The insect growth regulator insecticide cyromazine causes earlier emergence in Drosophila melanogaster

The insecticide cyromazine, classified as an insect growth regulator, inhibits the growth and development of Diptera. The precise mode of cyromazine action remains unknown. We investigated mortality and developmental time effects after exposing first instar Drosophila melanogaster larvae to cyromazine for 8-h intervals. Significant increases in mortality were only observed for cyromazine exposure within the first 16 h of the first instar stage, suggesting that cyromazine accumulates in the larvae over time and that there is a delay between cyromazine exposure and effect. We observed that exposure to cyromazine during the early first instar stage resulted in earlier eclosion of adults when compared to cyromazine exposure at later first instar stages. The presence of 20-hydroxyecdysone during cyromazine exposure significantly reduced the lethal effect of cyromazine. We raise the possibility that the mode of cyromazine action is related to the development hormone, 20-hydroxyecdysone.     

Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests

The ornamental tobacco (Nicotiana alata) produces one 6-kDa chymotrypsin inhibitor and four 6-kDa trypsin inhibitors from a single 40.3-kDa precursor protein. Three different approaches have been used to assess the potential of these proteinase inhibitors (PIs) in insect control. The first was an in-vitro approach in which all five inhibitors, the single chymotrypsin inhibitor or three of the four trypsin inhibitors were tested for their ability to inhibit gut protease activity in insects from four orders. The second approach was to incorporate the N. alata PIs in the artificial diet of the native budworm (Helicoverpa punctigera) and the black field cricket (Teleogryllus commodus). H. punctigera larvae and T. commodus nymphs had a significant (P<0.01) reduction in growth after ingestion of the PI and were more lethargic than insects on the control diet. Several of the H. punctigera larvae also failed to complete moulting at the third or fourth instar. The third approach was to express the N. alata PIs in transgenic tobacco under the control of the 35S CaMV promoter. When H. punctigera larvae were fed tobacco leaves expressing the N. alata PIs at 0.2% soluble protein, significant (P<0.01) differences in mortality and/or growth rate were observed.     

Assessment of insecticide resistance in five insect pests attacking field and vegetable crops in Nicaragua

Field populations of Hypothenemus hampei (Ferrari), Plutella xylostella (L.), Spodoptera exigua (Hübner), Helicoverpa zea (Boddie) and Bemisia tabaci (Gennadius) were tested for resistance to several insecticides commonly used in Nicariagua. Assays were conducted to estimate the LD50s or LC50s and the corresponding resistance ratios. A diagnostic concentration was used to discriminate between susceptible and resistant strains of H. hampei. The tests with >6,000 H. hampei adults collected from six different sites indicate the absence of resistance to endosulfan. Resistance to cypermethrin, deltamethrin, chlorfluazuron, thiocyclam, and methamidophos was documented in six field populations of P. xylostella. High levels of resistance to cypermethrin and deltamethrin, but moderate levels of resistance to chlorpyriphos and methomyl, were also documented in two field populations of S. exigua. Moderate levels of resistance to cypermethrin, deltamethrin and chlorpyriphos were also documented in three field populations of H. zea. Moderate to high levels of resistance to bifenthrin, methamidophos and endosulfan were documented in four field populations of B. tabaci. The presence of significant correlations between LD50s or LC50s suggests the occurrence of cross-resistance or simultaneous selection for resistance by different insecticides with different modes of action. Our data could not differentiate between these two possibilities. Because insecticides will continue being used in Nicaragua, a resistance management program is urgently needed. The implementation of integrated pest management tactics must be accompanied by specific regulations for pesticide registration. In the future, pesticide registration regulations in Nicaragua should include periodic resistance monitoring. The mechanisms to cover the costs of resistance monitoring and resistance management should also be established.     

