 100%, respectively. Conclusion: There are significant differences in serum DHEAS level and DHEAS ratio between ACS and CD patients, which might be used as indicators for the identification of the two main CS etiologies, especially in the identification of ACS patients without plasma ACTH suppression from CD patients.

[Clinical features and outcomes of surgical versus conservative management in patients with subclinical Cushing's syndrome].

Objective: To examine the clinical characteristics and metabolic features of subclinical Cushing's syndrome (SCS), and determine the effects of surgical or conservative approaches on the hormone levels and metabolic comorbidities in patients with SCS, thereby providing the evidence for decision-making in SCS management. Methods: A total of 56 consecutive SCS patients were selected in Drum Tower Hospital Affiliated to Nanjing University Medical School between 2010 and 2018, with 41 patients undergoing surgical treatment and 15 patients receiving conservative therapy. Meanwhile, 56 and 68 cases of sex-and age-matched patients diagnosed as nonfunctional adrenal adenoma (NFA) and adrenal Cushing's syndrome (CS) were included respectively. Clinical characteristics of patients in different groups were compared. Hormone levels and metabolic comorbidities were also observed during follow-up. Results: There were 56 SCS patients, including 15 males and 41 females, with an age of (52.0±12.6) years. The circadian rhythms of adrenocorticotropic hormone (ACTH) and cortisol disappeared in CS and SCS groups. Compared to NFA group, patients with SCS were characterized by suppressed plasma ACTH level [2.40 (1.11, 4.33) pmol/L vs 4.23 (2.74, 6.26) pmol/L], elevated midnight cortisol level [(240±121) nmol/L vs (59±8) nmol/L] and increased cortisol level after 1 mg overnight dexamethasone suppression test [(241±130) nmol/L vs (34±12) nmol/L] (all P<0.01). The derangement of ACTH-cortisol axis was more obvious in CS patients compared to SCS patients. The prevalence of hypertension, glucose intolerance, dyslipidemia and osteopenia/osteoporosis were higher in SCS patients compared to NFA patients (75.0% vs 41.1%, 33.9% vs 12.5%, 62.5% vs 28.6%, 35.7% vs 8.9%, all P<0.05). The 24-hour urine free cortisol correlated positively with systolic blood pressure, glycated hemoglobin A1c (HbA1c) and fasting blood glucose in SCS patients (r=0.335, 0.562 and 0.463, respectively, all P<0.05). In the surgical group, body weight, body mass index (BMI) and blood pressure decreased significantly after surgery (all P<0.05). Glucose intolerance/diabetes mellitus improved in 6 of 9 patients, BMI of 4 of 11 overweight/obesity patients normalized, and hypertension in 54.5% of patients (12/22) showed improvement after surgery. However, no alterations of hormone levels and metabolic parameters were observed in conservatively-managed patients. Conclusions: Patients with SCS are characterized by mild autonomous cortisol secretion and increased risk of metabolic comorbidities. Compared with conservative management, hormone abnormalities were corrected and metabolic abnormalities were improved in some SCS patients after surgery.

LmCDA1 organizes the cuticle by chitin deacetylation in Locusta migratoria.

Cells produce an extracellular matrix (ECM) with a stereotypic organization that is important for tissue function. The insect cuticle is a layered ECM that mainly consists of the polysaccharide chitin and associated proteins adopting a quasi-crystalline structure. Our understanding of the molecular mechanisms deployed during construction of the highly ordered protein-chitin ECM so far is limited. In this study, we report on the role of the chitin deacetylase 1 (LmCDA1) in the organization of the protein-chitin ECM in the migratory locust Locusta migratoria, and LmCDA1 localizes predominantly to the apical tier of the protein-chitin ECM, but it is also found in lower regions. Reduction of LmCDA1 function correlates with lower amounts of chitin and impedes conversion of chitin to chitosan by deacetylation. Establishment of the quasi-crystalline architecture of the protein-chitin ECM is, however, independent of LmCDA1 activity, but it is dependent on another chitin deacetylase, LmCDA2, which has no detectable effects on chitin deacetylation and, as shown previously, no influence on chitin content. Our data reveal that LmCDA1 and LmCDA2 act in parallel and independently from each other in defining the dimensions of the cuticle. Both enzymes are non-uniformly distributed within the protein-chitin matrix, suggesting a site-autonomous function.

Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae).

The purpose of this study was to examine whether the expression of two chitin synthase genes, AgCHS1 and AgCHS2, can be repressed by chitosan/AgCHS dsRNA-based nanoparticles through larval feeding in Anopheles gambiae. The AgCHS1 transcript level and chitin content were reduced by 62.8 and 33.8%, respectively, in the larvae fed on chitosan/AgCHS1 dsRNA nanoparticles compared with those of the control larvae fed on chitosan/GFP dsRNA nanoparticles. Our study suggested for the first time that RNA interference (RNAi) in mosquito larvae is systemic, and demonstrated that the larvae fed on the nanoparticles assembled from AgCHS1 and AgCHS2 dsRNA increased larval susceptibilities to diflubenzuron, and calcofluor white (CF) or dithiothreitol, respectively. These results suggest great potential for using such a nanoparticle-based RNAi technology for high-throughput screening of gene functions and for developing novel strategies for pest management.

Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen).

The susceptibilities to three organophosphate (OP) insecticides (malathion, chlorpyrifos, and phoxim), responses to three metabolic synergists [triphenyl phosphate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM)], activities of major detoxification enzymes [general esterases (ESTs), glutathione S-transferases (GSTs), and cytochrome P450 monooxygenases (P450s)], and sensitivity of the target enzyme acetylcholinesterase (AChE) were compared between a laboratory-susceptible strain (LS) and a field-resistant population (FR) of the oriental migratory locust, Locusta migratoria manilensis (Meyen). The FR was significantly resistant to malathion (57.5-fold), but marginally resistant to chlorpyrifos (5.4) and phoxim (2.9). The malathion resistance of the FR was significantly diminished by TPP (synergism ratio: 16.2) and DEM (3.3), but was unchanged by PBO. In contrast, none of these synergists significantly affected the toxicity of malathion in the LS. Biochemical studies indicated that EST and GST activities in the FR were 2.1- to 3.2-fold and 1.2- to 2.0-fold, respectively, higher than those in the LS, but there was no significant difference in P450 activity between the LS and FR. Furthermore, AChE from the FR showed 4.0-fold higher activity but was 3.2-, 2.2-, and 1.1-fold less sensitive to inhibition by malaoxon, chlorpyrifos-oxon, and phoxim, respectively, than that from the LS. All these results clearly indicated that the observed malathion resistance in the FR was conferred by multiple mechanisms, including increased detoxification by ESTs and GSTs, and increased activity and reduced sensitivity of AChE to OP inhibition.

Differential heat shock tolerance and expression of heat-inducible proteins in two stored-product psocids.

The recent recognition of psocids as a major concern in stored products and also the reemergence of heat treatment as a control tactic of stored-product insects led to the present investigation. The objectives of this study were to determine whether there are differences in heat shock tolerance of two species of stored-product psocids--Lepinotus reticulatus Enderlein (Trogiidae) and Liposcelis entomophila (Enderlein) (Liposcelididae)--and to determine whether heat shock proteins (HSPs) underlay such tolerance. Time-response bioassays were therefore carried out at increasing temperatures for both psocids. The lethal time (LT)50 and LT95 estimates were correlated with the expression of heat shock proteins after exposure at the same range of temperatures for 30 min. The expression of HSP was determined through Western blot analyses using HSP 70 antibody. Liposcelis entomophila was more than two-fold more tolerant than L. reticulatus for nearly all of the range of temperatures (> or = 40.0 degrees C). Expression of HSP 70 was not observed for either of the psocid species, but the expression of two low-molecular-mass heat-inducible proteins (HIPs; 23 and 27 kDa) was observed in L. entomophila. The expression of these small proteins was induced by exposure to higher temperatures, and the trend was particularly strong for HIP 27. In contrast, no expression of small heat-inducible proteins was detected in L. reticulatus, reflecting its higher susceptibility to heat treatments. The relatively high heat tolerance of L. entomophila might help explain its more common occurrence in grain stored in warmer regions of the world.

Biomechanical stability analysis of the lambda-model controlling one joint.

Computer modeling and control of the human motor system might be helpful for understanding the mechanism of human motor system and for the diagnosis and treatment of neuromuscular disorders. In this paper, a brief view of the equilibrium point hypothesis for human motor system modeling is given, and the lambda-model derived from this hypothesis is studied. The stability of the lambda-model based on equilibrium and Jacobian matrix is investigated. The results obtained in this paper suggest that the lambda-model is stable and has a unique equilibrium point under certain conditions.

