Antixenosis and larval and adult dispersal in the Mediterranean corn borer,Sesamia nonagrioides,in relation to Bt maize

Sesamia nonagrioides Lefèbvre (Lepidoptera: Noctuidae) is a key pest of maize [Zea mays L. (Poaceae)] and a main target of Bt maize in the Mediterranean area. To choose the most suitable non‐Bt refuge strategy for preventing or delaying resistance development in this maize borer, we examined its biology and behaviour. No antixenotic effects were found on numbers of eggs and egg batches per plant in choice (Bt vs. non‐Bt plants) and no‐choice assays. However, a greater ratio of young larvae dispersed from Bt than from non‐Bt plants. In addition, larvae that hatched on Bt plants tended to disperse more than those that hatched on non‐Bt plants, particularly during young growth stages. Many adults, especially females, could fly at least up to 400 m, as was found in a dispersal study with rubidium‐marked adults. The stimulation of larval dispersal by the Bt trait and the dispersal capacity of adults might compromise the efficacy of seed mixtures as an insecticide resistance management strategy.     

Dispersal capacity in the Mediterranean corn borer, Sesamia nonagrioides

Corn (Zea mays L.) borers are the primary target of Bacillus thuringiensis Berliner (Bt) transgenic maize. Management of corn borer resistance to Bt requires information on larval and adult dispersal capacities, a feature that is particularly unknown in Sesamia nonagrioides Lefèbvre (Lepidoptera: Noctuidae), the most damaging corn borer in Spain. Larval dispersal was studied over a 3 year period by infesting plants with egg masses and dissecting the neighbouring plants 7, 14, and 32 days later to measure larval dispersal at several ages. The number and age of larvae were recorded in the dissected plants. Only mature larvae dispersed in significant numbers; they moved at least to rows adjacent to those containing the infested plant, and down the row five plants. The percentage of larvae that dispersed from the infested plant was density‐dependent. Adult dispersal was studied with directional light and pheromone uni‐traps over 5 and 3 year periods, respectively. Directional light traps were placed in the margins between Bt and non‐Bt maize fields, half oriented towards each of the two kinds of maize field. Pheromone traps were placed in the Bt and non‐Bt fields at increasing distances (0–100 m) from the border. The numbers of males and females caught in directional light traps were not different in traps oriented towards Bt or non‐Bt fields, but the number of males caught in the third flight in Bt fields was lower than in non‐Bt fields. These results suggest that males from adjacent Bt and non‐Bt fields mate indiscriminately with females emerging in any of the two kinds of maize fields. However, male movement in the third flight may not be sufficient to randomly distribute males between the two fields.     

Nosema pyrausta and Cry1Ab‐incorporated diet led to decreased survival and developmental delays in European corn borer

The high dose/refuge strategy for delaying evolution of resistance to Bt maize [Zea mays L. (Poaceae)] relies on random mating between resistant European corn borers, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), and susceptible O. nubilalis from the refuge. However, differences in developmental rate caused by feeding on Bt maize, or infection with the microsporidium Nosema pyrausta Paillot (Microsporida: Nosematidae) may result in assortative mating. Developmental delays and mortality caused by infection with N. pyrausta and feeding on Bt maize were quantified alone and in combination in Cry1Ab‐resistant and susceptible O. nubilalis. Feeding on Cry1Ab‐incorporated diet significantly increased number of days from hatch to pupation and decreased survival in the resistant population. Infection with N. pyrausta increased mortality and lengthened development in both the resistant and susceptible populations. The combination of Cry1Ab‐incorporated diet and infection with N. pyrausta in resistant O. nubilalis lengthened development and increased mortality to a greater extent than either factor alone. Greater larval delays of resistant O. nubilalis feeding on Bt maize could lead to temporal isolation from adults emerging from refuge maize. The resulting assortative mating would hasten the evolution of resistance. Developmental delays caused by infection with N. pyrausta may increase the likelihood of mating between resistant and infected susceptible adults emerging from refuge maize, producing infected offspring that are also more susceptible to Bt maize.     

