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The Tn antigen-specific lectin from ground ivy is an insecticidal protein with an unusual physiology.

Leaves of ground ivy (Glechoma hederacea) contain a lectin (called Gleheda) that is structurally and evolutionary related to the classical legume lectins. Screening of a population of wild plants revealed that Gleheda accounts for more than one-third of the total leaf protein in some clones, whereas it cannot be detected in other clones growing in the same environment. Gleheda is predominantly expressed in the leaves where it accumulates during early leaf maturation. The lectin is not uniformly distributed over the leaves but exhibits a unique localization pattern characterized by an almost exclusive confinement to a single layer of palisade parenchyma cells. Insect feeding trials demonstrated that Gleheda is a potent insecticidal protein for larvae of the Colorado potato beetle (Leptinotarsa decemlineata). Because Gleheda is not cytotoxic, it is suggested that the insecticidal activity is linked to the carbohydrate-binding specificity of the lectin, which as could be demonstrated by agglutination assays with different types of polyagglutinable human erythrocytes is specifically directed against the Tn antigen structure (N-acetylgalactosamine O-linked to serine or threonine residues of proteins).

Aging↗

An insecticidal N-acetylglucosamine-specific lectin gene from Griffonia simplicifolia (Leguminosae).

Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.). A cDNA clone encoding G. simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system. The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties. These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.

Acetylglucosamine↗

Fitness costs of insecticide resistance in natural breeding sites of the mosquito Culex pipiens.

Genetic changes conferring adaptation to a new environment may induce a fitness cost in the previous environment. Although this prediction has been verified in laboratory conditions, few studies have tried to document this cost directly in natural populations. Here, we evaluated the pleiotropic effects of insecticide resistance on putative fitness components of the mosquito Culex pipiens. Experiments using different larval densities were performed during the summer in two natural breeding sites. Two loci that possess alleles conferring organophosphate (OP) resistance were considered: ace-1 coding for an acetylcholinesterase (AChE1, the OP target) and Ester, a ''super locus" including two closely linked loci coding for esterases A and B. Resistance ace-1 alleles coding for a modified AChE1 were associated with a longer development time and shorter wing length. The pleiotropic effects of two resistance alleles Ester1 and Ester4 coding for the overproduced esterases A1 and A4-B4, respectively, were more variable. Both A1 and A4-B4 reduced wing length, although only A1 was associated with a longer preimaginal stage. The fluctuating asymmetry (FA) of the wing did not respond to the presence or to the interaction of resistance alleles at the two loci at any of the density levels tested. Conversely, the FA of one wing section decreased when larval density increased. This may be the consequence of selection against less developmentally stable individuals. The results are discussed in relation to the local evolution of insecticide resistance genes.

Acetylcholinesterase↗

Species abundance and insecticide resistance of Anopheles gambiae in selected areas of Ghana and Burkina Faso.

The Ghanaian National Malaria Control Programme has prioritized insecticide-treated materials as a key strategy for malaria control. We report on a survey of the distribution of the molecular forms of Anopheles gambiae Giles (Diptera: Culicidae) and insecticide resistance (the kdr mutation), carried out by sampling mosquitoes from 11 locations in Ghana and one additional site in Burkina Faso. The molecular M and S forms of An. gambiae were found to occur in sympatry in southern Ghana. The S form predominated throughout its distribution in the coastal savannah, except at one location in the strand and mangrove zone where rice was cultivated. The M form was the only form collected in northern Ghana and was the predominant form (97.5%) in Burkina Faso. No M/S hybrids were detected. The kdr mutation was observed at very high frequencies (98-100%) within the S form but reached a maximum of only 3.38% in the M form in one population at an irrigation scheme in the Ghanaian coastal savannah zone.

Animals↗

The naturally derived insecticide spinosad is highly toxic to Aedes and Anopheles mosquito larvae.