Hydroprene prolongs developmental time and increases mortality in wandering-phase Indianmeal moth (Lepidoptera: Pyralidae) larvae.

Wandering phase Indianmeal moth, Plodia interpunctella (Hübner), larvae were exposed to the label rate of hydroprene (1.9 x 10(-3) mg [AI] /cm2) sprayed on concreted petri dishes. Larvae were exposed for 1, 3, 6, 12, 18, 24, and 30 h and maintained at 16, 20, 24, 28, and 32 degrees C and 57% RH until adult emergence. Larval developmental time and mortality were significantly influenced by temperature and exposure intervals. Maximum developmental time (47.2 +/- 1.3 d) occurred at 16 degrees C, and the minimum developmental time (7.0 +/- 0.5 d) occurred at 32 degrees C. Larval mortality generally increased at all of the five tested temperatures as exposure period increased. The greatest mortality (82.0 +/- 0.1%) occurred when larvae were exposed for 30 h at 28 degrees C, and minimum mortality (0.0 +/- 0.5%) occurred at 16 degrees C when larvae were exposed for 1 h. The relationships between temperature, exposure period, and developmental time were described by polynomial models, based on lack-of-fit tests. Hydroprene has potential to be an effective alternative to conventional insecticides in surface treatments for Indianmeal moth management. Response-surface models derived from this study can be used in simulation models to estimate the potential consequences of hydroprene on Indianmeal moth population dynamics.

Hydroprene prolongs developmental time and increases mortality of Indianmeal moth (Lepidoptera: Pyralidae) eggs.

Eggs of the Indianmeal moth, Plodia interpunctella (Hübner), were exposed to the labeled rate of hydroprene (1.9 x 10(-3) mg [AI]/cm2) sprayed on concreted petri dishes. These eggs were exposed for 1, 3, 6, 12, and 18 h and until hatching (continuous exposure) at temperatures of 16, 20, 24, 28, and 32 degrees C and 57% RH until the emergence of first instars. The developmental time and egg mortality were significantly influenced by temperature and exposure periods. At 16 degrees C, hydroprene did not cause differences in developmental time when eggs were exposed for different periods. At temperatures >16 degrees C, both exposure period and temperature influenced developmental time. The maximum developmental time (15.0 +/- 0.2 d) occurred at 16 degrees C, and the minimum developmental time (3.2 +/- 0.3 d) occurred at 32 degrees C. Mortality increased when eggs were exposed to hydroprene for longer periods at all of the five tested temperatures. The greatest mortality (81.6 +/- 2.1%) occurred when eggs were continuously exposed on treated surfaces at 32 degrees C. We used developmental time instead of rate (1/ developmental time) to fit simple linear or polynomial regression models to the development data. Appropriate models for developmental time and mortality were chosen based upon lack-of-fit tests. The regression models can be used in predictive simulation models for the population dynamics of Indianmeal moth to aid in optimizing use of hydroprene for insect management.

Effects of dietary conjugated linoleic acids on cellular immune response of piglets after cyclosporin A injection.

The present study investigated the effects of dietary conjugated linoleic acid (CLA) on the cellular immune response of piglets after cyclosporin A (CsA) treatment. The experimental study had a 2×2 factorial design, and the main factors consisted of diets (0% or 2% CLA) and immunosuppression treatments (CsA or saline injection). CsA injection significantly increased feed : gain (F : G) of piglets (P<0.05); however, dietary CLA significantly decreased F : G of piglets (P<0.05). Dietary CLA partly ameliorated the deterioration of the feed conversion rate caused by CsA treatment (P<0.01). CsA treatment significantly decreased the percentages of CD4+ and CD8+ T lymphocytes in the thymus (P<0.01). Dietary CLA increased the percentages of CD4+ CD8+ double-positive and CD8+ single-positive T lymphocytes in the thymus (P<0.05), and had the trend to inhibit the decrease of CD4+ T lymphocytes in the thymus after CsA injection (P=0.07). CsA treatment significantly depleted the peripheral blood CD3+, CD4+ and CD8+ T lymphocytes (P<0.01). Dietary CLA significantly increased the number of peripheral blood CD8+ T lymphocytes and interleukin-2 (IL-2) production (P<0.05), and inhibited the decreases of peripheral blood CD3+, CD4+ and CD8+ T lymphocytes counts (P<0.01) as well as IL-2 production (P<0.05) after CsA treatment. Dietary CLA partly rescued the decrease of lymphocyte proliferation after CsA injection (P<0.05). In summary, dietary CLA effectively ameliorated CsA-induced cellular immunosuppression in piglets.