Organophosphorus insecticide resistance inDrosophila melanogaster populations

Natural variation inDrosophila melanogaster populations for mixed function oxidase activity and organophosphorus resistance was studied by sampling iso-chromosomal lines and laboratory selection. A 20-fold variation in malathion LC₅₀ was found among a sample of 25 third chromosomes from a Raleigh, North Carolina, population. These chromosomes were combined in a population that was selected for malaoxon (a toxic metabolite of malathion) resistance over 12 generations. Response to selection was rapid—within three generations—but small, less than two-fold increase in malathion LC₅₀. Mixed function oxidase activity, as assayed by 7-ethoxycoumarin-O-deethylase, increased in parallel with malathion resistance in the selected population. In spite of the fact that this population was initially formed from strains which were homozygous for chromosome III, after 12 generations of selection for malaoxon resistance only a minority of third chromosomes could be isolated as homozygous combinations. This suggests that selection favoured heterozygous combinations of alleles with decreased fitness in the homozygous state. In a second study, a sample of 39 iso-female lines were collected from a Vineland, Ontario, population. Imidan? (phosmet) LC₅₀ varied 20-fold among these iso-female lines and was strongly correlated with increased 7-ethoxycoumarin-O-deethylase activity. The distribution of 7-ethoxycoumarin-O-deethylase activity was bimodal and estimates of the effective number of segregating factors by Wright’s formula were consistent with a single gene controlling extreme 7-ethoxycoumarin-O-deethylase activity differences. Vineland flies responded rapidly to selection for imidan resistance, but as with malaoxon selection only to a small degree. The 7-ethoxycoumarin-O-deethylase activity increased in imidan-selected flies to the level of the most resistant iso-female line from the sampled population. The major part of selected imidan resistance and all of the increased 7-ethoxycoumarin-O-deethylase activity were attributed to third chromosomal genes. The results suggest that theseDrosophila populations contained a polymorphism for a major factor on chromosome III controlling elevated mixed function oxidase activity together with associated organophosphorus resistance. This polymorphism provided the immediate response to insecticide selection. Other genes have minor effects and combine to give a multifactorial response to selection over longer periods of time.     

Generic insect repellent detector from the fruit fly Drosophila melanogaster

Background

Insect repellents are prophylactic tools against a number of vector-borne diseases. There is growing demand for repellents outperforming DEET in cost and safety, but with the current technologies R&D of a new product takes almost 10 years, with a prohibitive cost of $30 million dollar in part due to the demand for large-scale synthesis of thousands of test compounds of which only 1 may reach the market. R&D could be expedited and cost dramatically reduced with a molecular/physiological target to streamline putative repellents for final efficacy and toxicological tests.

Methodology

Using olfactory-based choice assay we show here that the fruit fly is repelled by not only DEET, but also IR3535 and picaridin thus suggesting they might have “generic repellent detector(s),” which may be of practical applications in new repellent screenings. We performed single unit recordings from all olfactory sensilla in the antennae and maxillary palps. Although the ab3A neuron in the wild type flies responded to picaridin, it was unresponsive to DEET and IR3535. By contrast, a neuron housed in the palp basiconic sensilla pb1 responded to DEET, IR3535, and picaridin, with apparent sensitivity higher than that of the DEET detectors in the mosquitoes Culex quinquefasciatus and Aedes aegypti. DmOr42a was transplanted from pb1 to the “empty neuron” and showed to be sensitive to the three insect repellents.

Conclusions

For the first time we have demonstrated that the fruit fly avoids not only DEET but also IR3535 and picaridin, and identified an olfactory receptor neuron (ORN), which is sensitive to these three major insect repellents. We have also identified the insect repellent-sensitive receptor, DmOr42a. This generic detector fulfils the requirements for a simplified bioassay for early screening of test insect repellents.     

Protein–protein interaction network analysis of insecticide resistance molecular mechanism in Drosophila melanogaster

The problem of resistance has not been solved fundamentally at present, because the development speed of new insecticides can not keep pace with the development speed of resistance, and the lack of understanding of molecular mechanism of resistance. Here we collected seed genes and their interacting proteins involved in insecticide resistance molecular mechanism in Drosophila melanogaster by literature mining and the String database. We identified a total of 528 proteins and 13514 protein–protein interactions. The protein interaction network was constructed by String and Pajek, and we analyzed the topological properties, such as degree centrality and eigenvector centrality. Proteasome complexes and drug metabolism—cytochrome P450 were an enrichment by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. This is the first time to explore the insecticide resistance molecular mechanism of D. melanogaster by the methods and tools of network biology, it can provide the bioinformatic foundation for further understanding the mechanisms of insecticide resistance.     

Can natural enemies of current insect pests provide biotic resistance to future pests?