Regulation of pheromone biosynthesis in the "Z strain" of the European corn borer, Ostrinia nubilalis

The regulation of pheromone biosynthesis by the neuropeptide PBAN in the Z strain of the European corn borer, Ostrinia nubilalis, was investigated using labeled intermediates. Injection of radiolabeled acetate showed PBAN did not influence the de novo synthesis of saturated fatty acids in the gland. When deuterium-labeled myristic acid was topically applied to the gland, females injected with PBAN produced more labeled pheromone than did control females, indicating that PBAN controls one of the later steps of pheromone biosynthesis. Although more myristic acid was Delta11-desaturated in the gland in the presence of PBAN, this was counterbalanced by less Delta11-desaturation of palmitic acid, indicating that desaturase activity did not change overall. This change in flux of myristic acid through to pheromone was shown to be caused by increased reduction of fatty acid pheromone precursors occurring in the presence of PBAN.     

Resistance monitoring of field populations of the corn borers Sesamia nonagrioides and Ostrinia nubilalis after 5 years of Bt maize cultivation in Spain

Approximately 22 000 hectares (5% of the total maize growing area) of transgenic maize expressing the Cry1Ab toxin from Bacillus thuringiensis (Bt maize) have been planted annually in Spain since 1998. Changes in the susceptibility to Cry1Ab of Spanish populations of the Mediterranean corn borer (MCB), Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae), and the European corn borer (ECB), Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), were assessed by annual monitoring on Bt maize fields. No increase in resistance was detected in the MCB populations from Ebro, Albacete, and Badajoz, nor in the ECB populations from Ebro and Badajoz during the period 1999–2002. The susceptibility of the MCB population from Madrid fluctuated from year to year, but a gradual trend towards higher levels of tolerance was not observed. Laboratory selection assays for eight generations yielded selected strains of MCB and ECB that were 21‐ and 10‐fold significantly more tolerant to Cry1Ab than the corresponding unselected strains, respectively. Nevertheless, none of the field‐collected or laboratory‐selected larvae were able to survive on Bt maize. Considering these data, no consistent shifts in susceptibility were found for Spanish populations of MCB nor ECB after 5 years of Bt maize cultivation, but systematic field monitoring needs to be continued.     

Electroantennogram, wind tunnel and field responses of male Mediterranean corn borer, Sesamia nonagrioides, to several blends of its sex pheromone components

The electrophysiological and behavioural responses of male Sesamia nonagrioides Lefèbvre (Lepidoptera: Noctuidae) to several synthetic blends of its sex pheromone components were investigated. Electroantennogram (EAG) recordings showed the greatest response to a 77:8:10:5 blend of the four compounds found in the female glands: (Z)-11-hexadecen-1-yl acetate, (Z)-11-hexadecen-1-ol, (Z)-11-hexadecenal and dodecyl acetate. A logistic equation was fitted to the dose-response data and differences in relative EAG responses were analysed by deriving five parameters of this equation. Flight responses of males were also evaluated in a wind tunnel where the proposed blend (77:8:10:5) also elicited the best response. Field tests confirmed the laboratory findings as the 77:8:10:5 blend captured more S. nonagrioides males than other pheromone mixtures and showed more selectivity for male S. nonagrioides over other noctuid species, Discestra trifolii Rottenberg and Lacanobia oleracea L., but not for Mythimna unipuncta Haworth, a species with a sex pheromone composition almost identical to that of S. nonagrioides.     

Genetic structure of European and Mediterranean maize borer populations on several wild and cultivated host plants

