Fitness costs associated with Cry1F resistance in the European corn borer

Crops producing insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are widely planted to manage insect pests. Bt crops can provide an effective tool for pest management; however, the evolution of Bt resistance can diminish this benefit. The European corn borer, Ostrinia nubilalis Hübner, is a significant pest of maize and is widely managed with Bt maize in the Midwest of the United States. When Bt crops are grown in conjunction with non‐Bt refuges, fitness costs of Bt resistance can delay the evolution of resistance. Importantly, fitness costs often vary with ecological factors, including host‐plant genotype and diapause. In this study, we examined fitness costs associated with Cry1F resistance in O. nubilalis when insects were reared on three maize lines. Fitness costs were tested in two experiments. One experiment assessed the fitness costs when Cry1F‐resistant and Cry1F‐susceptible insects were reared on plants as larvae and experienced diapause. The second experiment tested resistant, susceptible and F1 heterozygotes that were reared on plants but did not experience diapause. Despite some evidence of greater adult longevity for Cry1F‐resistant insects, these insects produced fewer fertile eggs than Cry1F‐susceptible insects, and this occurred independent of diapause. Reduced fecundity was not detected among heterozygous individuals, which indicated that this fitness cost was recessive. Additionally, maize lines did not affect the magnitude of this fitness cost. The lower fitness of Cry1F‐resistant O. nubilalis may contribute to the maintenance of Cry1F susceptibility in field populations more than a decade after Cry1F maize was commercialized.     

Evidence for a robust sex-specific trade-off between cold resistance and starvation resistance in Drosophila melanogaster

In insects changes in lipid metabolism may underlie a trade-off between cold resistance and starvation resistance. To test this we examined correlated responses in independent sets of Drosophila melanogaster lines selected for increased cold resistance and increased starvation resistance. The starvation lines showed correlated patterns found in other D. melanogaster populations selected for this trait, including higher lipid levels and increased resistance to desiccation, although the selected lines did not show a longer development time as found in some other studies. Consistent with the trade-off hypothesis, selected lines with increased starvation resistance showed decreased resistance to a cold stress as measured by mortality, whereas selected lines with increased cold resistance showed a decrease in starvation resistance. To counter the possibility of inadvertent selection accounting for these patterns, selected and control lines from both selection regimes were crossed to form mass bred populations, which were left for four generations prior to establishing isofemale lines. By scoring starvation and cold resistance in these lines derived from both sets of selection regimes, we confirmed the negative association between resistance to these stresses in females but not in males. Potential implications of this trade-off for surviving cold conditions when food resources are limiting are discussed.     

Advances in Transgenic Research for Insect Resistance in Sugarcane

The first phase of transgenic research in sugarcane concentrated on the development and evaluation of transgenic lines transformed for resistance to biotic stresses, particularly diseases and insect pests. Sugarcane is attacked by a range of insects including tissue borers, sucking pests and canegrubs. Losses due to these pests are estimated to be around 10%. Although chemical control and integrated pest management are regularly practiced for the control of insect pests, success is often limited due to practical difficulties. The genetic complexity of sugarcane coupled with the non-availability of resistance genes in the germplasm has made conventional breeding for insect resistance difficult. In this context, transgenic technology has become a handy tool for imparting insect resistance to an elite variety which is otherwise superior for most other agronomic traits. A number of transgenic sugarcane lines have been developed with genes expressing Cry proteins, proteinase inhibitors or lectins resistant to borers, sucking insects or grubs. While commercializing transgenic lines, issues such as higher and stable transgene expression, preparedness for resistance management and non-target effects need to be addressed. To manage the constant threat of resistance development in target insects, it is imperative to deploy field-level strategies taking clues from other crops coupled with the search for new potent replacement molecules for transformation.     