Comparative susceptibility of western corn rootworm (Coleoptera: Chrysomelidae) adults to selected insecticides in Kansas.

Susceptibility of adult populations of the western corn rootworm, Diabrotica virgifera virgifera LeConte, to several insecticides was evaluated in seven Kansas counties, including Dickinson, Ford, Finney, Pottawatomie, Republic, Riley, and Stevens, between 1996 and 2002. All populations surveyed were highly susceptible to methyl parathion with the largest difference in susceptibility of only three-fold based on 16 complete bioassays for the populations from six counties over a 5-yr period. Noticeable decreases in carbaryl susceptibility were found in populations collected from Republic County between 1997 and 2001 when the cucurbitacin-carbaryl-based bait SLAM was widely used as an areawide management approach for adult corn rootworm control. However, the lowered carbaryl susceptibility returned to previous levels 1 yr after the use of SLAM was halted in the managed (treated) cornfields. This change implies possible dispersal of insects into the relatively small managed area from surrounding untreated cornfields and / or some fitness cost associated with carbaryl resistance within the population. Relative susceptibility of western corn rootworm adults also was evaluated for seven commonly used insecticides, including bifenthrin, carbaryl, chlorpyrifos, cypermethrin, fipronil, malathion, and methyl parathion. They were tested with corn rootworm adults collected from a single cornfield. Methyl parathion and bifenthrin were highly toxic to corn rootworm adults, and cypermethrin, chlorpyrifos, carbaryl, and malathion were only slightly less toxic. Although fipronil was highly toxic to adult rootworms, its activity was much slower than that of other insecticides. Thus, bifenthrin and methyl parathion were among the most effective in killing corn rootworm adults.

An acetylcholinesterase purified from the greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics.

An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the greenbug, Schizaphis graminum (Rondani). The maximum velocities (Vmax) for hydrolyzing acetylthiocholine (ATC), acetyl-(beta-methyl) thiocholine (AbetaMTC), propionylthiocholine, and S-butyrylthiocholine were 78.0, 67.0, 37.4, and 2.3 micromol/min/mg, and the Michaelis constants (Km) were 57.6, 60.6, 31.3, and 33.4 microM, respectively. More than 98% of AChE activity was inhibited by 10 microM eserine or BW284C51, but only 7% of the activity was inhibited by ethopropazine at the same concentration. Based on the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nondenaturing polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing of the purified AChE revealed three molecular forms. The isoelectric points were 7.3 for the major form and 6.3 and 7.1 for two minor forms. The major form of purified AChE showed molecular masses of 129 kDa for its native protein and 72 kDa for its subunits on SDS-PAGE. However, the purified AChE exhibited some distinctive characteristics including: (1) lack of affinity to the affinity ligand 3-(carboxyphenyl) ethyldimethyl ammonium, which has been used widely in purification of AChE from various insect species; and (2) 20-200-fold higher substrate-inhibition thresholds for ATC and AbetaMTC than AChE from other insect species. These biochemical properties may reflect structural differences of AChE purified from the greenbug compared with that from other insect species.

Cloning and sequencing of a cDNA encoding acetylcholinesterase in Colorado potato beetle, Leptinotarsa decemlineata (Say).

A cDNA encoding acetylcholinesterase (AChE, EC 1.1.1.7) was cloned from a cDNA library constructed from an insecticide-susceptible strain of Colorado potato beetle, Leptinotarsa decemlineata (Say). The complete amino acid sequence of AChE deduced from the cDNA consisted of 29 residues for the putative signal peptide and 600 residues for the mature protein with a predicted molecular weight of 67,994. Northern blot analysis of poly(A) RNA showed an approx 13.1-kb transcript. The mature protein sequence had 57 and 61% of amino acid residues identical to those of Drosophila melanogaster and Anopheles stephensi, respectively, and produced a remarkably similar hydropathy profile when compared to those of the two dipterous species. The three residues (Ser, Glu and His) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds were completely conserved when compared to the other seven AChEs from a broad range of animal species reported to date. Other properties of the deduced protein of AChE, including molecular weight and amino acid composition, agreed well with those of a previously reported study on the purified AChE from the same insect species. All these features firmly established that the cloned cDNA encodes AChE in Colorado potato beetle.