Target pests may become resistant to Bacillus thuringiensis (Bt) toxins produced by trangenic maize (Zea mays L.). Untreated refuge areas are set aside to conserve high frequencies of susceptibility alleles: a delay in resistance evolution is expected if susceptible individuals from refuges mate randomly with resistant individuals from Bt fields. In principle, refuges can be toxin‐free maize or any other plant, provided it hosts sufficiently large pest populations mating randomly with populations from Bt‐maize fields. Our aim was to examine the suitability of several cultivated or weedy plants [pepper (Capsicum frutescens L.), sorghum (Sorghum spec.), sunflower (Helianthus annuus L.), cocklebur (Xanthium spec.), cantaloupe (Cucumis melo L.), and hop (Humulus lupulus L.)] as refuges for Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae), two major maize pests in southern Europe. Larvae of both species were collected on these plants. Their genetic population structure was examined at several allozyme loci. We found little or no evidence for an influence of geographic distance, but detected a significant host‐plant effect on the genetic differentiation for both species. Ostrinia nubilalis populations from sunflower, pepper, cocklebur, and sorghum appear to belong to the same genetic entity as populations collected on maize, but to differ from populations on hop. Accordingly, females from pepper and cocklebur produced exclusively the ‘Z’ type sexual pheromone, which, in France, characterizes populations developing on maize. Qualitatively, these plants (except hop) could thus serve as refuges for O. nubilalis; however, they may be of little use quantitatively as they were found much less infested than maize. Sesamia nonagrioides populations on maize and sorghum reached comparable densities, but a slight genetic differentiation was detected between both. The degree of assortative mating between populations feeding on both hosts must therefore be assessed before sorghum can be considered as a suitable refuge for this species.     

Distribution of chemo- and mechanoreceptors on the antennae and maxillae of Busseola fusca larvae

The stem borer Busseola fusca (Fuller) (Lepidoptera: Noctuidae) is a major pest of maize, Zea mays L., and sorghum, Sorghum bicolor (L.) Moench (both Poaceae), in sub-Saharan Africa. Like in many other lepidopteran insects, the success of B. fusca in recognizing and colonizing a limited variety of plants is based on the interaction between its sensory systems and the physicochemical characteristics of its immediate environment. The sensilla on the maxillary galeae of B. fusca larvae are typical of Lepidoptera and comprise two uniporous styloconic sensilla, which are contact chemoreceptors, three basiconic sensilla, and two aporous sensilla chaetica. The maxillary palp is two-segmented and has eight small basiconic sensilla at the tip, which were also found to be gustatory. The antennae of B. fusca larvae are short and simple. The sensilla of the antenna are composed of two aporous sensilla chaetica, three multiporous cone-shaped basiconic sensilla, three small basiconic sensilla, and one aporous styloconic sensillum. The basiconic sensillum located on the third antennal segment displayed a contact chemoreception response. The other basiconic sensilla did not show any action potential activity in tip-recording tests. The significant and positive dose–response curve obtained for the antennal basiconic sensillum with sucrose indicated for the first time the presence of gustatory chemoreceptors on the antennae of a lepidopteran larva.     

Sex pheromone blend discrimination in two races and hybrids of the European corn borer moth, Ostrinia nubilalis

Abstract. The response specificity of males of two races, and hybrids, of the European corn borer moth, to a large series of doses/ratios of E- and Z11-14:OAc, was determined in a sustained-flight tunnel. For both races an area of peak response was determined, which included the natural ratio eliciting peak levels of source contact, and other treatments eliciting response profiles over the behavioural sequence not significantly different from the peak. Consistent with studies on other moth species response specificity was controlled by two threshold effects, one affecting locking-on to odour plumes of lower doses and off-ratios containing lower proportions of the E or Z isomer than the natural blend, and the other resulting in arrestment of upwind flight to higher doses and off-ratios containing higher proportions of the E or Z isomer man the natural blend. A comparison of the size of the areas of peak response showed that males of the univoltine Z race (UZ) using a 3:97 E:Z mix displayed greater specificity and sensitivity than did males of the bivoltine E race (BE) responding to a 99:1 E:Z pheromone mix. At doses higher than those eliciting peak response (<100 μg) response specificity was lower for both races, but especially for the BE race, with increased levels of upwind flight and source contact occurring to off-ratios. Finally, consistent with a previous behaviour/genetic study on this species, F₁ hybrid males displayed peak levels of source contact not only to their natural isomer ratio (65:35 E:Z), but also to an expanded range of doses of ratios ranging from 2% to 98% E.     

