Baseline susceptibility of Asian corn borer (Ostrinia furnacalis (Guenée)) populations in Vietnam to Cry1Ab insecticidal protein

Susceptibility of Asian corn borer (ACB), Ostrinia furnacalis (Guenée) to Bacillus thuringiensis (Bt) Cry1Ab protein was studied between 2015 and 2016 with 11 ACB populations, collected from various geographical regions in Vietnam. A concentration range of Cry1Ab from 0.20 to 26.10 ng/cm² of diet was evaluated against F₁ ACB neonates using diet surface-overlay bioassays. Mortality data was recorded daily until seven days after infestation. Growth inhibition was recorded at the end of seven days. The median lethal concentration (LC₅₀) varied ≈3-fold among the different populations, ranging from 0.58 to 1.83 ng/cm² of diet with an overall mean of 0.86 ng/cm² of diet. Even the lowest concentration of 0.20 ng/cm² caused 73.53% growth inhibition. >90% growth inhibition was achieved at 0.82 ng/cm² or higher concentrations. The results reflect natural variation in Bt susceptibility among ACB populations rather than variation caused by prior exposure to selection pressures. LC₉₉ value (17.26 ng/cm²) was generated by pooling mortality data across different populations. The upper fiducial limit of LC₉₉ (24.38 ng/cm²) could be a potential diagnostic dose for future resistance monitoring programs. The findings from this study suggest that ACB populations in Vietnam are highly susceptible to Cry1Ab protein. This is the first report of Cry1Ab susceptibility of different ACB populations in Vietnam and will serve as a baseline for future resistance monitoring work.     

A short-type peptidoglycan recognition protein from Asian corn borer,Ostrinia furnacalis (Guenée) exhibits antibacterial activity,and regulates the expression of antimicrobial peptides

Peptidoglycan recognition proteins (PGRPs) discriminate and bind peptidoglycans by acting as pattern recognition receptors (PRRs) in insects, and function pivotal roles in innate immune response. In the present study, we cloned a full-length PGRP gene designed as OfPGRP8 from the Asian corn borer, Ostrinia furnacalis (Guenée). Its mRNA exhibited the highest abundance in fat body, and its expression level upregulated dramatically after bacterial challenges. Purified recombinant OfPGRP8 exhibited intensive binding capacity to peptidoglycans from Staphylococcus aureus and Micrococcus luteus. Additionally, recombinant OfPGRP8 could inhibit the growth of S. aureus, M. luteus. However, recombinant OfPGRP8 could not cause agglutination of S. aureus, M. luteus or Escherichia coli. Furthermore, we also demonstrated that OfPGRP8 may be involved in modulating the signaling pathway of antimicrobial peptides (AMPs) synthesis. In sum, our results provided evidence that OfPGRP8 discriminates peptidoglycans from microbes and acts as a PRR to initiate downstream immune signaling pathways.     

Tissue Distribution and Purification of Prophenoloxidase in Larvae of Asian Corn Borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae)

Phenoloxidase (PO) plays an important role in the de-fense response to the foreign invaders, e.g. pathogens andparasites, in the formation of melanin as well as in cuticlesclerotization in insects [1]. PO is synthesized as azymogen, prophenoloxidase (PPO)…     

Types of Antennal Sensilla of the Asian Corn Borer,Ostrinia furnacalis (Guenéee)

This study was carried out to investigate the types of sensilla and their distribution on the antennae of the Asian corn borer, Ostrinia furnacalis (Guenéee). O. furnacalis had six types of antennal sensilla: sensilla trichodea, sensilla chaetica, sensilla coeloconica, sensilla auricillica, sensilla styloconica and Böohms bristle. S. auricillica, single-walled multiporous sensilla, were presumed to be involved in chemical reception. S. coeloconica were double-walled, multiporous sensilla and could detect chemicals also. S. chaetica appeared to be mechano-receptors. S. styloconica were poreless and were thought to have thermo- and hygro-receptors. S. trichodea were single-walled chemosensilla and two subtypes were identified by transmission electron microscopy. The scape and the pedicel had Böohms bristles, which seem to function as mechanical stimulus receptors.     