Molecular cloning and characterization of a greenbug (Schizaphis graminum) cDNA encoding acetylcholinesterase possibly evolved from a duplicate gene lineage.

An acetylcholinesterase (AChE, EC 3.1.1.7) cDNA was cloned and characterized from a greenbug (Schizaphis graminum (Rondani)) cDNA library. The complete cDNA (3283 bp) contains a 2028-bp open reading frame encoding 676 amino acid residues. The putative AChE preproenzyme has a 17 amino acid signal peptide, a 78 amino acid activation peptide and a mature enzyme of 581 amino acid residues. The first nine amino acid residues (YTSDDPLII) that were determined by sequencing the N-terminus of a 72-kDa AChE purified from the greenbug matched the nine residues deduced from the cDNA. The key amino acid residues, including the three residues Ser206 (200 in Torpedo), Glu332 (327) and His446 (440) forming a catalytic triad, three pairs of cysteine putatively forming intrachain disulfide bonds, and 10 out of the 14 aromatic residues lining the active site gorge of the Torpedo AChE, are conserved. However, Ser336 (Phe331) in the greenbug substituted an aromatic amino acid residue that is conserved in all other known AChEs. Northern blot analysis of mRNA revealed a 3.7-kb transcript, and Southern blot analysis suggested a single copy of this gene in the greenbug. The deduced amino acid sequence is most similar to AChE1 of the nematodes Caenorhabditis briggsae and C. elegans with 43% identity. Phylogenetic analysis showed that the greenbug AChE formed a cluster with those of nematodes, a squid and ticks, and grouped out of the insect cluster. This result suggests that the cloned gene evolved from a different duplicate gene lineage of insect AChEs.

Comparative toxicity of selected organophosphate insecticides against resistant and susceptible clones of the greenbug, Schizaphis graminum (Homoptera: aphididae).

Comparative toxicity of selected organophosphate (OP) insecticides against resistant and susceptible clones of the greenbug, Schizaphis graminum, were studied both in vitro and in vivo. Two resistant (OR-1 and OR-2) clones of the greenbug showed marginal to high levels of resistance to all seven OPs tested, ranging from 11- to 327-fold greater than those of a susceptible (OSS) clone. The OR-1 clone showed lower levels of resistance to phenyl (parathion and parathion-methyl) and heterocyclic (chlorpyrifos) OPs than to aliphatic OPs (dimethoate, omethoate, disulfoton, and demeton-S-methyl), whereas the OR-2 clone showed a rather broad spectrum of resistance to nearly all OP insecticides examined. In vitro inhibition of acetylcholinesterase (AChE) using six selected OP oxon analogues showed that alterations of AChE were involved in resistance to all OP compounds examined in both the OR-1 and OR-2 clones. Although the levels of insensitivity of AChE to these OPs were relatively low, ranging from 1.1- to 3.8-fold, the insensitivity spectrum of AChE to different OPs was rather broad. The general esterase activity in the OR-1 and OR-2 clones was 1.3-8. 4-fold higher than that in the OSS clone, depending on the substrates used. The AChE activity in both the OR-1 and OR-2 clones was 1.8-fold higher than that in the OSS clone. High resistance levels of the OR-2 clone to phenyl and heterocyclic OPs appeared to be associated with the ability of the esterases to hydrolyze beta-naphthyl acetate and more hydrophobic substrates.

Characterization of acetylcholinesterase purified from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae).

Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the lesser grain borer (Rhyzopertha dominica) was significantly inhibited by higher concentrations of the substrates acetylthiocholine (ATC), acetyl-(beta-methyl) thiocholine (A beta MTC) and propionylthiocholine (PTC). 2. The efficiency of AChE for hydrolyzing different substrates was ATC > A beta MTC > PTC > S-butyrylthiocholine. The enzyme activity was completely inhibited by 10(-5) M eserine or BW284C51, but was only partially inhibited by ethopropazine at the same concentration. These results confirmed that the purified enzyme was an typical insect AChE. 3. Non-denaturing and SDS polyacrylamide gel electrophoresis (PAGE) showed only one major molecular form in the purified AChE with a molecular weight of about 107,000 prior to reduction and about 56,000 after reduction, suggesting the homodimer of AChE linked with disulfide bonds.