High basal defense gene expression determines sorghum resistance to the whorl‐feeding insect southwestern corn borer

Southwestern corn borer (SWCB, Diatraea grandiosella) and fall armyworm (FAW, Spodoptera frugiperda) are major pests of sorghum in the southern United States. Host plant resistance is a desirable means for reducing plant damage and yield losses from both insects. In this study, we evaluated 12 sorghum lines for whorl‐stage resistance to leaf‐feeding SWCB and FAW in greenhouse and laboratory bioassays. Differential plant responses were detected against the two insects. Among 12 lines tested, CM1821, Della and PI196583 were resistant to both insects, while BTx2752 was largely susceptible. Line R.09110 was resistant to SWCB, but susceptible to FAW, whereas Redbine‐60 was susceptible to SWCB, but not to FAW. In addition, we quantified various chemical components in the plants and determined their association with insect resistance. Tannin and chlorophyll in leaves did not show any significant correlation with resistance to either insects, but contents of soluble protein in general were negatively correlated with resistance to both insects. Endogenous soluble sugar and dhurrin were only positively correlated with resistance to SWCB, but not with FAW resistance. To gain some molecular insight into resistance mechanism of sorghum to SWCB, we performed qPCR reactions for key genes encoding enzymes involved in dhurrin and jasmonic acid (JA) biosynthesis on selected resistant or susceptible lines. Although these genes were rapidly and strongly induced by insect feeding in all lines, the observed resistance is likely explained by higher constitutive dhurrin contents in some resistant lines and higher basal JA biosynthesis in others. Our results suggest that sorghum utilizes multiple strategies to defend itself against SWCB.     

Cytochromes P450 and insecticide resistance.

The cytochrome P450-dependent monooxygenases (monooxygenases) are an extremely important metabolic system involved in the catabolism and anabolism of xenobiotics and endogenous compounds. Monooxygenase-mediated metabolism is a common mechanism by which insects become resistant to insecticides as evidenced by the numerous insect species and insecticides affected. This review begins by presenting background information about P450s, the role of monooxygenases in insects, and the different techniques that have been used to isolate individual insect P450s. Next, insecticide resistance is briefly described, and then historical information about monooxygenase-mediated insecticide resistance is reviewed. For any case of monooxygenase-mediated resistance, identification of the P450(s) involved, out of the dozens that are present in an insect, has proven very challenging. Therefore, the next section of the review focuses on the minimal criteria for establishing that a P450 is involved in resistance. This is followed by a comprehensive examination of the literature concerning the individual P450s that have been isolated from insecticide resistant strains. In each case, the history of the strain and the evidence for monooxygenase-mediated resistance are reviewed. The isolation and characterization of the P450(s) from the strain are then described, and the evidence of whether or not the isolated P450(s) is involved in resistance is summarized. The remainder of the review summarizes our current knowledge of the molecular basis of monooxygenase-mediated resistance and the implications for the future. The importance of these studies for development of effective insecticide resistance management strategies is discussed.     

Intraspecific variation of thermal reaction norms for development in insects: New approaches and prospects

The main parameters (thermal constants) characterizing the effects of constant temperatures on the duration and rate of development in ectotherms are considered. The advantages and drawbacks of the linear and nonlinear models describing the dependence of the development rate on temperature are discussed. The main patterns of interspecific variation of thermal constants in insects are described. The development of new ideas in the study of intraspecific variation of thermal reaction norms for development in insects is examined. The discrepancies in the data and their interpretation in this field are shown to result mostly from using the traditional methods of determining thermal constants. According to this method, the development rates calculated as reciprocals of the mean development times at each experimental temperature are used for regression analysis. The standard errors calculated according to this method are usually too large. Instead, a fundamentally new method is employed by the authors, according to which the individual development rates calculated for each individual are used for regression analysis of the reciprocals of the mean development times. This method reduces the standard errors of thermal constants by about an order and reveals significant differences between populations and other groups of insects. New data on intraspecific variation of the thermal reaction norms for development in Myrmica ants, the linden bug Pyrrhocoris apterus, and Calliphora blowflies are briefly described and discussed. The possible adaptive significance of these forms of intraspecific variation is discussed. The prospects of research in this new field are outlined.     

