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PubMed1999

Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase.

Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1. 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity. The EKI1 gene was isolated and used to construct eki1Delta and eki1Delta cki1Delta mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1Delta mutant, cki1Delta mutant, and eki1Delta cki1Delta double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells. The EKI1 gene product also exhibited choline kinase activity. Biochemical analyses of the enzyme expressed in insect cells, in eki1Delta mutants, and in cki1Delta mutants indicated that ethanolamine was the preferred substrate. The eki1Delta mutant did not exhibit a growth phenotype. Biochemical analyses of eki1Delta, cki1Delta, and eki1Delta cki1Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S. cerevisiae. In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway. Unlike cki1Delta mutants, eki1Delta mutants did not suppress the essential function of Sec14p.

Amino Acid Sequence↗
PubMed2001

Pattern generation for walking and searching movements of a stick insect leg. II. Control of motoneuronal activity.

In the stick insect, Cuniculina impigra, intracellular recordings from mesothoracic motoneurons that control flexion and extension of the tibia and depression and levation of the trochantero-femur were made while the leg performed walking-like movements on a treadband or stereotyped rhythmic searching movements. We were interested in how synaptic input and intrinsic properties contribute to form the activity pattern of motoneurons during rhythmic leg movements without sensory feedback from other legs. During searching and walking, motoneurons expressed a rhythmic bursting pattern that was formed by a depolarizing input followed by a hyperpolarizing input in the inter-burst interval. This basic pattern was similar in all fast, semi-fast, and slow motoneurons that were recorded. Hyperpolarizations were in synchrony with activity in the antagonistic motoneurons. De- and hyperpolarizations were associated with a decrease in input resistance. All motoneurons showed spike frequency adaptation when depolarized by current injection to a membrane potential similar to that observed during walking. In the hyperpolarizing phase of fast flexor motoneurons, the initial maximum hyperpolarization was followed by a sag in potential toward more depolarized values. Consistent with this observation, only fast flexor motoneurons developed a depolarizing sag potential when hyperpolarized by injection of constant negative current.

Action Potentials↗
PubMed1993

Glutamate receptor inhibitors as potential insecticides.

Philanthotoxin (PhTX) is a neurotoxic constituent of the paralytic venom of the digger wasp, Philanthus triangulum. PhTX inhibits glutamate receptors of insect muscles mostly as a channel blocker, thereby producing muscle paralysis. Since glutamate receptor blockers may be of value as selective insect control agents, numerous derivatives of PhTX were synthesized and tested for their potencies as inhibitors of insect skeletal muscle glutamate receptors. Structure-activity relationship studies revealed that shortening the polyamine chain length reduced potency, and quaternarization of the nitrogen destroyed it. The potency was increased by a bulky anchoring group with moderate hydrophobicity at the end of the polyamine chain. The conversion of the tryosyl moiety to 3,5-diiodo-tyrosyl also increased potency and so did lengthening the butyryl chain from 4 to 10 carbons. Not only did PhTXs inhibit different subtypes of glutamate receptors, including the mammalian N-methyl-D-aspartate receptor, but also nicotinic receptors of insects and vertebrates. Because of this low selectively, and the hydrophilicity of the derivatives tested, which interferes with their penetration to the target receptor, these compounds cannot be used as insecticides. Nevertheless, the insect skeletal muscle glutamate receptor is a viable target for selective insecticides and major changes in PhTX structure may possibly produce derivatives that can be potential insecticides.

Animals↗
PubMed1999

Development of insect cell lines: virus susceptibility and applicability to prawn cell culture.

Insect cells have been successfully cultured in vitro as continuous cell lines for over 35 years. The media, culture methodology and conditions have been well resolved such that, for many insects, new cells lines can be routinely developed. Factors that are considered important for developing insect cell cultures are described as well as some of the history that led to the success. One of the major rationales for developing insect cell lines was for the study of insect viruses. This was particularly true for species of Lepidoptera from which over 900 viruses have been reported. Since many species of Lepidoptera are serious agricultural and forestry pests, effects have been made to utilize some of these pathogens as biological pesticides. Cell cultures are important in this endeavor since viruses require a living cell to reproduce. Of the known insect viruses, the most intensely studied have been the baculoviruses. In addition to their potential for controlling insect pests, they also have been used as expression vectors for producing recombinant proteins. Details of some of these experiments are described. Finally, experiences with insect cells are considered in relation to efforts to develop prawn cell cultures.

Animals↗
PubMed1998

Isolation and characterization of the Saccharomyces cerevisiae LPP1 gene encoding a Mg2+-independent phosphatidate phosphatase.

The DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase enzyme accounts for half of the Mg2+-independent phosphatidate (PA) phosphatase activity in Saccharomyces cerevisiae. The LPP1 (lipid phosphate phosphatase) gene encodes a protein that contains a novel phosphatase sequence motif found in DGPP phosphatase and in the mouse Mg2+-independent PA phosphatase. A genomic copy of the S. cerevisiae LPP1 gene was isolated and was used to construct lpp1Delta and lpp1Delta dpp1Delta mutants. A multicopy plasmid containing the LPP1 gene directed a 12.9-fold overexpression of Mg2+-independent PA phosphatase activity in the S. cerevisiae lpp1Delta dpp1Delta double mutant. The heterologous expression of the S. cerevisiae LPP1 gene in Sf-9 insect cells resulted in a 715-fold overexpression of Mg2+-independent PA phosphatase activity relative to control insect cells. The Mg2+-independent PA phosphatase activity encoded by the LPP1 gene was associated with the membrane fraction of the cell. The LPP1 gene product also exhibited lyso-PA phosphatase and DGPP phosphatase activities. The order of substrate preference was PA > lyso-PA > DGPP. Like the dpp1Delta mutant, the lpp1Delta mutant and the lpp1Delta dpp1Delta double mutant were viable and did not exhibit obvious growth defects. Biochemical analyses of lpp1Delta, dpp1Delta, and lpp1Delta dpp1Delta mutants showed that the LPP1 and DPP1 gene products encoded nearly all of the Mg2+-independent PA phosphatase and lyso-PA phosphatase activities and all of the DGPP phosphatase activity in S. cerevisiae. Moreover, the analyses of the mutants showed that the LPP1 and DPP1 gene products played a role in the regulation of phospholipid metabolism and the cellular levels of phosphatidylinositol and PA.

Animals↗
PubMed2001

Role of superoxide and reactive nitrogen intermediates in Rhodnius prolixus (Reduviidae)/Trypanosoma rangeli interactions.

This study compares aspects of the superoxide, nitric oxide and prophenoloxidase pathways in Rhodnius prolixus hemolymph, measured in parallel, in response to Trypanosoma rangeli inoculation. Responses to two strains of T. rangeli, and two developmental forms, were studied, and the results obtained were correlated with the ability of the parasites to survive, multiply, and complete their life cycles in the hemolymph of the host. T. rangeli H14 strain parasites, which fail to complete their life cycle in Rhodnius by invading the salivary glands, stimulated high levels of superoxide and prophenoloxidase activity, which peaked 24 h after inoculation. Simultaneously, the concentration of hemolymph nitrites and nitrates increased, indicative of nitric oxide activity, but parasite numbers remained low. T. rangeli Choachi strain parasite inoculation also stimulated superoxide and prophenoloxidase activity, which, though significantly lower than the equivalent responses to the H14 strain, also peaked at 24 h. However, nitrate and nitrite levels in Choachi strain-inoculated hemolymph remained low, and this parasite strain multiplied rapidly, especially following peak superoxide activity, and eventually invaded the salivary glands for transmission to a vertebrate host. In both strains, short form epimastigotes stimulated greater superoxide and prophenoloxidase responses than long form epimastigotes. Injection of the NADPH oxidase inhibitor N-ethylmaleimide or the inducible nitric oxide synthase inhibitor S-methyl isothiourea sulfate caused significantly higher insect mortalities in groups of R. prolixus inoculated with either parasite strain compared with those of uninfected control insects. This indicates that both NADPH oxidase and nitric oxide synthase activity may be involved in the immune response of R. prolixus to infection by T. rangeli. Finally, Western blotting of R. prolixus hemocyte lysates revealed the presence of a protein immunologically related to the human NADPH oxidase complex, the initiator enzyme of the respiratory burst.

Animals↗
PubMed2001

Adult survival, maturation, and reproduction of the desert locust Schistocerca gregaria infected with the fungus Metarhizium anisopliae var acridum.