Molecular Cloning,Characterization and Expression Profiling of a Ryanodine Receptor Gene in Asian Corn Borer,Ostrinia furnacalis (Guenée)

Ryanodine receptor (RyR) Ca²⁺ release channel is the target of diamide insecticides, which show selective insecticidal activity against lepidopterous insects. To study the molecular mechanisms underlying the species-specific action of diamide insecticides, we have cloned and characterized the entire cDNA sequence of RyR from Ostrinia furnacalis (named as OfRyR). The OfRyR mRNA has an Open Reading Frame of 15324 bp nucleotides and encodes a 5108 amino acid polypeptide that displays 79–97% identity with other insects RyR proteins and shows the greatest identity with Cnaphalocrocis medinalis RyR (97%). Quantitative real-time PCR showed that the OfRyR was expressed at the lowest level in egg and the highest level in adult. The relative expression level of OfRyR in first, third and fifth-instar larva were 1.28, 1.19 and 1.99 times of that in egg. Moreover, two alternative splicing sites were identified in the OfRyR gene. One pair of mutually exclusive exons (a/b) were present in the central part of the predicted SPRY domain, and an optional exon (c) was located between the third and fourth RyR domains. Diagnostic PCR demonstrated that exons a and b existed in all developmental stages of OfRyR cDNA, but exon c was not detected in the egg cDNA. And the usage frequencies of these exons showed a significant difference between different developmental stages. These results provided the crucial basis for the functional expression of OfRyR and for the discovery of compound with potentially selective insect activtity.     

Enhanced Resistance of Snowdrop Lectin (Galanthus nivalis L. Agglutinin)-Expressing Maize to Asian Corn Borer (Ostrinia furnacalis Guenée)

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through the Agrobacterium tumefaciens-mediated also studied. Thirty-six independently derived plants were subjected to molecular analyses. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of three GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to ACB. These plants synthesized GNA at levels above 0.24% total soluble protein and enhanced resistance to ACB was demonstrated by exposing the plants to insects under greenhouse conditions. Semi-artificial diet bioassays also showed the toxic effect of GNA on ACB. Field evaluation of the transgenic plants supported the results from the artificial trial. In the present study, we have obtained new insect-resistant maize material for further breeding work and have found that GNA-expressing plants not only gained significant resistance to homopterans, but also showed toxicity to ACB, which is a type of Lepidoptera.     

Monoamine oxidase in the brain of European corn borer larvae,Ostrinia nubilalis (Hübner)

Monoamine oxidase (MAO) activity was demonstrated in intact brains of larvae from the European corn borer, Ostrinia nubilalis using 2-(2′-benzothiazolyl)-5-stryl-3-(4′-phthalhydrazidyl) tetrazolium chloride (BSPT). With tryptamine as the substrate, MAO specific activity was restricted to mitochondria within perineurial cells. The basic BSPT methodology was modified by the substitution of 2% dimethylsulfoxide for dimethylformamide in the incubation medium. This yielded increased permeability of the brain to the incubation medium, presumably by disrupting the integrity of the blood-brain barrier. A discussion of possible reasons for the previous inability to demonstrate insect neuronal MAO activity is presented.     

Developmental consequences of water and temperature in the European corn borer — maize interaction

Maize plants were grown under four moisture regimes (wet to extreme deficit) and three constant temperatures (20°, 25° & 30°C) in a phytotron. Each plant was infested with one E-race European corn borer [Ostrinia nubilalis (Hubn.)] (ECB) egg mass at pollen shed. ECB development, location, and establishment were recorded over the course of 12 destructive sample dates (4/temperature). ECB developmental rates were not significantly affected by soil moisture treatments, but were significantly affected by temperature. In spite of successful establishment of four distinctly different soil moisture regimes, the maize stalk tissue water levels were not significantly different among soil water treatments. Instead, the maize plants exhibited accelerated leaf senescence in response to the water deficit conditions. Among the soil water treatments, differences were found in larval establishment, vertical distribution and dispersion, and feeding site selection; however, those effects were slight and could not explain the similarity in ECB developmental rates observed in these treatments.In maize, the larval environment within the stalk was effectively insulated from changes in the external environment by the plant's ability to maintain a relatively high and stable stalk tissue water content. Thus, large changes to the soil environment had essentially no effect on ECB development, though drastic consequences for the plant. This study indicates that ECB rates of development are relatively insensitive to changes in the soil water environment as well as the associated changes in the maize plant that accompany severe drought stress. The significance of these findings to insect modelling, crop physiology, and insect-crop interactions is discussed.

