Worker Size, Geographical Distribution, and Introgressive Hybridization of Invasive Solenopsis invicta and Solenopsis richteri (Hymenoptera: Formicidae) in Tennessee.

Worker size and geographical distribution of red imported fire ants (Solenopsis invicta Buren), black imported fire ants (Solenopsis richteri Forel), and their hybrid (S. invicta × S. richteri) (Hymenoptera: Formicidae) were evaluated from colonies sampled across Tennessee. The fire ant species and hybrid status were determined using cuticular hydrocarbon and venom alkaloid indices obtained from gas chromatography and mass spectrometry. Hybrids were the most common fire ant throughout Tennessee. With the exception of a few isolated S. invicta samples, only hybrids were found in east Tennessee, and hybrids predominated in middle Tennessee. In west Tennessee, mixed populations of S. richteri and hybrids were found. Hybrids were more common in west Tennessee than a survey performed a decade earlier. No statistical differences were detected in the average inter-colonial worker size of S. richteri and hybrids. Likewise, average worker size was not related to geographic location in Tennessee. The similarity in average worker size among hybrid colonies with a wide range of cuticular hydrocarbon and venom alkaloid values suggests introgression was not impacting ant size in colonies sampled throughout Tennessee.

ICTV Virus Taxonomy Profile: Dicistroviridae.

Dicistroviridae is a family of small non-enveloped viruses with monopartite, linear, positive-sense RNA genomes of approximately 8-10 kb. Viruses of all classified species infect arthropod hosts, with some having devastating economic consequences, such as acute bee paralysis virus in domesticated honeybees and taura syndrome virus in shrimp farming. Conversely, the host specificity and other desirable traits exhibited by several members of this group make them potential natural enemies for intentional use against arthropod pests, such as triatoma virus against triatomine bugs that vector Chagas disease. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Dicistroviridae which is available at www.ictv.global/report/dicistroviridae.

ICTV Virus Taxonomy Profile: Iflaviridae.

Iflaviridae is a family of small non-enveloped viruses with monopartite, positive-stranded RNA genomes of approximately 9-11 kilobases. Viruses of all classified species infect arthropod hosts, with the majority infecting insects. Both beneficial and pest insects serve as hosts, and infections can be symptomless (Nilaparvatalugens honeydew virus 1) or cause developmental abnormalities (deformed wing virus), behavioural changes (sacbrood virus) and premature mortality (infectious flacherie virus). The host range has not been examined for most members. The most common route of infection for iflaviruses is the ingestion of virus-contaminated food sources. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Iflaviridae, which is available at www.ictv.global/report/iflaviridae.

Novel sodium channel gene mutations in Blattella germanica reduce the sensitivity of expressed channels to deltamethrin.

Pyrethroid insecticides alter the normal gating of voltage-gated sodium channels in the nervous system. Three sodium channel mutations (E434K, C764R, L993F) were recently identified in pyrethroid resistant German cockroach populations. In this report, we show that the L993F mutation decreased sodium channel sensitivity to the pyrethroid, deltamethrin, by five-fold in Xenopus oocytes. In contrast, neither E434K nor C764R alone decreased channel sensitivity to deltamethrin. However, E434K or C764R combined with L993F reduced deltamethrin sensitivity by 100-fold. Furthermore, concomitant presence of all three mutations (KRF) reduced channel sensitivity to deltamethrin by 500-fold. None of the mutations significantly affected channel gating. However, sodium current amplitudes from the mutant sodium channel carrying either E434K or C764R alone were much reduced compared to those of the wild-type channel or the channel carrying the double or triple mutations (KF, RF and KRF). These results indicated that evolution of sodium channel insensitivity in the German cockroach is achieved by sequential selection of a primary mutation L993F and two secondary mutations E434K and C764R, and concomitant presence of all three mutations dramatically reduced sodium channel sensitivity to deltamethrin.

Effects of the synergists piperonyl butoxide and S,S,S-tributyl phosphorotrithioate on propoxur pharmacokinetics in Blattella germanica (Blattodea: Blattellidae).