Studies were conducted with two different doses of Metarhizium anisopliae var acridum to examine the effects on survival and reproductive potential of adult Schistocerca gregaria under conditions that either limited thermoregulation or enabled optimal thermoregulation. Adult S. gregaria infected with the fungal pathogen showed either a rapid and high mortality at relatively constant temperatures or a much reduced mortality and lengthened survival time when allowed to thermoregulate. Mortality rate varied from >90% after 10 days under constant temperature conditions to 66% after 70 days under optimal thermoregulatory conditions. Effects of infection on maturation and reproduction depended on the age of the adults at the time of inoculation, the nighttime temperature regime, the fungal dose, and the length of time of the monitoring period. No difference in reproductive behaviors in treated and control insects were found in one experiment that utilized older adults and was conducted over 25 days. In a second experiment with newly fledged locusts, differences in maturation rates and total reproductive output were observed due to infection. The results from these experiments are discussed in terms of the potential of M. anisopliae var acridum to alter the balance of insect endocrine systems and the importance of the assessment of behavioral changes and their impact on microbial control agents in the long term.

Aging↗
PubMed1993

Hormonal control of transmitter plasticity in insect peptidergic neurons. II. Steroid control of the up-regulation of bursicon expression.

Each abdominal ganglion of the central nervous system of the tobacco hawkmoth, Manduca sexta contains four individually identified lateral neurosecretory cells (LNCs) that undergo a postembryonic transmitter switch in vivo. In the embryonic and caterpillar stages, the primary LNC transmitter is cardioacceleratory peptide 2 (CAP2), a myoregulatory peptide. During metamorphosis, these cells stop expressing CAP2 and instead produce bursicon, a classic insect peptide hormone responsible for cuticular tanning. We have previously reported that this transmitter plasticity is under the control of the insect steroid hormone 20-hydroxyecdysone (20-HE), which surges twice during the last larval instar. In that report we showed that the CAP2 decline is indirectly regulated by the first 20-HE rise, the commitment pulse (CP). Here we provide evidence that the rise in bursicon levels in the LNCs is directly triggered by the second 20-HE surge, the prepupal peak (PP). We performed several experimental manipulations that exposed LNCs to the PP without the CP; cells treated in this manner exhibited a significant rise in bursicon content. In contrast, bursicon levels remained unchanged in those LNCs exposed only to the CP. Exposure to the PP triggered a precocious increase in bursicon expression in LNCs from the penultimate larval stage. Increased bursicon levels in the LNCs were also induced by direct infusion of 20-HE. Taken together, the results of these experiments suggest that the rise in bursicon in the LNCs during metamorphosis is due to the direct action of the PP on the LNCs. Thus, the two 20-HE surges combine to regulate the CAP2-to-bursicon switch in the LNCs, the first acting indirectly to cause a decline in CAP2 levels and the second triggering a rise in bursicon expression, possibly by a direct action on the LNCs.

Animals↗
PubMed2000

Determination of spinosad and its metabolites in food and environmental matrices. 1. High-performance liquid chromatography with ultraviolet detection.

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidoptera larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection have been described for determining spinosad and its metabolites in environmental and food matrices. These residue methods typically involve an extraction with organic solvents, followed by purification using liquid-liquid partitioning and/or solid phase extraction prior to measurement by HPLC-UV. The residue methods determine the active ingredients (spinosyns A and D) and up to three minor metabolites (spinosyn B, spinosyn K, and N-demethylspinosyn D). The methods have validated limits of quantitation ranging from 0.010 to 0.040 microgram/g. This paper briefly reviews the residue methodology for spinosad and metabolites in food and environmental matrices and provides a summary of method validation results for 61 different sample types, including newly published results for 37 additional crop matrices and processed commodities.

Agriculture↗
PubMed2000

Determination of spinosad and its metabolites in food and environmental matrices. 3. Immunoassay methods.

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidopteran larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing a magnetic particle-based immunoassay (IA) test kit have been developed and validated for determining spinosad in environmental and food matrices. These methods involve an extraction of the residues from the matrices with appropriate solvents. For some matrices, the sample extracts can be diluted and measured directly by IA without any cleanup. For other matrices, sample extracts are purified using liquid-liquid partitioning and/or solid phase extraction prior to measurement by IA. The methods determine the total residue of spinosad, which includes the active ingredients (spinosyns A and D) and several minor metabolites, including spinosyn B, spinosyn K, and N-demethylspinosyn D. The methods have validated limits of quantitation of 0.0001 microgram/mL in water, 0.05 microgram/g in sediment, and 0.010 microgram/g in crops, crop processed commodities, and animal tissues. This paper briefly summarizes the residue methodology and method validation data for spinosad in 34 food, feed, and environmental matrices.

Animals↗
PubMed2000

Residues of spinosad in meat, milk, and eggs.