Resistance to 16 diverse species of herbivorous insects within a population of goldenrod,Solidago altissima: genetic variation and heritability

Summary Genetic variation in resistance to 16 species of herbivorous insects was studied in 18 clones of Solidago altissima growing in an old field near Ithaca, New York, USA. Resistance to each insect, defined as the abundance of a species attacking a particular host genotype relative to other genotypes, was measured in both the natural stand and in two experimental gardens. The heritability of resistance was estimated by parent-offspring regression and sibcorrelation. The primary result was that clones differed in resistance to 15 of 16 insect species. The resistance of genotypes to these insect species remained relatively constant over the four years of the study. However, for only 10 of these resistances were the heritability estimates significantly different from zero. Thus the common assumption of plant-insect studies — that phenotypic variation in insect abundance is closely correlated with underlying genetic variation — is only conditionally true. There is heritable variation in resistance to many insects, but not all. The insects for which we observed heritable variation in plant resistance represent five different orders and several functional groups, including leaf chewers, phloem and xylem feeders, and gall formers. There was no apparent pattern between the degree of heritability of plant resistance and the destructiveness, feeding method, breadth of host range, or taxonomic group of the insects. The lack of marked heritable variation in resistance to some insects may be the result of (a) reduced variation caused by strong selection during prolonged or repeated insect outbreaks, and (b) genotype-environment interactions that obscure differences among genotypes.     

Are there effective resistance management strategies for vectors of human disease?*

The rotation of insecticides used by the Onchocerciasis Control Programme in West Africa is reviewed and the motives for this rotation are shown to be not only management of temephos resistance in the Simulium vectors but also constraints on what compounds are usable at particular seasons. A computer model indicates that without these seasonal constraints there is unlikely to be an advantage in a pre-planned rotation of insecticides, as compared with the prompt switching of compounds as dictated by detection of build up of resistance and switching back to the original compound if the regression of resistance is found to give the opportunity to do so. The latter sequence of events can hardly be called a ‘strategy’ for resistance management, but is what any well-managed pest control programme would be expected to do. The use of an insecticide mixture is different in principle from the use of rotation and depends on the idea that if the mixture is used from the outset, when resistance to both components of the mixture is likely to be rare, the double resistance combination would be so rare as to be dwarfed in numbers by those insects which avoid exposure altogether. The prospects for successful use of a mixture depend on each component killing a very high percentage of the exposed insects which are genetically susceptible to it. Whether this condition is met could be tested, for example, in the case of exposure of mosquitoes to insecticide-treated bednets.     

Resistance and cross-resistance to four insecticides in populations of obliquebanded leafroller (Lepidoptera: Tortricidae)

Populations of obliquebanded leafroller, Choristoneura rosaceana (Harris), were collected from organic and conventionally managed orchards located in the Okanagan and Similkameen Valleys of British Columbia Neonate F1 progeny were assayed for resistance to azinphosmethyl, tebufenozide, methoxyfenozide, and indoxacarb using a leaf disk bioassay. Significant differences in resistance levels among populations were observed for all four insecticides. Insects collected from organic sites were more susceptible to all insecticides than were insects collected from conventional sites. Resistance to the benzoylhydrazine insect growth regulators tebufenozide and methoxyfenozide was highly correlated with resistance to azinphosmethyl across populations, indicating cross-resistance between these compounds. The highest levels of resistance were observed with indoxacarb, but resistance levels to indoxacarb did not correlate with those for azinphosmethyl. Dose-response regression lines for tebufenozide were parallel across populations, suggesting that the resistance mechanism(s) were quantitatively, but not qualitatively, different. Cross-resistance between azinphosmethyl and benzoylhydrazine insecticides indicates that a resistance management strategy for obliquebanded leafroller involving the rotation of these materials is not likely to be successful.     

THE EFFECT OF REPEATED SPRAYING OF INSECTS IN INCREASING THEIR RESISTANCE TO INSECTICIDES: I. DEVELOPMENT OF RESISTANCE TO DDT IN A STRAIN OF DROSOPHILA MELANOGASTER MEIG.

