Extraordinary resistance to insecticides reveals exotic Q biotype of Bemisia tabaci in the New World

A strain of the whitefly Bemisia tabaci (Gennadius) possessing unusually high levels of resistance to a wide range of insecticides was discovered in 2004 in the course of routine resistance monitoring in Arizona. The multiply resistant insects, collected from poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) plants purchased at a retail store in Tucson, were subjected to biotype analysis in three laboratories. Polyacrylamide gel electrophoresis of naphthyl esterases and sequencing of the mitochondrial cytochrome oxidase I gene (780 bp) confirmed the first detection of the Q biotype of B. tabaci in the New World. This U.S. Q biotype strain, referred to as Poinsettia'04, was highly resistant to two selective insect growth regulators, pyriproxyfen and buprofezin, and to mixtures of fenpropathrin and acephate. It was also unusually low in susceptibility to the neonicotinoid insecticides imidacloprid, acetamiprid, and thiamethoxam, relative to B biotype whiteflies. In 100 collections of whiteflies made in Arizona cotton (Gossypium spp.), vegetable, and melon (Cucumis melo L.) fields from 2001 to 2005, no Q biotypes were detected. Regions of the United States that were severely impacted by the introduction of the B biotype of B. tabaci in the 1980s would be well advised to promote measures that limit movement of the Q biotype from controlled environments into field systems and to formulate alternatives for managing this multiply-resistant biotype, in the event that it becomes more widely distributed.     

Establishment of baseline susceptibility data to various insecticides for Homalodisca coagulata (Homoptera: Cicadellidae) by comparative bioassay techniques

Homalodisca coagulata Say, adults from three locations in California were subjected to insecticide bioassays to establish baseline toxicity. Initially, two bioassay techniques, petri dish and leaf dip, were compared to determine the most useful method to establish baseline susceptibility data under laboratory and greenhouse conditions. Comparative dose-response data were determined by both techniques to endosulfan, dimethoate, cyfluthrin, and acetamiprid. Toxic values were similar to some insecticides with both techniques but not for all insecticides, revealing susceptibility differences among the three populations of H. coagulata. In subsequent tests, the petri dish technique was selected to establish baseline susceptibility data to various contact insecticides. A systemic uptake bioassay was adapted to estimate dose-mortality responses to a systemic insecticide, imidacloprid. A 2-yr comparison of toxicological responses showed all three populations of H. coagulata to be highly susceptible to 10 insecticides, including chlorpyrifos, dimethoate, endosulfan, bifenthrin, cyfluthrin, esfenvalerate, fenpropathrin, acetamiprid, imidacloprid, and thiamethoxam. In general, two pyrethroids, bifenthrin and esfenvalerate, were the most toxic compounds, followed by two neonicotinoids, acetamiprid and imidacloprid. The LC50 values for all insecticides tested were lower than concentrations used as recommended field rates. Baseline data varied for the three geographically distinct H. coagulata populations with the petri dish technique. Adult H. coagulata collected from San Bernardino County were significantly more susceptible to select pyrethroids compared with adults from Riverside or Kern counties. Adults from San Bernardino County also were more sensitive to two neonicotinoids, acetamiprid and imidacloprid. The highest LC50 values were to endosulfan, which nonetheless proved highly toxic to H. coagulata from all three regions. In the majority of the tests, mortality increased over time resulting in increased susceptibility at 48 h compared with 24 h. These results indicate a wide selection of highly effective insecticides that could aid in managing H. coagulata populations in California.     

Interactions Between Pattern Formation and Domain Growth

In this paper we develop a theoretical framework for investigating pattern formation in biological systems for which the tissue on which the spatial pattern resides is growing at a rate which is itself regulated by the diffusible chemicals that establish the spatial pattern. We present numerical simulations for two cases of interest, namely exponential domain growth and chemically controlled growth. Our analysis reveals that for domains undergoing rapid exponential growth dilution effects associated with domain growth influence both the spatial patterns that emerge and the concentration of chemicals present in the domain. In the latter case, there is complex interplay between the effects of the chemicals on the domain size and the influence of the domain size on the formation of patterns. The nature of these interactions is revealed by a weakly nonlinear analysis of the full system. This yields a pair of nonlinear equations for the amplitude of the spatial pattern and the domain size. The domain is found to grow (or shrink) at a rate that depends quadratically on the pattern amplitude, the particular functional forms used to model the local tissue growth rate and the kinetics of the two diffusible species dictating the resulting behaviour.     