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and has a high level of activity against insects that infest a variety of crops. Dairy and poultry feeding studies were conducted to determine the magnitude of spinosad residues in animal products that would result from the consumption of typical feed commodities containing residues of spinosad. Dairy cows were dosed for 28 days with spinosad at rates equivalent to 0, 1, 3, and 10 microg/g in the diet. Chicken hens were dosed for 42 days with spinosad at rates equivalent to 0, 0.1, 0.3, 1, and 5 microg/g in the diet. Milk, eggs, and tissue samples were analyzed by high-performance liquid chromatography and/or immunoassay methods. Spinosad residues occurred in all of the sample types but were lowest in eggs, skim milk, and lean meat and were highest in the fat.

Animals↗
PubMed2000

Field evaluation of deet and a piperidine repellent against Aedes communis (Diptera: Culicidae) and Simulium venustum (Diptera: Simuliidae) in the Adirondack Mountains of New York.

Repellent efficacy of N,N-diethyl-3-methyl-benzamide (deet), the piperidine, 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and a 1:1 ratio of deet + AI3-37220 were evaluated topically (0.25 mg/cm2 applied in ethanol solution) on human volunteers against the mosquito Aedes communis (DeGeer) and the black fly Simulium venustum Say. The average repellency of all three formulations was > 95% at 4 h. For both mosquitoes and black flies, deet alone provided < 90% protection at 6 h, whereas AI3-37220 provided > 95% protection. Although repellent treatments were not significantly different overall, the contrasts between AI3-3720 versus deet were significant at 6 and 8 h. The 95% confidence interval on percent repellency at 6 h ranged from 90.1 to 98.9% for AI3-37220 versus 64.3 to 82.2% for deet, and at 8 h ranged 76.1 to 88.5% for AI3-37220 versus 47.8 to 64.0% for deet. Similarly, the confidence interval for protection against black flies at 6 h by (AI3-37220 ranged from 86.3 to 99.5% and did not overlap with the confidence interval provided by deet alone (51.2 to 78.8%). There was no evidence of synergistic repellency from a combination of the two compounds; i.e., protection from combined compounds was no better than either repellent used alone.

Aedes↗
PubMed1999

Bacillus sphaericus interferes with the development of Brugia malayi in Aedes aegypti.

Aedes aegypti (black-eyed Liverpool strain) were exposed to a sublethal dose (LD25) of Bacillus sphaericus and were fed to Mastomys coucha infected with Brugia malayi. The development of the filarial parasite was found to be arrested mostly at the second larval stage. The infection (P < 0.05), infectivity rates (P < 0.001) and L3 load (P < 0.001) were found to be reduced significantly in the treated group.

Aedes↗
PubMed2001

[The prescription of repellents].

Repellents are chemical products designed to ward off insects. They are effective against a wide range of arthropods including mosquitoes and flies but not against bees and wasps. Choosing the proper repellent can be difficult because no single product repels all pests. This article compares the performance and toxicity of various repellents based on reports describing systemic, topical, or physical repellents made from natural or synthetic ingredients. Essential oils are the most common natural ingredient. Synthetic ingredients range from dimethyphtalate, ethylexanediol, and diethyltoluamide (DEET) to new molecules such as 35/35, piperidine, bayerepel, and KBR 3023. Our findings indicate that DEET, used as the gold standard in most studies, is still the most effective repellent. There is no synergy between any of these products but combinations (e.g. ethylhexanediol and DEET) can optimize protection. In conclusion we propose guidelines for prescribing repellents to achieve the required level of protection in function of the duration of exposure and benefit-to-risk ratio. Several recommendations are made on the safe use repellents for adults and children.

Animals↗
PubMed1999

Genetic mapping of resistance to Bacillus thuringiensis toxins in diamondback moth using biphasic linkage analysis.

Transgenic plants producing environmentally benign Bacillus thuringiensis (Bt) toxins are deployed increasingly for insect control, but their efficacy will be short-lived if pests adapt quickly. The diamondback moth (Plutella xylostella), a worldwide pest of vegetables, is the first insect to evolve resistance to Bt toxins in open-field populations. A recessive autosomal gene confers resistance to at least four Bt toxins and enables survival without adverse effects on transgenic plants. Allelic variants of this gene confer resistance in strains from Hawaii, Pennsylvania, and the Philippines. Here we exploited the biphasic nature of Lepidopteran genetic linkage to map this gene in diamondback moth with 207 amplified fragment length polymorphisms as DNA markers. We also cloned and sequenced an amplified fragment length polymorphism marker for the chromosome containing the Bt resistance gene. The results provide a powerful tool for facilitating progress in understanding, monitoring, and managing resistance to Bt.

Animals↗