Successive spraying with DDT suspensions of the adults of a wild colony of Drosophila melanogaster and the progeny of survivors enhanced the resistance to that insecticide.
The rate at which resistance increased depended on: (1) the relative proportion of resistants to susceptible individuals, or (2) the intensity of selection as measured by the concentration of DDT, the proportion killed or on both. The resistance of the populations of the insects fluctuated considerably whether subjected to successive sprayings or not, and in one sprayed series there was some indication of a rhythm with peaks of susceptibility occurring at regular intervals.
Enhanced resistance may show a change of slope in the probit log concentration regression line, leading to different relative values at different levels of mortality, or by a parallel shift of the regression line. The former appears to be a preliminary stage of selection and indicates a change in the frequency distribution within a population.
Increasing the concentration of DDT, slowly or rapidly, may have enhanced resistances at an increased rate, but the series sprayed with the lower initial concentration reached finally the same end point, as judged by the values of log lc . 50.
During the course of these experiments the insects developed sensitivity to carbon dioxide (used in anaesthesis). Its bearing on our work is considered in Part II.     

Trade-off associated with selection for increased ability to resist parasitoid attack in Drosophila melanogaster.

Costs of resistance are widely assumed to be important in the evolution of parasite and pathogen defence in animals, but they have been demonstrated experimentally on very few occasions. Endoparasitoids are insects whose larvae develop inside the bodies of other insects where they defend themselves from attack by their hosts'' immune systems (especially cellular encapsulation). Working with Drosophila melanogaster and its endoparasitoid Leptopilina boulardi, we selected for increased resistance in four replicate populations of flies. The percentage of flies surviving attack increased from about 0.5% to between 40% and 50% in five generations, revealing substantial additive genetic variation in resistance in the field population from which our culture was established. In comparison with four control lines, flies from selected lines suffered from lower larval survival under conditions of moderate to severe intraspecific competition.     

Glasshouse tests and field selection for heritable resistance to Myzus persicae in sugar beet

A technique is described to assess and select in the field for resistance to Myzus persicae in sugar beet. Lines developed by this method were aphid resistant when compared with commercial cultivars. The resistance was inherited by progeny lines. Variation between beet lines in glasshouse tests suggested segregation of a number of resistance genes with varying effects. Integration of selection for aphid-resistance and agronomic performance during beet breeding is discussed.     

The rph2 gene is responsible for high level resistance to phosphine in independent field strains of Rhyzopertha dominica

The lesser grain borer Rhyzopertha dominica (F.) is one of the most destructive insect pests of stored grain. This pest has been controlled successfully by fumigation with phosphine for the last several decades, though strong resistance to phosphine in many countries has raised concern about the long term usefulness of this control method. Previous genetic analysis of strongly resistant (SR) R. dominica from three widely geographically dispersed regions of Australia, Queensland (SR(QLD)), New South Wales (SR(NSW)) and South Australia (SR(SA)), revealed a resistance allele in the rph1 gene in all three strains. The present study confirms that the rph1 gene contributes to resistance in a fourth strongly resistant strain, SR2(QLD), also from Queensland. The previously described rph2 gene, which interacts synergistically with rph1 gene, confers strong resistance on SR(QLD) and SR(NSW). We now provide strong circumstantial evidence that weak alleles of rph2, together with rph1, contribute to the strong resistance phenotypes of SR(SA) and SR2(QLD). To test the notion that rph1 and rph2 are solely responsible for the strong resistance phenotype of all resistant R. dominica, we created a strain derived by hybridising the four strongly resistant lines. Following repeated selection for survival at extreme rates of phosphine exposure, we found only slightly enhanced resistance. This suggests that a single sequence of genetic changes was responsible for the development of resistance in these insects.     