Oviposition deterrents from the egg masses of the adzuki bean borer, Ostrinia scapulalis and Asian corn borer, O. furnacalis

Bioassays using gravid females of the adzuki bean borer, Ostrinia scapulalis (Walker), and the Asian corn borer, O. furnacalis (Guenée) (Lepidoptera: Crambidae), showed that the presence of an egg mass of a conspecific deters oviposition. Volatile chemicals emanating from the egg mass were responsible for the deterrence, and these deterrents could be extracted from the egg mass with hexane. When fractionated using a Sep‐Pak® Plus NH₂ cartridge, the deterrents were eluted with a 98 : 2 mixture of diethyl ether and acetic acid (polar lipid fraction). The polar lipid fraction contained free fatty acids with 14–20 carbons, and palmitic acid, palmitoleic acid, and oleic acid were predominant. A blend of all identified fatty acids, a blend of six major fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic, and linolenic acids), a blend of the two Z‐9‐alkenoic acids (palmitoleic and oleic acids), palmitoleic acid alone, and oleic acid alone showed deterrence against O. scapulalis which was comparable to that provoked by the full egg extract. The dose‐dependency of the deterrent effects of palmitoleic acid and oleic acid was verified in O. scapulalis. The binary blend of palmitoleic acid and oleic acid was also confirmed to deter oviposition in O. furnacalis.     

Frequency of alleles conferring resistance to Bacillus thuringiensis maize in Louisiana populations of the southwestern corn borer

Transgenic maize [Zea mays L. (Poaceae)] expressing Bacillus thuringiensis proteins (Bt maize) has become the most important tool for managing stalk borers in maize in the USA. The current strategy for delaying the evolution of resistance in target insects for Bt maize is referred to as high dose/refuge strategy. A key requirement of the strategy is that initial resistance allele frequencies in field insect populations are low (e.g., <0.001). More than 200 iso‐line families of the southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera: Crambidae), a major target stalk borer pest of Bt maize, were developed from Louisiana populations and evaluated for Bt resistance using a modified F₂ screening method during 2005. No major resistance alleles were detected in these populations. The results showed that the expected Bt resistance allele frequency in the Louisiana populations was <0.0035 with 95% probability and a detection power of 83.9 ± 0.6%. The F₂ screen indicates that Bt resistance allele frequencies in D. grandiosella are low among the Louisiana populations and should meet the rare resistance allele requirement of the ‘high dose/refuge’ strategy.     

Dynamics of Nosema pyrausta in natural populations of the European corn borer, Ostrinia nubilalis: A six-year study

Nosema pyrausta (Paillot) (Microsporida: Nosematidae) is an obligatory intracellular parasite of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae). This pathogen is maintained in natural populations of O. nubilalis by both horizontal and vertical transmission. The impact of N. pyrausta on fecundity of adults and survival of larvae has been well documented in laboratory and field research. In an extensive study covering a 6 year period at one site, we described the effect of N. pyrausta within O. nubilalis populations in a continuous corn following corn ecosystem. We documented the presence of the pathogen through all life stages of O. nubilalis (egg, larva, pupa, adult), by collecting throughout the crop season and examining each insect stage in the laboratory for the frequency of infection with N. pyrausta. The percentage of infection with N. pyrausta and magnitude of the O. nubilalis population fluctuated throughout generation 1 and generation 2. Both horizontal and vertical transmission played a role in maintaining N. pyrausta in the population in both generations. There were strong correlations between percentage adults with N. pyrausta and percentage larvae with N. pyrausta, and between percentage eggs with N. pyrausta and percentage larvae with N. pyrausta. There was a weak correlation between percentage adults with N. pyrausta and percentage eggs with N. pyrausta. The percentage of insects infected with N. pyrausta was always lowest in the egg.     

Selection for Cry1F resistance in the European corn borer and cross-resistance to other Cry toxins

Evolution of resistance by insect pests is the greatest threat to the continued success of Bacillus thuringiensis (Bt) toxins used in insecticide formulations or expressed by transgenic crop plants such as Cry1F‐expressing maize [(Zea mays L.) (Poaceae)]. A strain of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), obtained from field collections throughout the central US Corn Belt in 1996 was selected in the laboratory for resistance to Cry1F by exposure to the toxin incorporated into artificial diet. The selected strain developed more than 3000‐fold resistance to Cry1F after 35 generations of selection and readily consumed Cry1F expressing maize tissue; yet, it was as susceptible to Cry1Ab and Cry9C as the unselected control strain. Only a low level of cross‐resistance (seven‐fold) to Cry1Ac was observed. These lacks of cross‐resistance between Cry1F and Cry1Ab suggest that maize hybrids expressing these two toxins are likely to be compatible for resistance management of O. nubilalis.     