Effects of the synergists piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) on propoxur pharmacokinetics were examined in the German cockroach, Blattella germanica (L.). Treatment of adult male German cockroaches with the cytochrome P450 monooxygenase inhibitor, PBO, or the esterase inhibitor, DEF, increased propoxur toxicity by 2- and 6.8-fold, respectively, implicating hydrolysis as a major detoxification route of propoxur in the German cockroach. However, significant hydrolytic metabolism could not be demonstrated conclusively in vitro resulting in a conflict between in situ bioassay data and in vitro metabolic studies. In vitro propoxur metabolism with NADPH-fortified microsomes produced at least nine metabolites. Formation of metabolites was NADPH-dependent; no quantifiable metabolism was detected with cytosolic fractions. However, microsomal fractions lacking an NADPH source did produce a low, but detectable, quantity of metabolites (1.6 pmol). PBO inhibited NADPH-dependent propoxur metabolism in a dose-dependent fashion, implicating cytochrome P450 monooxygenases as the enzyme system responsible for the metabolism. Interestingly, DEF also inhibited the NADPH-dependent metabolism of propoxur, albeit to a lower extent. Treatment with PBO or DEF also caused a significant reduction in the cuticular penetration rate of propoxur. The data demonstrate that unanticipated effects are possible with synergists and that caution must be exercised when interpreting synergist results.

Purification and characterization of trans-permethrin metabolizing microsomal esterases from workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar).

Three alpha-naphthyl acetate hydrolyzing esterase isozymes were purified from microsomes prepared from Reticulitermes flavipes workers. The two step process involved sequential preparative IEF followed by continuous elution preparative electrophoresis on a 5% non-denaturing polyacrylamide gel. The first IEF run resulted in 5.4-fold purification with a yield of 46.1%. Subsequent IEF further purified the esterases 14.3-fold and 12% yield. Preparative electrophoresis of the pooled IEF fractions produced three major peaks of alpha-naphthyl acetate hydrolyzing activity. The esterases were correspondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on increasing molecular retention on a native PAGE gel. ME 1, ME 2, and ME 3 were acidic proteins with pI values of 4.61, 4.70, and 4.77, respectively. Molecular mass as determined by gel filtration chromatography of ME 1, ME 2, and ME 3 was 69, 64, and 62 kDa, respectively. SDS-PAGE gels produced a single band for each of the isozymes with a molecular mass of 63 kDa indicating that the esterases were monomers. Specific activities of ME 1, ME 2, and ME 3 increased with increasing pH and the enzymes were active over a broad temperature range (25-55 degrees C). The three purified isozymes were inhibited at low concentration by paraoxon (10(-10) M), chlorpyrifos (10(-6) M), DEF (10(-6) M), and PMSF (10(-6) M) indicating that they were "B" type serine esterases. Conversely, inhibition was not observed at 10(-4) M eserine, PHMB, or CaCl(2), further supporting the conclusion that the microsomal esterases were of the "B" type. None of the isozymes was inhibited by 10(-4) M imidacloprid, fipronil, or PBO. Quantitatively, ME 1, ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metabolites.

Toxicity and in vitro metabolism of t-permethrin in eastern subterranean termite (Isoptera: Rhinotermitidae).

Toxicity and metabolism of t-permethrin were evaluated in two colonies (UF and ARS) of the eastern subterranean termite, Reticulitermes flavipes (Kollar), collected in Gainesville, FL. The UF colony (LC50 = 1.86 micrograms per vial) was approximately twofold more tolerant of t-permethrin than the ARS colony (LC50 = 0.89 microgram per vial) at the LC50. The synergists piperonyl butoxide and S,S,S-tributylphosphorotrithioate increased t-permethrin toxicity four- and threefold (at the LC50) in the UF and ARS colonies, respectively. Despite these differences in t-permethrin susceptibility, microsomal oxidase activities toward surrogate substrate (aldrin epoxidase, and methoxyresorufin O-demethylase), cytochrome P450 content, and microsomal esterase activity toward alpha-naphthyl acetate did not differ significantly between the colonies. Moreover, no significant differences in qualitative and quantitative metabolism of [14C]t-permethrin were observed between the UF and ARS colonies for three enzyme sources (microsomal oxidase, microsomal esterase, and cytosolic esterase). Based on in vitro metabolism assays, the major detoxification route of t-permethrin in the UF and ARS termite colonies appears to be hydrolysis catalyzed by microsomal esterases.

Novel point mutations in the German cockroach para sodium channel gene are associated with knockdown resistance (kdr) to pyrethroid insecticides.

Knockdown resistance (kdr) to pyrethroid insecticides has been attributed to point mutations in the para sodium channel gene in more than a half dozen insect pest species. In this study, we identified two novel para mutations in five highly resistant kdr-type German cockroach strains. The two mutations, from glutamic acid (E434) to lysine (K434) and from cysteine (C764) to arginine (R764), respectively, are located in the first intracellular linker connecting domains I and II. E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II). Two additional mutations from aspartic acid (D58) to glycine (G58), and from proline (P1880) to leucine (L1888), respectively, were found in one of the resistant strains. The four mutations coexist with the previously identified leucine to phenylalanine (L993F) kdr mutation in IIS6, and are present only in the highly resistant individuals of a given strain. These findings suggest that these mutations might be responsible for high levels of knockdown resistance toward pyrethroid insecticides in the German cockroach.