Viscoelastic study of the mechanical unfolding of a protein by AFM

We have applied a dynamic force modulation technique to the mechanical unfolding of a homopolymer of immunoglobulin (Ig) domains from titin, (C47S C63S I27)5, [(I27)5] to determine the viscoelastic response of single protein molecules as a function of extension. Both the stiffness and the friction of the homopolymer system show a sudden decrease when a protein domain unfolds. The decrease in measured friction suggests that the system is dominated by the internal friction of the (I27)5 molecule and not solvent friction. In the stiffness-extension spectrum we detected an abrupt feature before each unfolding event, the amplitude of which decreased with each consecutive unfolding event. We propose that these features are a clear indication of the formation of the known unfolding intermediate of I27, which has been observed previously in constant velocity unfolding experiments. This simple force modulation AFM technique promises to be a very useful addition to constant velocity experiments providing detailed viscoelastic characterization of single molecules under extension.     

Morphological and genetic variation indicate cryptic species within Lamarck's little sea star, Parvulastra (=Patiriella) exigua

The asterinid sea star Parvulastra exigua (Lamarck) is a common member of temperate intertidal marine communities from geographically widespread sites around the southern hemisphere. Individuals from Australian populations lay benthic egg masses (through orally directed gonopores) from which nonplanktonic offspring hatch and metamorphose without a dispersing planktonic larval phase. Scattered reports in the taxonomic literature refer to a similar form in southern Africa with aborally directed gonopores (and possibly broadcast spawning of planktonic eggs and larvae); such differences would be consistent with cryptic species variation. Surveys of morphology and mtDNA sequences have revealed cryptic species diversity in other asterinid genera. Here we summarize the taxonomic history of Lamarck's "Astérie exigu?" and survey morphological variation (the location of the gonopores) for evidence that some P. exigua populations include cryptic species with a different mode of reproduction. We found strong evidence for multiple species in the form of two phenotypes and modes of reproduction (oral and aboral gonopore locations) in populations from southern Africa and islands in the Atlantic and Indian oceans. Both modes of reproduction have broad geographic ranges. These results are consistent with previously published genetic data that indicate multiple species in African and island (but not Australian) populations.     

Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor

Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium ( approximately 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe(2) hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe(C) hTF), and a mutant (designated Mut-Fe(C) hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small ( approximately 20%) but consistent difference is noted for the binding of Fe(C) hTF and the Mut-Fe(C) hTF to the sTFR N317D mutant. The rate of iron release from Fe(C) hTF and Mut-Fe(C) hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.     

Fermentation, purification, and efficacy of a recombinant vaccine candidate against botulinum neurotoxin type F from Pichia pastoris

A recombinant vaccine candidate was developed that protected mice against botulinum neurotoxin serotype F (BoNTF) intoxication. A synthetic gene encoding BoNTF fragment C (rBoNTF(H(c))) was designed, constructed, and inserted into a plasmid for expression in the yeast Pichia pastoris. A total cell protein content of 2.9 g was obtained per liter of fermentation broth. Recombinant rBoNTF(H(c)) was purified from the soluble yeast extract in two chromatographic steps. The process employed Mono S cation exchange chemistry followed by Alkyl-Superose hydrophobic interaction chromatography, producing material judged to be greater than 98% pure by SDS-PAGE. The recovery of purified product from cell extract was estimated to be greater than 42%, with a yield of 140 mg/kg of cell paste. rBoNTF(H(c)) was also purified from the insoluble fraction of the yeast cell lysate. Because the fragment C in the pellet was 35% of the total insoluble protein, only a Mono S cation exchange chromatography step was necessary to achieve a purity greater than 98%. Mice that received three injections of 0.2 microgram of purified soluble rBoNTF(H(c)) were completely protected when challenged with 1000 mouse ip LD(50) of BoNTF toxin. Similarly, three doses of 1 microgram of purified resolubilized rBoNTF(H(c)) completely protected mice from a challenge of 5000 mouse ip LD(50) of BoNTF toxin. Individual serum antibody ELISA titers of mice injected with soluble rBoNTF(H(c)) correlated with survival as all 34 mice with ELISA titers of 100 or greater survived toxin challenge. The work presented here demonstrates that purified rBoNTF(H(c)) is able to protect against a high challenge dose of neurotoxin.