Changes in calling behaviour and mating success in the European corn borer (Ostrinia nubilalis), caused by relative humidity

We examined the calling behaviour of O. nubilalis virgin females, held at three different constant conditions of relative humidity from the time of emergence through their sixth night of calling, as well as the incidence of mating over time at low or high relative humidity conditions. The mean age at which females initiated calling in their life was independent of relative humidity conditions, although more variable at low humidity. On successive nights of calling, fewer virgin European corn borer females called under low compared with high humidity conditions, and those that did began later in scotophase, had fewer calling bouts, and spent less time calling. These changes in the female prereproductive behaviour were reflected on the incidence of mating which, in the first two days following emergence, was significantly less at low than at high humidity conditions. The ecological significance of these results is discussed in relation with the susceptibility of O. nubilalis to desiccation.

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Résumé Nous avons étudié le comportement d'appel des femelles vierges d'Ostrinia nubilalis maintenues sous trois conditions constantes d'humidité relative, depuis l'émergence jusqu'à la sixième nuit d'appel, ainsi que l'incidence d'accouplement d'individus placés en milieu sec ou humide pour diverses périodes de temps. Les conditions d'humidité n'ont pas significativement affecté l'âge moyen auquel les femelles ont commencé à appeler au cours de leur vie, bien qu'il ait été plus variable à 53% h.r. Les nuits subséquentes, le comportement d'appel des femelles maintenues à basse humidité a débuté plus tard dans la nuit, s'est exprimé pour une période de temps plus courte avec moins de séquences d'appel que celui de leurs congénères placés en milieu humide, et la proportion de femelles en appel a toujours été plus faible à 53% h.r. Ces changements du comportement préreproducteur des femelles se sont répercutés sur l'incidence d'accouplement qui, les deux premiers jours suivant lémergence, a été significativement plus faible à basse humidité qu'elle ne l'a été en milieu humide. Ces résultats seront discutés en relation avec l'écologie de O. nubilalis et associés à sa susceptibilité à la dessication.

     

Two multiplex sets of eight and five microsatellite markers for the European corn borer,Ostrinia nubilalis Hübner (Lepidoptera: Crambidae)

Primer sequence and polymorphism data are presented for 13 microsatellite loci isolated from the European corn borer moth, Ostrinia nubilalis, as part of a project to construct a linkage map for the two pheromone strains. Experimental conditions are described for polymerase chain reaction (PCR) multiplexing, which allows genotyping in two electrophoresis runs of eight and five markers each. In a sample of 27 individuals coming from one European locality, the number of alleles per locus ranged from one to 12, and gene diversity from 0 to 0.859. Seven loci showed a deficit of heterozygotes. Eleven loci cross‐amplify in the related Ostrinia furnacalis.     

Proteolytic processing of native Cry1Ab toxin by midgut extracts and purified trypsins from the Mediterranean corn borer Sesamia nonagrioides

The proteolytic processing of native Cry1Ab toxin by midgut extracts from the Mediterranean corn borer, Sesamia nonagrioides, takes place in successive steps. Several cuts occur until a 74 kDa protein is obtained; this is further digested to give rise to an active form of 69 kDa, which can be again processed to fragments of 67, 66 and 43 kDa. We have shown that three different trypsins (TI, TIIA and TIII) purified from the S. nonagrioides midgut were able to digest Cry1Ab protoxin to obtain the active form of 69 kDa. Interestingly, TI and TIII further hydrolyzed the 69 kDa protein to a fragment of slightly lower molecular mass (67 kDa), while TIIA was able to continue digestion to give fragments of 46 and 43 kDa. These results contrast with those obtained using bovine trypsin, in which the main product of Cry1Ab digestion is a 69 kDa protein. The digestion of the toxin with a "non-trypsin" fraction from S. nonagrioides midgut lumen, mostly containing chymotrypsins and elastases and free of trypsin-like activity, resulted in a different processing pattern, yielding fragments of 79, 77, 71, 69 and 51 kDa. Our results indicate that trypsins and other proteases are involved in the first steps of protoxin processing, but trypsins play the most important role in obtaining the 74 and 69 kDa proteins. All the digestion products, including the proteins of 46 and 43 kDa obtained from the digestion of Cry1Ab by TIIA, were toxic to neonate larvae, indicating that none of the tested proteases contribute to toxin degradation in a significant manner.     