Effect of insecticides used in corn, sorghum, and alfalfa on the predator Orius insidiosus (Hemiptera: Anthocoridae).

Orius insidoisus (Say) is an important predator in corn, sorghum, and alfalfa. Foliar insecticides commonly used on corn (permethrin, bifenthrin, and fipronil); sorghum (chlorpyrifos, carbofuran, dimethoate, and cyfluthrin); and both crops (A-cyhalothrin and ethyl parathion) were evaluated in 1998 and 1999 for their residual effects on O. insidiosus by caging adults on treated plants at several time intervals: at application (day 0) and 2, 3, and 6 d after application. In addition, imidacloprid, fipronil, and thiamethoxam used as seed treatments on corn and sorghum were tested for their effects on O. insidiosus by caging adults on plants in the presence and absence of greenbugs, Schizaphis graminum (Rondani). Finally, six of the same insecticides that also are used on alfalfa were evaluated in the field for their effects on O. insidiosus and other insects. On day 0, ethyl parathion. bifenthrin, and [lambda]-cyhalothrin on corn caused significantly higher mortality to O. insidiosus than permethrin and fipronil. Ethyl parathion and carbofuran on sorghum caused significantly higher mortality than chlorpyrifos, dimethoate, and A-cyhalothrin, which differed significantly from the control. Mortality with cyfluthrin did not differ significantly from that in the control. Insecticides had no significant effects on O. insidiosus 3 and 6 d after application in 1998 with the exception of permethrin on day 3. Similar patterns of mortality were observed in 1999 experiments. No significant differences in mortality of adults occurred with fipronil and thiamethoxam in the presence and absence of greenbugs. Imidachloprid caused significantly higher mortality to O. insidiosus adults than thiamethoxam or fipronil in some instances when greenbugs were not supplied as food. In alfalfa, the insecticides caused significant mortality to most of the insects evaluated. Ethyl parathion, permethrin, chlorpyrifos, and cyfluthrin caused significantly higher mortality to O. insidiosus than carbofuran and A-cyhalothrin, which differed significantly from the control in 1998. In 1999, all treatments significantly reduced O. insidiosus numbers and did not differ significantly from each other.

Host plant-induced changes in detoxification enzymes and susceptibility to pesticides in the twospotted spider mite (Acari: Tetranychidae).

Adult female twospotted spider mites, Tetranychus urticae Koch, reared on lima bean plants were moved to cucumber, maize, or new lima bean plants (the latter being a control) and evaluated after 24 h or 7 d for changes in susceptibility to three pesticides and in levels of related detoxification enzymes. The largest and most consistent changes were observed in mites feeding on cucumber. Susceptibility of mites on cucumber to the synthetic pyrethroids bifenthrin and lambda-cyhalothrin was greater than that of mites reared on lima bean and maize after only 24 h on the plants, and remained higher after 7 d. Mites on cucumber also were more susceptible to the organophosphate dimethoate than were mites on lima bean, but only after 7 d on the host. Susceptibility was inversely related to activities of both general esterase and glutathione S-transferase (GST) in mites on cucumber; general esterase and GST activities were 60 and 25% lower, respectively, than activities of twospotted spider mite on lima bean after 7 d of feeding. Mites on maize were slightly but significantly more susceptible than those on lima bean to bifenthrin, but not to lambda-cyhalothrin, after 7 d and to dimethoate after 24 h but not after 7 d. General esterase and GST activities in twospotted spider mite fed on maize for 24 h were 20 and 16% higher, respectively, than activities in twospotted spider mite on lima bean, but general esterase activity was 30% lower than lima bean-fed mites and GST was not different after 7 d. Thus, plant-induced changes in general esterase activity, perhaps in combination with GST activity, in twospotted spider mite appear to be inversely related to, and possibly responsible for, changes in susceptibility of twospotted spider mite to several pesticides, particularly the synthetic pyrethroids. General esterases appear to play less of a role in the detoxification of the organophosphate insecticide dimethoate.