Spinosad is a naturally derived biorational insecticide with an environmentally favourable toxicity profile, so we investigated its potency against mosquito larvae (Diptera: Culicidae). By laboratory bioassays of a suspension concentrate formulation of spinosad (Tracer), the 24 h lethal concentration (LC50) against Aedes aegypti (L.) third and fourth instars was estimated at 0.025 p.p.m. following logit regression. The concentration-mortality response of third- and fourth-instar Anopheles albimanus Weidemann did not conform to a logit model. The LC50 value of spinosad in Anopheles albimanus was 0.024 p.p.m. by quadratic linear regression. A field trial in southern Mexico demonstrated that spinosad 1 p.p.m. compared with the standard temephos (Abate) 1% granules 100 g/m3 water prevented Ae. aegypti breeding in plastic containers of water for 8 weeks; at 10 p.p.m. spinosad prevented breeding for > 22 weeks. In another field trial, spinosad at 5 p.p.m. and temephos both completely eliminated reproduction of Ae. aegypti for 13 weeks. In contrast, the bacterial insecticide Bacillus thuringiensis var. israelensis (Bti, Vectobac) AS) performed poorly with just 2 weeks of complete inhibition of Ae. aegypti breeding. Spinosad also effectively prevented breeding of Culex mosquitoes and chironomids in both trials to a degree similar to that of temephos. We conclude that spinosad merits evaluation as a replacement for organophosphate or Bti treatment of domestic water tanks in Mesoamerica. We also predict that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.

Aedes↗

Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover.

We have identified two mutations in the ace1 gene of Aphis gossypii that are associated with insensitivity of acetylcholinesterase (AChE) to carbamate and organophosphate insecticides. The first of these, S431F (equivalent to F331 in Torpedo californica), is associated with insensitivity to the carbamate insecticide pirimicarb in a range of A. gossypii clones. The S431F mutation is also found in the peach-potato aphid, Myzus persicae (Sulzer), and a rapid RFLP diagnostic allows the identification of individuals of both aphid species with a resistant genotype. This diagnostic further revealed the presence of S431 in several other pirimicarb-susceptible aphid species. The serine at this position in the wild-type enzyme has only been reported for aphids and provides a molecular explanation of why pirimicarb has a specific aphicidal action. A less specific insensitivity to a wide range of carbamates and organophosphates is associated with a second mutation, A302S (A201 in T. californica).

Acetylcholinesterase↗

Organophosphate insecticide poisoning.

Organophosphate insecticides are widely used agents which are quickly absorbed through the skin and mucous membranes. The effects of acute exposure to these agents can be severe and intensive therapy may be required. Specific drugs are available to reverse the muscarinic, nicotinic and central effects of these poisons. When given early they are very effective and early diagnosis and treatment may therefore be life-saving. A case of acute poisoning with an organophosphate anticholinesterase insecticide is reported. The signs and symptoms of acute poisoning are described and a rational approach to specific treatment is discussed.

Acute Disease↗

The unique mutation in ace-1 giving high insecticide resistance is easily detectable in mosquito vectors.

High insecticide resistance resulting from insensitive acetylcholinesterase (AChE) has emerged in mosquitoes. A single mutation (G119S of the ace-1 gene) explains this high resistance in Culex pipiens and in Anopheles gambiae. In order to provide better documentation of the ace-1 gene and the effect of the G119S mutation, we present a three-dimension structure model of AChE, showing that this unique substitution is localized in the oxyanion hole, explaining the insecticide insensitivity and its interference with the enzyme catalytic functions. As the G119S creates a restriction site, a simple PCR test was devised to detect its presence in both A. gambiae and C. pipiens, two mosquito species belonging to different subfamilies (Culicinae and Anophelinae). It is possibile that this mutation also explains the high resistance found in other mosquitoes, and the present results indicate that the PCR test detects the G119S mutation in the malaria vector A. albimanus. The G119S has thus occurred independently at least four times in mosquitoes and this PCR test is probably of broad applicability within the Culicidae family.

Acetylcholinesterase↗

Flight activity of insecticide resistant and susceptible Anopheles stephensi mosquitoes in actograph chambers lined with malathion, gamma HCH or dieldrin.