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Résumé Des plants de maïs se développent dans un phytotron dans 4 conditions d'humidité du sol (de la saturation à la dessication) et à 3 températures constantes (20°, 25° & 30°C). Chaque pied est contaminé au moment de l'émission du pollen, par une ooplaque d'O. nubilalis Hübn. (ECB) de race européenne E. L'installation, la colonisation et le développement des chenilles sont notés lors de 12 périodes de prélèvements destructifs (4 par température). La vitesse de développement d'O. nubilalis est affectée par la température, main non par l'humidité du sol. Les 4 niveaux d'humidité du sol n'ont aucun effet sur la teneur en eau des tiges de maïs. En fait, les feuilles de maïs présentent une senescence précoce lorsqu'il y a déficit en eau dans le sol. La teneur en eau du sol agit sur l'installation, sur la distribution verticale, la dispersion et le lieu d'alimentation des chenilles; mais ces effets sont légers et ne modifient pas la vitesse de développement.L'environnement larvaire dans la tige de maïs est efficacement isolé des variations externes par l'aptitude de la plante à maintenir la teneur en eau des tiges relativement élevée et stable. Ainsi, des changements importants au niveau du sol n'ont pratiquement pas d'effets sur le développement d'O. nubilalis, malgré les conséquences brutales pour la plante. Cette étude montre que la vitesse de développement d'O. nubilalis est relativement insensible aux modifications de la teneur en eau du sol ainsi qu'aux effets de ce stress de sécheresse sévère sur le pied de maïs. La discussion porte sur l'importance de ces résultats pour la modélisation de la dynamique de l'insecte, la physiologie de la culture et les interactions entre insecte et plante.

     

High temperature tolerance and thermal-adaptability plasticity of Asian corn borer (Ostrinia furnacalis Guenée) after a single extreme heat wave at the egg stage

Due to ongoing climate change, short-term extreme heat waves in the summer are expected to be more frequent. Insect eggs are sensitive to thermal stress. This raises the question of whether herbivore insects' thermal adaptability would be changed after a single extreme heat wave at the egg stage. In this study, we examined the developmental performance of Ostrinia furnacalis Guenée at 25?°C, 27?°C, 29?°C or 31?°C after a single extreme heat wave (42?°C) for 0?h (control), 1?h, 2?h, or 3?h at the egg stage. The results showed that O. furnacalis at the egg or larval stage was more sensitive to a single heat wave than it was at the pupae or adult stage. After a single heat wave, O. furnacalis showed a reduced egg-hatching rate or reduced larval survival rate, but the optimum temperature for egg hatching and larval survival was higher than that in the control. The upper temperature threshold and optimum temperature for larval development in the control were higher than that after a single extreme heat wave. Both male and female pupal weight decreased with increasing temperature, and pupal weight decreased faster in females than in males. The Cox proportional hazard model showed that when O. furnacalis developed at 25?°C, the instantaneous death risk of adults with a 3?h heat wave at the egg stage was higher than that of the control, but when O. furnacalis developed at 29?°C and 31?°C, the instantaneous death risk of adults after a heat wave was significantly lower than that of the control. Our study highlights the effect of a single heat wave on O. furnacalis eggs and the subsequent development of survival individuals.     

Comparative ultrastructure and blood-brain barrier in diapause and nondiapause larvae of the European corn borer Ostrinia nubilalis (Hübner)

Ultrastructural examination of diapause and nondiapause larval brains of the European corn borer disclosed anatomical differences that may be related to the insect's "blood-barrier". The perineurial type I cells are quite closely appressed in the diapause brain, but thrown into extensive folds with large intercellular spaces in the nondiapause brain. The perineurial type II cells of diapause and nondiapause larvae are basically similar in general ultrastructure, and most likely form the basis for the "blood-brain barrier." Horseradish peroxidase penetration studies indicated that the outer margin of the perineurial type II cells constitute the limits of infiltration into the brain. An enzymatic component of the "blood-brain barrier" is postulated in this insect. The localization of ATPase in the perineurial type II cells indicates that energy-requiring regulatory mechanisms may be localized here. Metabolic studies with isolated brains, coupled with recent evidence from mammalian systems, suggest that glial cells may be of importance in an enzymatic "blood-brain barrier."     

Fatty acid and sex pheromone changes and the role of glandular lipids in the Z-strain of the European corn borer, Ostrinia nubilalis (Hübner)