Behavioural and electrophysiological responses of the European corn borer Ostrinia nubilalis to host‐plant volatiles and related chemicals

The European corn borer Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) is a worldwide pest of maize (Zea mays L.) and other crops. The semiochemicals released by maize plants and structurally‐related compounds can be used by adult female O. nubilalis for host‐plant location and oviposition. Headspace volatile compounds emitted by watered and water‐deprived maize plants are collected and identified by their retention indices and mass spectra. The most abundant compounds from watered plants are limonene, linalool, benzoic acid, indole, β‐caryophyllene and acetophenone, whereas, in water‐deprived plants, limonene, acetophenone, hexanoic acid, benzoic acid and indole are dominant. In addition, (E)‐4,8‐dimethyl‐1,3,7‐nonatriene, 6‐methyl‐5‐hepten‐2‐one, anisole and 1‐carvone are undetected in the water‐deprived plants. Some of the identified compounds show electrophysiological activity (electroantennogram) in the antennae of both sexes, with the responses elicited by tridecane, tetradecane, dodecane, nonanal, decanal and 2‐ethylhexanol on males being particularly noteworthy. In a dual‐choice olfactometer, adult females show a preference for 2‐hexanol, heptanal, methyl salicylate, hexyl acetate, nonanal, methyl dodecanoate, β‐pinene and (E)‐2‐hexenyl acetate over hexane controls. Tetradecane, linalool, methyl hexanoate, methyl nonanoate, (Z)‐3‐hexenyl benzoate, tridecane, 2‐cyclopentylcyclopentanone, 3‐methylbutyl acetate, β‐myrcene and (Z)‐3‐hexenyl butanoate result in fewer females in the test arm compared with the control arm. No single compound displays an activity similar to watered maize plants, supporting the hypothesis that blends of volatiles in specific ratios are more effective than single volatile chemicals. The results of the present study suggest that methyl salicylate, which elicits also one of the highest electrophysiological responses in female antennae, plays a role in host preference by O. nubilalis females.     

Intercrop effects on the distribution of populations of the European corn borer, Ostrinia nubilalis, in maize

Field investigations conducted from 1982 to 1984 to assess intercrop effects on the distribution of Ostrinia nubilalis (Hübner), Lepidoptera, Pyralidae, in maize revealed that the incidence of damage by O. nubilalis was influenced significantly by the proximity of maize to potato. Maize fields adjacent to potato fields had a lower incidence of early season damage by O. nubilalis and a higher incidence of late-season damage than maize isolated from potato.     

Gene genealogies reveal differentiation at sex pheromone olfactory receptor loci in pheromone strains of the European corn borer, Ostrinia nubilalis

Males of the E and Z strains of the European corn borer Ostrinia nubilalis (Lepidoptera: Crambidae) are attracted to different blends of the same pheromone components. The difference in male behavioral response is controlled by the sex-linked locus Resp. The two types of males have identical neuroanatomy but their physiological specificity is reversed, suggesting that variation at the periphery results in behavioral change. Differences in the olfactory receptors (ORs) could explain the strain-specific antennal response and blend preference. Gene genealogies can provide insights into the processes involved in speciation and allow delineation of genome regions that contribute to reproductive barriers. We used intronic DNA sequences from five OR-encoding genes to investigate whether they exhibit fixed differences between strains and therefore might contribute to reproductive isolation. Although two genealogies revealed shared polymorphism, molecular polymorphism at three genes revealed nearly fixed differences between strains. These three OR genes map to the sex chromosome, but our data indicate that the distance between Resp and the ORs is >20 cM, making it unlikely that variation in pheromone-sensitive OR genes is directly responsible for the difference in behavioral response. However, differences in male antennal response may have their origin in the selection of strain-specific alleles.