The activity and resting behaviour of resistant and susceptible Anopheles stephensi Liston were recorded in acoustic actograph chambers lined with residual deposits of malathion, dieldrin or gamma HCH. In gamma HCH-treated flight chambers, SS and RS mosquitoes became active only after picking up lethal doses of insecticide, which explains why few SS and RS mosquitoes survive release into gamma HCH-treated experimental huts. Similar results were obtained in flight chambers treated with dieldrin; however, more mosquitoes would be expected to survive dieldrin under field conditions because resistance to this insecticide is greater than to gamma HCH. Mosquitoes in contact with malathion showed a three-phase activity pattern: an initial active phase, an inactive phase, and hyper-activity/convulsions. Initial activity or irritability was especially pronounced in SS and RS but absent in RR mosquitoes. Whether or not irritability would protect RS mosquitoes from malathion would probably depend on the ratio of sprayed to unsprayed surfaces in treated huts.

Animals↗

Insecticide resistance gene frequencies in Anopheles sacharovi populations of the Cukurova plain, Adana Province, Turkey.

In Turkey, the mosquito Anopheles sacharovi has been under field selection pressure sequentially with DDT, dieldrin, malathion and pirimiphosmethyl over a period of 30 years for the purpose of malaria control. In 1984, the field population of An.sacharovi in the malarious Cukurova plain of Adana Province contained an altered acetylcholinesterase-based resistance gene giving broad spectrum resistance against organophosphorus and carbamate insecticides. The cross-resistance spectrum from this mechanism conferred resistance to malathion but not to the organophosphorus insecticide pirimiphos-methyl. Over the 6 years that pirimiphos-methyl has been applied for malaria vector control in this area, the frequency of the altered acetylcholinesterase resistance gene has declined, although in 1989 and 1990 it was still present at measurable frequencies in An.sacharovi from Cukurova. In addition to the acetylcholinesterase resistance mechanism there is evidence of an increased level of glutathione S-transferase in some of the An.sacharovi populations tested. This is known to be correlated with DDT resistance in other anophelines. In Turkish An.sacharovi, DDT resistance and elevated glutathione S-transferase occur in the same populations at similar frequencies. The continued prevalence of resistance to DDT and dieldrin, long after the 1971 cessation of DDT spraying for malaria control in Turkey, suggests that the DDT resistance gene has insufficient reduced fitness associated with it to have been lost from the field population during the past two decades. The implications of the slow decline in resistance gene frequencies in this field population are discussed in relation to mathematical models for managing resistance.

Acetylcholinesterase↗

Changes in enzyme titres with age in four geographical strains of Aedes aegypti and their association with insecticide resistance.

The enzymes acetylcholinesterase, glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PD), and general esterases were assayed in four strains of Aedes aegypti mosquitoes aged between 1 and 30 days. Microtitre plate methods were used to assay activity in the homogenates of individual mosquitoes. The levels of GST and G6PD declined with the age of the mosquitoes, while the activity for the other enzymes remained constant. Soluble protein content was also found to decline with mosquito age in all the strains. Insecticide bioassays showed that two strains (Trinidad and Virtudes) of Ae. aegypti were resistant to DDT, deltamethrin and malathion, whereas two other strains (Bangkok and Indian) were susceptible to all four classes of insecticides tested. Higher esterase activity levels in the resistant compared to the susceptible strains were assumed to be the cause of organophosphate resistance. The combination of DDT and deltamethrin resistance in two strains with normal GST and G6PD characteristics suggests that a kdr-type nerve insensitivity mechanism may be involved.

Acetylcholinesterase↗

Laboratory evaluation of desiccants and insecticidal soap applied to various substrates to control the deer tick Ixodes scapularis.