Comparative insecticide susceptibility and detoxification enzyme activities among pestiferous blattodea.

Topical bioassays using propoxur, chlorpyrifos, and lambda-cyhalothrin were conducted on eight cockroach species. Based on lethal dose values, the relative toxicities of the insecticide classes were generally pyrethroid > carbamate > organophosphorous. Lambda-Cyhalothrin and propoxur were more toxic toward the Blattidae as compared with the Blattellidae. The order of lambda-cyhalothrin toxicity was Periplaneta americana > Periplaneta brunnea = Periplaneta australasiae = Periplaneta fuliginosa = Blatta orientalis > Blattella asahinai = Blattella germanica > Blattella vaga. The order of propoxur toxicity was B. orientalis > P. americana > P. brunnea = P. australasiae > B. asahinai > P. fuliginosa = B. germanica > B. vaga. The order of chlorpyrifos toxicity was P. americana > B. asahinai = B. vaga > B. orientalis = P. australasiae = P. brunnea > B. germanica = P. fuliginosa. Detoxification enzyme activities for each species also were measured and compared with insecticide toxicity. Propoxur LD50 was significantly (P = 0.01; r = 0.81) correlated with glutathione S-transferase activity. Lambda-Cyhalothrin LD50 correlated with methoxyresorufin O-demethylase activity (P = 0.01; r = 0.81), carboxylesterase activity (P = 0.03; r = - 0.75), general esterase activity (P = 0.02; r = - 0.79), and cockroach weight (P = 0.01; r = -0.95).

Influence of carbon dioxide anesthesia on chlorpyrifos toxicity in the German cockroach (Dictyoptera: Blattellidae).

Insecticide-susceptible (Orlando normal) and multi-insecticide-resistant (Village Green) adult male German cockroaches, Blattella germanica (L.), were exposed to carbon dioxide (CO2) gas in either varying durations or multiple exposures of fixed duration followed by topical treatment with acetone or chlorpyrifos (LD25) to evaluate the effect of CO2 anesthesia on chlorpyrifos toxicity. CO2 anesthesia did not increase chlorpyrifos toxicity in either strain regardless of exposure duration up to 1 h. However, multiple CO2-induced knockdowns alone caused mortality and increased chlorpyrifos toxicity. Twenty-four-hour weight loss was independent of CO2 duration exposure by strain. Village Green and Orlando cockroaches lost 5.3 and 7.6% of their weight in 24 h when treated with acetone and 10.5 and 12.8% when treated with chlorpyrifos, respectively. Conversely, 0- and 24-h weight loss increased with increasing CO2-induced knockdowns regardless of strain or topical treatment. Based on these results, CO2 used either once of limited duration (< or = 15 min) or multiple times of short duration, not to exceed four total knockdowns, does not increase chlorpyrifos toxicity in the adult male German cockroach.

Mechanism of agonist-induced downregulation of muscarinic receptors in rat pancreatic acini.

Exposure of pancreatic acini to cholinergic agonists reduces their muscarinic acetylcholine receptor content. To investigate the mechanism of this reduction, rat pancreatic acini were cultured in the presence of carbachol and its effects on binding of [N-methyl-3H] scopolamine (NMS), which labels only cell surface receptors, and [3H]scopolamine, which also accesses intracellular receptors, were determined. Carbachol (0.1 mM) caused disappearance of 90% of binding sites for [3H]NMS and [3H]-scopolamine with half-time values of 3.9 +/- 0.4 and 5.7 +/- 0.7 h, respectively. Nocodazole and cytochalasin D, ouabain, ionophore A23187, 12-O-tetradecanoylphorbol 13-acetate (TPA), bombesin, cholecystokinin octapeptide (CCK-8), secretin, and vasoactive intestinal peptide (VIP) had no effect on the carbachol-induced decrease in binding sites for either antagonist, although staurosporine and the calmodulin inhibitor W-7 each caused slight inhibition. By contrast, the lysosomotropic agents methylamine and ammonium chloride caused 80% inhibition of disappearance of binding sites for [3H]scopolamine, although they only slightly inhibited disappearance of [3H]NMS binding sites. These results implicate the endosomal/lysosomal pathway in cholinergic agonist-evoked downregulation of muscarinic receptors in the pancreatic acinar cell.