Ecdysteroid signaling in ecdysteroid-resistant cell lines from the polyphagous noctuid pest Spodoptera exigua

Although dibenzoylhydrazine-type non-steroidal ecdysone agonists such as methoxyfenozide (RH-2485) have an excellent performance record, the emergence of resistance could severely compromise the efficacy of these compounds in integrated pest management programs. To investigate possible mechanisms of resistance, cell lines derived from the polyphagous noctuid pest Spodoptera exigua (Se4 cells) were selected for continuous growth in the presence of high concentrations of 20-hydroxyecdysone (20E) or methoxyfenozide. Here we describe an analysis of ecdysteroid receptor signaling in the ecdysteroid-resistant Se4 cell lines. In contrast to other ecdysteroid-resistant cell lines described in literature, our data support the existence of a normal functioning ecdysteroid receptor complex in the resistant Se4 cell lines: (1) using a recombinant BmNPV baculovirus as a transduction tool, activation of an ecdysone-responsive luciferase cassette was demonstrated; (2) the early gene HR3 is constitutively expressed in the resistant cell lines that are grown in the presence of 20E or methoxyfenozide. Quantitative RT-PCR experiments indicated that expression levels of SeEcR mRNA were comparable among sensitive and resistant cell lines. Sequencing of PCR fragments also revealed the presence of SeEcR mRNA with a wild-type ligand-binding domain in resistant cells. Finally, a possible role for the gene FTZ-F1, whose expression correlates with the absence of circulating ecdysteroids during insect development, in the resistance mechanism was investigated. However, it was observed that FTZ-F1, in contrast to what is observed during insect development, is constitutively expressed in Se4 cells and that its expression is not regulated by the addition of ecdysteroid. It is proposed that the resistance mechanism in Se4 cells resides at the coupling between the conserved hierarchical cascade of early and early-late gene expression and the differentiation program in the Se4 cell line. The use of insect cell lines for the investigation of resistance against dibenzoylhydrazine ecdysone agonists and their relevance for uncovering resistance mechanisms in insects during pest control programs is discussed.     

Direct and correlated responses to artificial selection for high and low knockdown resistance to high temperature in Drosophila buzzatii

Knockdown resistance to high temperature (KRHT) is a genetically variable trait for thermal adaptation in insects. Selection for KRHT may affect a number of fitness components as well as resistance to several forms of environmental stress. To test for heritable (co)-variation in KRHT, we examined direct and correlated responses to bi-directional selection on this trait in Drosophila buzzatii. Replicated lines were artificially selected for decreased and increased KRHT. After 12 generations of artificial selection, lines diverged significantly for high KRHT only. Starvation resistance increased in two lines that strongly responded to selection for high KRHT, and these two lines also showed relatively longer chill-coma recovery time. Developmental time and body size showed no correlated responses to KRHT-selection. These results suggest that KRHT is a heritable trait that can evolve towards increased thermotolerance with no genetic trade-offs associated to starvation resistance, developmental time and body size.     

Immune function responds to selection for cuticular colour in Tenebrio molitor

Cuticular colour in the mealworm beetle (Tenebrio molitor) is a quantitative trait, varying from tan to black. Population level variation in cuticular colour has been linked to pathogen resistance in this species and in several other insects: darker individuals are more resistant to pathogens. Given that cuticular colour has a heritable component, we have taken an experimental evolution approach: we selected 10 lines for black and 10 lines for tan adult cuticular phenotypes over at least six generations and measured the correlated responses to selection in a range of immune effector systems. Our results show that two immune parameters related to resistance (haemocyte density and pre-immune challenge activity of phenoloxidase (PO)) were significantly higher in selection lines of black beetles compared to tan lines. This may help to explain increased resistance to pathogens in darker individuals. Cuticular colour is dependent upon melanin production, which requires the enzyme PO that is present in its inactive form inside haemocytes. Thus, the observed correlated response to selection upon cuticular colour and immune variables probably results from these traits' shared dependence on melanin production.     