Desiccant and soap pest control products were tested against Ixodes scapularis nymphs on sod, pachysandra, landscaping stones and pinebark woodchips in a laboratory study. High mortality (91-100%) was obtained with the silica-based desiccant Drione and Safer's insecticidal soap (SIS) treatments except for SIS treated woodchips. SIS and Drione contain 0.2 and 1% pyrethrins, respectively. SIS may be absorbed by the woodchips and not picked up by the nymphs as mortality decreased to 78%. Diatomaceous earth and Dri-die reduced nymphs by only 10-41%; neither desiccant contains insecticides. Nymphal mortality increased 15-17% after 20 or 100 ml of water was sprayed over sod plugs treated with SIS or Drione. The addition of isopropyl alcohol (ROH) to SIS increased the efficacy of SIS against nymphal I.scapularis on treated sod plugs but not on treated landscaping substrates. Drione, SIS, SIS-ROH and chlorpyrifos 50WP treated landscaping stones and pinebark woodchips resulted in 88-95% and 72-96% nymphal mortality, respectively. Unexpectedly, these substrates and treatments affected tick movement as well. Effective pest control products applied to xeric landscaping substrates present in maintained beds and borders near wooded areas could deter tick movement and provide significant levels of tick control.

Animals↗

Regulation of insecticidal crystal protein production in Bacillus thuringiensis.

The production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis normally coincides with sporulation, resulting in the appearance of parasporal crystalline inclusions within the mother cell. In most instances, the temporal and spatial regulation of ICP gene expression is determined at the transcriptional level by mother-cell-specific sigma factors that share homology with sigma E and sigma K from Bacillus subtilis. The cryIII ICP genes are a notable exception; these genes are transcribed from sigma A-like promoters during vegetative growth, are induced or derepressed at the onset of stationary phase, and are overexpressed in sporulation mutants of B. thuringiensis blocked in the phosphorylation of Spo0A, a key regulator of sporulation initiation. Transcription alone, however, cannot account for the impressive ability of this bacterium to accumulate insecticidal proteins. A variety of post-transcriptional and post-translational mechanisms also contribute to the efficient production of ICPs in B. thuringiensis, thus making this bacterium a cost-effective biological control agent.

Amino Acid Sequence↗

Low temperature-induced insecticidal activity of Yersinia enterocolitica.

The insecticidal toxin complexes (Tcs) are produced by several Enterobacteriaceae associated with insects, such as Photorhabdus luminescens, Serratia entomophila and Xenorhabdus nematophilus. Genome sequences revealed tc-like genes in Yersinia spp., but insecticidal activity of this genus associated with the toxins has not been described. Through the search for genes upregulated at low growth temperatures in Yersinia enterocolitica strain W22703, a genomic island of 19 kb termed tc-PAI(Ye) with homologues of the toxin genes tcaA, tcaB, tcaC and tccC was identified. Southern blot and polymerase chain reaction (PCR) analysis of 34 strains demonstrated that the tc-PAI(Ye) is present in biovars 2, 3 and 4, but neither in biovars 1A and 1B, nor in five Yersinia species apathogenic in humans. Using the luxCDABE operon as reporter, the expression of the toxin genes was shown to be completely repressed in cells cultured at 37 degrees C, and to increase by 4.6 orders of magnitude when the growth temperature was decreased gradually to 10 degrees C. These data provide the first indication that temperature is a critical parameter for induction or repression of tc gene transcription. Whole-cell extracts of Y. enterocolitica strain W22703 cultivated at 10 degrees C, but not at 30 degrees C, led to insect mortality when fed to Manduca sexta larvae, in contrast to an insertional tcaA mutant. Overall the results suggest that the tc-PAI(Ye) could play an important role in the transmission and survival of pathogenic Y. enterocolitica strains outside mammalian hosts.

Animals↗

Experience of targeting subsidies on insecticide-treated nets: what do we know and what are the knowledge gaps?