1. Economic pests jeopardize agricultural production worldwide. Classical biological control, comprising the import of exotic natural enemies to control target pest populations, has a successful history in many countries. However, little is known about how these natural enemies contribute to the suppression of pests that are yet to arrive. Biotic resistance theory, though, posits that communities resist species invasions as a result of natural enemies.
2. We assessed the potential of the resident exotic parasitoid wasp fauna in New Zealand (intentionally‐introduced biological control agents and unintentionally‐introduced species) to provide biotic resistance against possible future pests. A dataset was generated containing resident exotic parasitoid species (Ichneumonoidea: Braconidae; Ichneumonidae) in New Zealand, as well as their known global host ranges and the pest status of host species, to infer the potential for biotic resistance.
3. The known exotic ichneumonoid fauna in New Zealand comprises 65 species. These species associate with 107 host species in New Zealand, of which 54 species are pests. However, the current exotic species could potentially suppress 442 pest species not yet occurring in New Zealand.
4. This approach could be used to inform pest management programmes worldwide. Future research should consider how biotic resistance from the established parasitoid fauna can be used to inform specific decisions with respect to classical biological control.

     

Ether resistance in Drosophila melanogaster

Summary Strains set up from single inseminated females of D. melanogaster from the wild differ in their resistance to the anaesthetics, ether and chloroform. The main differences between four selected extreme strains could be explained by additive genes, which in the case of ether resistance were located to regions of chromosomes 2 and 3. The lack of correspondence between ether and chloroform resistance between strains indicates that although the type of genetic architecture controlling the traits is similar, the actual genes differ, which is reasonable in view of their differing chemical structures. Quite high heritabilities were found for resistance to ether based on five inbred strains. No significant associations between resistance to ether and body weight, developmental rate or longevity were found.It is clear that resistance to both anaesthetics would be amenable to more detailed genetic analyses. It is pointed out that the general conclusions reached from such studies will have implications with respect to the effect of chemicals such as insecticides, not naturally present in nature.     

The endosymbiotic bacterium Wolbachia enhances the nonspecific resistance to insect pathogens and alters behavior of Drosophila melanogaster

To determine biologically important effects of the cytoplasmic endosymbiont Wolbachia, two substrains of the same Drosophila melanogaster strain have been studied, one of them infected with Wolbachia and the other treated with tetracycline to eliminate the bacterium. Female D. melanogaster infected with Wolbachia are more resistant to the fungus Bauveria bassiana (an insect pathogen) than uninfected females; infected females also exhibited changes in oviposition substrate preference. Males infected with the bacterium are more competitive than uninfected males. The possible role of Wolbachia in the formation of alternative ecological strategies of D. melanogaster is discussed.     

Using experimental evolution to study the physiological mechanisms of desiccation resistance in Drosophila melanogaster

Data from populations undergoing experimental evolution can be used to make comparisons between physiologically differentiated populations and to determine evolutionary trajectories. Comparisons of long-established laboratory populations of Drosophila melanogaster that are strongly differentiated with respect to desiccation resistance are used to test alternative hypotheses concerning the mechanisms that fruit flies use to survive bouts of extreme desiccation. This comparative study supports the hypothesis that, in at least one case, D. melanogaster can evolve increased resistance to desiccation by decreasing water loss rates and by increasing bulk water content but not by increasing metabolic water content or dehydration tolerance. While glycogen was involved in water storage, its primary role was in water binding, not the production of metabolic water. Measurement of the trajectories of these component mechanisms during selection for desiccation resistance is used to demonstrate that water loss rate quickly plateaus in response to selection, while water content continues to improve. This disparity reveals the value of studying evolutionary trajectories and the need for longer-term selection studies in evolutionary physiology.     

Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.     

Evaluating the insecticide resistance potential of eight Drosophila melanogaster cytochrome P450 genes by transgenic over-expression

In Drosophila melanogaster, the increased expression of Cyp6g1 results in resistance to chemically unrelated insecticides including DDT, neonicotinoids and insect growth regulator insecticides. To determine the insecticide resistance capacity of other D. melanogaster cytochrome P450s, we used the GAL4/UAS system to express individual P450s in the midgut, Malpighian tubules and fat body of transgenic flies. Drosophila over-expressing Cyp6g1, Cyp6g2, Cyp6t3, Cyp6a2, Cyp6a8, Cyp6a19, Cyp6a23 and Cyp12d1 were screened for resistance to four insecticides--DDT, nitenpyram, dicyclanil and diazinon. Increased survival on insecticides is detected for Cyp6g1 (DDT, nitenpyram and dicyclanil), Cyp6g2 (nitenpyram and diazinon) and Cyp12d1 (DDT and dicyclanil) over-expression lines. No increased survival on any insecticide was detected for flies over-expressing either Cyp6a2, Cyp6a8, Cyp6t3, Cyp6a19 or Cyp6a23.     