Pinellia ternata agglutinin expression in chloroplasts confers broad spectrum resistance against aphid, whitefly, Lepidopteran insects, bacterial and viral pathogens

Broad spectrum protection against different insects and pathogens requires multigene engineering. However, such broad spectrum protection against biotic stress is provided by a single protein in some medicinal plants. Therefore, tobacco chloroplasts were transformed with the agglutinin gene from Pinellia ternata (pta), a widely cultivated Chinese medicinal herb. Pinellia ternata agglutinin (PTA) was expressed up to 9.2% of total soluble protein in mature leaves. Purified PTA showed similar hemagglutination activity as snowdrop lectin. Artificial diet with purified PTA from transplastomic plants showed marked and broad insecticidal activity. In planta bioassays conducted with T0 or T1 generation PTA lines showed that the growth of aphid Myzus persicae (Sulzer) was reduced by 89%-92% when compared with untransformed (UT) plants. Similarly, the larval survival and total population of whitefly (Bemisia tabaci) on transplastomic lines were reduced by 91%-93% when compared with UT plants. This is indeed the first report of lectin controlling whitefly infestation. When transplastomic PTA leaves were fed to corn earworm (Helicoverpa zea), tobacco budworm (Heliothis virescens) or the beet armyworm (spodoptera exigua), 100% mortality was observed against all these three insects. In planta bioassays revealed Erwinia population to be 10,000-fold higher in control than in PTA lines. Similar results were observed with tobacco mosaic virus (TMV) challenge. Therefore, broad spectrum resistance to homopteran (sap-sucking), Lepidopteran insects as well as anti-bacterial or anti-viral activity observed in PTA lines provides a new option to engineer protection against biotic stress by hyper-expression of an unique protein that is naturally present in a medicinal plant.     

Induced Resistance to 6-Methylpurine and Cycloheximide in Tetrahymena. I. Germ Line Mutants of T. THERMOPHILA

Two new mutant lines of Tetrahymena thermophila ( T. pyriformis, syngen 1), each conferring resistance to a different agent, are described. Resistance to cycloheximide and 6-methylpurine are each determined by dominant genes, ChxA2 and Mpr; the traits show phenotypic assortment. The method used to select these mutations, the critical importance of backcrossing to wild type following mutagenesis, and the utility of these marker genes in further mutagenic selection schemes and studies of the sexual cycle of Tetrahymena are noted.     

The analysis of a factorial series of insecticide tests

When a set of insecticidal toxicity tests yields parallel regression lines for the relationship between mortality probit and log dose, the potencies of the materials or conditions under test may be compared purely in terms of log L.D. 50's. The purpose of this paper is to suggest that, when tests have been made with all combinations of several different factors, standard methods for the statistical analysis of factorial experiments may be adapted to the examination of the relative potencies.
Data obtained by Potter & Gillham (1046), in a 2' factorial experiment on alternative storage conditions for insects before and after spraying and the adjuvant action of terpineol in a pyrethrins spray, are used in an example of the computations. Details are given of the test of parallelism of the regression lines, the factorial analysis of the log L.D. '50s, the estimation of the mean effects and interactions and their standard errors, the significance tests, and the preparation of summary tables.     

Genetic analysis of the Drosophila ellipsoid body neuropil: organization and development of the central complex.

The central complex is an important center for higher-order brain function in insects. It is an intricate neuropil composed of four substructures. Each substructure contains repeated neuronal elements which are connected by processes such that topography is maintained. Although the neuronal architecture has been described in several insects and the behavioral role investigated in various experiments, the exact function of this neuropil has proven elusive. To describe the architecture of the central complex, we study 15 enhancer-trap lines that label various ellipsoid body neuron types. We find evidence for restriction of gene expression that is correlated with specific neuronal types: such correlations suggest functional classifications as well. We show that some enhancer-trap patterns reveal a single ellipsoid body neuron type, while others label multiple types. We describe the development of the ellipsoid body neuropil in wild-type animals and propose developmental mechanisms based on animals displaying structural mutations of this neuropil. The experiments performed here demonstrate the degree of resolution possible from the analysis of enhancer-trap lines and form a useful library of tools for future structure/function studies of the ellipsoid body.