Widespread coverage of vulnerable populations with insecticide-treated nets (ITNs) constitutes an important component of the Roll Back Malaria (RBM) strategy to control malaria. The Abuja Targets call for 60% coverage of children under 5 years of age and pregnant women by 2005; but current coverage in Africa is unacceptably low. The RBM 'Strategic Framework for Coordinated National Action in Scaling-up Insecticide-Treated Netting Programmes in Africa' promotes coordinated national action and advocates sustained public provision of targeted subsidies to maximise public health benefits, alongside support and stimulation of the private sector. Several countries have already planned or initiated targeted subsidy schemes either on a pilot scale or on a national scale, and have valuable experience which can inform future interventions. The WHO RBM 'Workshop on mapping models for delivering ITNs through targeted subsidies' held in Zambia in 2003 provided an opportunity to share and document these country experiences. This paper brings together experiences presented at the workshop with other information on experiences of targeting subsidies on ITNs, net treatment kits and retreatment services (ITN products) in order to describe alternative approaches, highlight their similarities and differences, outline lessons learnt, and identify gaps in knowledge. We find that while there is a growing body of knowledge on different approaches to targeting ITN subsidies, there are significant gaps in knowledge in crucial areas. Key questions regarding how best to target, how much it will cost and what outcomes (levels of coverage) to expect remain unanswered. High quality, well-funded monitoring and evaluation of alternative approaches to targeting ITN subsidies is vital to develop a knowledge base so that countries can design and implement effective strategies to target ITN subsidies.

Africa↗

Deltamethrin, a pyrethroid insecticide, is a potent inducer for the activity-dependent gene expression of brain-derived neurotrophic factor in neurons.

The mRNA expression of brain-derived neurotrophic factor (BDNF) is controlled in an activity-dependent manner through Ca(2+) influx into neurons. Pyrethroids are widely used insecticides of low acute toxicity in mammals, but their effects on sodium channels are known to lead to hyperexcitation in neuronal cells of insects. In this study, we found that deltamethrin, a type II pyrethroid insecticide, was highly effective in inducing BDNF expression in culture and in the rat brain. Addition of deltamethrin to rat cortical cells in culture markedly increased the expression of BDNF exon III-V mRNA and protein, dependent upon the neuronal activity accompanying the influx of Ca(2+) into neurons and the Ca(2+) influx-dependent phosphorylation of extracellular signal-regulated kinases 1/2. The elevated expression was maintained for at least 48 h, even after deltamethrin was withdrawn from the culture medium. Comparison of the effects of selected pyrethroids on the expression revealed that type II but not type I pyrethroids effectively induced BDNF mRNA expression. In addition, administration of deltamethrin to rats increased the level of BDNF protein in the cerebral cortex and hippocampus. These results indicate that deltamethrin is a potent inducer of BDNF expression in neurons and that it may induce neuronal hyperexcitation if it reaches the brain.

Anesthetics, Local↗

Evolution of supergene families associated with insecticide resistance.

The emergence of insecticide resistance in the mosquito poses a serious threat to the efficacy of many malaria control programs. We have searched the Anopheles gambiae genome for members of the three major enzyme families- the carboxylesterases, glutathione transferases, and cytochrome P450s-that are primarily responsible for metabolic resistance to insecticides. A comparative genomic analysis with Drosophila melanogaster reveals that a considerable expansion of these supergene families has occurred in the mosquito. Low gene orthology and little chromosomal synteny paradoxically contrast the easily identified orthologous groups of genes presumably seeded by common ancestors. In A. gambiae, the independent expansion of paralogous genes is mainly a consequence of the formation of clusters among locally duplicated genes. These expansions may reflect the functional diversification of supergene families consistent with major differences in the life history and ecology of these organisms. These data provide a basis for identifying the resistance-associated enzymes within these families. This will enable the resistance status of mosquitoes, flies, and possibly other holometabolous insects to be monitored. The analyses also provide the means for identifying previously unknown molecules involved in fundamental biological processes such as development.

Animals↗

Uterotrophic action of the insecticide methoxychlor.

Dusting of rats and mice, as well as oral treatment with the insecticide methoxychlor, produced uterine weight increase in these rodents. Ablation of the ovaries, adrenals, or pituitary did not alter this effect, thus indicating a direct trophic action of this agent on the uterus. It is apparent that the application of this insecticide to animals used for hormonal experiments introduces an additional variable.

Animals↗