Determination of metabolic resistance mechanisms in pyrethroid-resistant and fipronil-tolerant brown dog ticks.

Rhipicephalus sanguineus (Latreille) (Ixodida: Ixodidae) is a three-host dog tick found worldwide that is able to complete its' entire lifecycle indoors. Options for the management of R. sanguineus are limited and its' control relies largely on only a few acaricidal active ingredients. Previous studies have confirmed permethrin resistance and fipronil tolerance in R. sanguineus populations, commonly conferred by metabolic detoxification or target site mutations. Herein, five strains of permethrin-resistant and three strains of fipronil-tolerant ticks were evaluated for metabolic resistance using synergists to block metabolic enzymes. Synergist studies were completed with triphenyl phosphate (TPP) for esterase inhibition, piperonyl butoxide (PBO) for cytochrome P450 inhibition, and diethyl maleate (DEM) for glutathione-S-transferase inhibition. Additionally, increased esterase activity was confirmed using gel electrophoresis. The most important metabolic detoxification mechanism in permethrin-resistant ticks was increased esterase activity, followed by increased cytochrome P450 activity. The inhibition of metabolic enzymes did not have a marked impact on fipronil-tolerant tick strains.

The Thermal Breadth of Nylanderia fulva (Hymenoptera: Formicidae) Is Narrower Than That of Solenopsis invicta at Three Thermal Ramping Rates: 1.0, 0.12, and 0.06°C min-1.

Determining the upper (CTmax) and lower (CTmin) critical thermal limits of invasive ants provides insight into how temperature could shape their distribution, seasonality, and daily activity. Understanding the potential distribution of invasive ants is imperative to improving quarantine and management efforts. Nylanderia fulva (Mayr) (tawny crazy ant) and Solenopsis invicta (Buren) (red imported fire ant) are invasive ants that are established throughout the southeastern United States. Recent studies have found that body size and thermal ramping rate can affect the estimation of critical thermal limits. However, the effects of both variables and their interactions on the thermal limits of N. fulva and S. invicta have not previously been described. Thus, we evaluated the impacts of body size and ramping rate on the critical thermal limits of N. fulva and S. invicta Overall, N. fulva had a narrower thermal breadth than S. invicta (Nf CTmin = 7.3°C and Nf CTmax = 41.3°C vs. Si CTmin = 4.1°C and Si CTmax = 45.3°C). For both species, slower ramping rates resulted in lower CTmax values and ants with smaller head capsules had a narrower thermal breadth than ants with larger head capsules. These data improve our understanding of the critical thermal limits of both species and could be useful for developing predictive models that estimate the future spread of these invasive ants in nonnative ranges.

Two hexamerin genes from the termite Reticulitermes flavipes: Sequence, expression, and proposed functions in caste regulation.

Previous molecular studies on the termite Reticulitermes flavipes have revealed that two hexamerin proteins serve an important status quo role in the regulation of juvenile hormone (JH)-dependent caste differentiation. Here, we report sequence data and other experimental evidence suggesting how these two hexamerins function in achieving caste regulation. The two hexamerin genes, named Hex-1 and Hex-2, encode highly unique sequence features relative to the 100+ other known insect hexamerins. These features include a long hydrophobic tail and prenylation motif in Hex-1, and a long hydrophilic insertion plus several putative protease cleavage sites in Hex-2. Both hexamerin genes are primarily expressed in fat body tissue, but only Hex-2 expression is substantially induced by JH. SDS-PAGE showed that the hexamerin proteins constitute a major proportion of total soluble termite protein. Also, although each protein occurs in both the membrane and soluble protein fractions, Hex-2 has stronger membrane affinity. Anti-JH antiserum specifically recognizes hemolymph-soluble Hex-1 protein, supporting that the unique prenylation site in Hex-1 facilitates covalent JH binding to the primary amino acid chain. Finally, increased ratios of Hex-2 to Hex-1 transcription occur in caste phenotypes and developmental stages that differentiate in response to rising JH titers. Two main conclusions can be taken from these studies. First, elevated ratios of Hex-2 to Hex-1 expression are associated with caste phenotypes that differentiate in response to rising JH titers (i.e., workers, presoldiers and soldiers). Second, due to their unique structural features and other observed characteristics, our findings support the hypothesis that the two hexamerins participate in the regulation of caste-differentiation by modulating JH availability.

Juvenile hormone and colony conditions differentially influence cytochrome P450 gene expression in the termite Reticulitermes flavipes.

In lower termites, the worker caste is a totipotent immature stage that is capable of differentiating into other adult caste phenotypes. We investigated the diversity of family 4 cytochrome P450 (CYP4) genes in Reticulitermes flavipes workers, with the specific goal of identifying P450s potentially involved in regulating caste differentiation. Seven novel CYP4 genes were identified. Quantitative real-time PCR revealed the tissue distribution of expression for the seven CYP4s, as well as temporal expression changes in workers in association with a release from colony influences and during juvenile hormone (JH)-induced soldier caste differentiation. Several fat-body-related CYP4 genes were differentially expressed after JH treatment. Still other genes changed expression in association with removal from colony influences, suggesting that primer pheromones and/or other colony influences impact their expression. These findings add to a growing database of candidate termite caste-regulatory genes, and provide explicit evidence that colony factors influence termite gene expression.

Acoustic detection of termite infestations in urban trees.

A portable, low-frequency acoustic system was used to detect termite infestations in urban trees. The likelihood of infestation was rated independently by a computer program and an experienced listener that distinguished insect sounds from background noises. Because soil is a good insulator, termite sounds could be detected easily underneath infested trees, despite the presence of high urban background noise. Termite sounds could be detected also in trunks, but background noise often made it difficult to identify termite signals unambiguously. High likelihoods of termite infestation were predicted at four live oak (Quercus virginiana Mill, Fagacae), two loblolly pine (Pinus taeda L., Pinacae), and two baldcypress (Taxodium distichum Rich. Pinacae) trees that wood-baited traps had identified as infested with Coptotermes formosanus Shiraki. Infestations also were predicted at two pine trees with confirmed recoveries of Reticulitermes flavipes (Kollar). Low likelihoods of infestation were predicted in four oak trees where no termites were found. Additional tests were conducted in anechoic environments to determine the range of acoustic detectability and the feasibility of acoustically estimating termite population levels. There was a significant regression between the activity rate and the number of termites present in a wood trap block, with a minimum detectable number of approximately 50 workers per liter of wood. The success of these field tests suggests that currently available acoustic systems have considerable potential to detect and monitor hidden infestations of termites in urban trees and around building perimeters in addition to their present uses to detect and monitor termite infestations in buildings.

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.