Behavioural response to combined insecticide and temperature stress in natural populations of Drosophila melanogaster

Insecticide resistance evolves extremely rapidly, providing an illuminating model for the study of adaptation. With climate change reshaping species distribution, pest and disease vector control needs rethinking to include the effects of environmental variation and insect stress physiology. Here, we assessed how both long‐term adaptation of populations to temperature and immediate temperature variation affect the genetic architecture of DDT insecticide response in Drosophila melanogaster. Mortality assays and behavioural assays based on continuous activity monitoring were used to assess the interaction between DDT and temperature on three field‐derived populations from climate extremes (Raleigh for warm temperate, Tasmania for cold oceanic and Queensland for hot tropical). The Raleigh population showed the highest mortality to DDT, whereas the Queensland population, epicentre for derived alleles of the resistance gene Cyp6g1, showed the lowest. Interaction between insecticide and temperature strongly affected mortality, particularly for the Tasmanian population. Activity profiles analysed using self‐organizing maps show that the insecticide promoted an early response, whereas elevated temperature promoted a later response. These distinctive early or later activity phases revealed similar responses to temperature and DDT dose alone but with more or less genetic variance depending on the population. This change in genetic variance among populations suggests that selection particularly depleted genetic variance for DDT response in the Queensland population. Finally, despite similar (co)variation between traits in benign conditions, the genetic responses across population differed under stressful conditions. This showed how stress‐responsive genetic variation only reveals itself in specific conditions and thereby escapes potential trade‐offs in benign environments.     

Seasonal fluctuation in susceptibility to insecticides within natural populations of Drosophila melanogaster: empirical observations of fitness costs of insecticide resistance

To investigate genetic variation and seasonal fluctuation in susceptibility to insecticides, natural populations of Drosophila melanogaster were collected from Katsunuma in mid summer and late fall for two consecutive years. After isofemale lines of each population collected in each season had been established in a laboratory, the susceptibility of each line to five insecticides, including permethrin, malathion, prothiophos, fenitrothion, and DDT, was examined. Lines of each population exhibited the broad ranges of variation in susceptibility to all chemicals. Comparison between populations in different seasons indicated that genetic variation in susceptibility to organophosphates fluctuated in consistency with the population size, in which the susceptibility increased in fall. In addition, highly significant correlations were observed among responses to organophosphates, and the correlations also fluctuated with seasons. On the other hand, genetic variation in susceptibility to permethrin and DDT was less fluctuated. These results suggest that not only a common resistance factor for organophosphate resistance but also different resistance factor(s) for each insecticide could be involved within a natural population, and that the fluctuation observed in the susceptibility to organophosphates could be associated with fitness costs of organophosphate resistance factor(s).     

Overexpression of Methoprene-tolerant, a Drosophila melanogaster gene that is critical for juvenile hormone action and insecticide resistance

The Methoprene-tolerant (Met) gene of Drosophila melanogaster is involved in both juvenile hormone (JH) action and resistance to JH insecticides, such as methoprene. Although the consequences of Met mutations on development and methoprene resistance are known, no studies have examined Met+ overexpression. Met+ was overexpressed in transgenic lines with various promoters that drive overexpression to different levels. Flies expressing either genomic or cDNA Met+ transgenes showed higher susceptibility to both the morphogenetic and toxic effects of methoprene, consistent with the hormone-binding property of MET. Both the sensitive period and lethal period were the same as seen for non-overexpressing Met+ flies. However, continual exposure of high-overexpressing Met+ larvae to borderline-toxic or higher methoprene doses advanced the sensitive period from prepupae to first instar and the lethal period from pharate adults to larvae and early pupae. When expression of transgenic UAS-Met+ was driven to high levels by either an actin-GAL4 or tubulin-GAL4 promoter, larvae showed high mortality in the absence of methoprene, indicating that high MET titer is lethal, perhaps resulting from expression in an inappropriate tissue. Adults overexpressing Met+ did not show enhanced oogenesis, ruling out MET as a limiting factor for this hormone-driven physiology.