Lipids in the sex pheromone gland of females of the Z-strain of Ostrinia nubilalis were analyzed for fatty acyl pheromone analogs (FAPAs) and other potential biosynthetic intermediates. More than 80% of the FAPAs were found in the triacylglycerols (TGs), with smaller amounts found in the phosphatidyl cholines, ethanolamines, and serines. Analysis of the TGs by lipase revealed that the two FAPAs were distributed fairly evenly among all three stereospecific positions. Comparison of changes in titers of key glandular fatty acids with those of pheromone components, with respect to photoperiodic time and age of females, showed that both FAPA and pheromone titers exhibited a cyclical pattern with peaks in the scotophase and valleys in the photophase. However, whereas pheromone titer tended to peak in the first half of the scotophase, FAPA titer peaked at the end of the scotophase. Significantly, the titer of the FAPA of the minor component, (E)-11-tetradecenyl acetate (3% of pheromone), was always much greater than the titer of the FAPA of the major component, (Z)-11-tetradecenyl acetate (97%), of the pheromone. Titer of myristate, an intermediate in pheromone biosynthesis, was also higher during the scotophase than the photophase. However, myristate titer showed a pronounced dip in the middle of the scotophase. These data suggest two roles for glandular lipids in sex pheromone biosynthesis in O. nubilalis. Firstly, they remove excess FAPA of the minor component so the fatty acid reductase system is not presented with a high ratio of this isomer (which would otherwise result from the reductase's own selectivity), which could cause changes in the final pheromone ratio. Secondly, hydrolysis of the large amounts of stored saturated fatty acids from the TGs may provide substrate for pheromone biosynthesis.     

Passive evasion of encapsulation in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae), a polyembryonic parasitoid of Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae)

The hymenopteran Macrocentrus cingulum usually deposits one egg into the larval body cavity of lepidopteran Ostrinia furnacalis, and the egg subsequently splits into several dozens of embryos during its development. How the parasitoid eggs and embryos avoid encapsulation by the host's immune response remains unknown. We compared hemocyte counts, morphologies and behaviors between unparasitized O. furnacalis larvae, and larvae parasitized by M. cingulum. No distinct differences were observed. Sephadex A-25 beads elicited a strong encapsulation response when injected into the parasitized host larvae, which indicates that parasitism by M. cingulum does not affect host's cellular immunity. However, there were significant differences in the host's encapsulation reactions towards injected eggs from different sources. Injected M. cingulum mature eggs excised from the lateral oviducts of the female wasps were not encapsulated, while immature eggs or driselase treated mature ones provoked an encapsulation response within 2 h after injection. Inspection of eggs by transmission electron microscopy revealed that the driselase collapsed the surface fibrous layer of the eggs, indicating that surface fibrous layer may play a role in protecting eggs from host's immune attack.     

Anatomy of the neurosecretory cells in the cerebral and subesophageal ganglia of the female European corn borer moth,Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae)

The anatomy of the neurosecretory cells in the brain-subesophageal ganglion complex of female European corn borer moth Ostrinia nubilalis (Lepidoptera: Pyralidae) was studied using histological and cobalt backfilling techniques. Histological staining revealed the presence of 2 median and one lateral neurosecretory cell groups in the brain. These brain neurosecretory cells are made up of mainly type A cells with a few type B cells in the median group. Three type C neurosecretory cell clusters occupy the apparent mandibular, maxillary, and labial neuromeres at the ventral median aspect of the subesophageal ganglion. Axonal pathways of the neurosecretory cell groups were delineated by retrograde cobalt filling from the corpora cardiaca. Fibers of the 3 brain neurosecretory cell groups merged to form a distinct axonal tract that exits the brain via the fused nervi corporis cardiaci-1 + 2. Cobalt backfilling from the corpora cardiaca filled 4 groups of cell bodies in the subesophageal ganglion. The presence in the subesophageal ganglion of extensive dendritic arborizations derived from the brain suggests interactions between neurosecretory cell groups in the 2 head ganglia.     

Divergence in behaviour between the European corn borer,Ostrinia nubilalis,and its sibling species Ostrinia scapulalis: adaptation to human harvesting?

Divergent adaptation to host plant species may be the major mechanism driving speciation and adaptive radiations in phytophagous insects. Host plants can differ intrinsically in a number of attributes, but the role of natural enemies in host plant specialization is often underappreciated. Here, we report behavioural divergence between the European corn borer (ECB, Ostrinia nubilalis) and its sibling species Ostrinia scapulalis, in relation to a major enemy: humans. Harvesting maize imposes selective mortality on Ostrinia larvae: those located above the cut-off line of the stalk face almost certain death. We show that ECB larvae diapause closer to the ground than those of O. scapulalis, which is sympatric but feeds mainly on weeds. The difference in diapause height results from genetically determined differences in geotactic behaviour. ECB larvae descend towards the ground specifically at harvest time, increasing their chances of surviving harvesting by about 50 per cent over O. scapulalis larvae. Natural enemies appear as a major driver of host-plant specialization in this example, stressing the need to consider ‘tri-trophic’ ecological niches to understand insect diversification. Our results also strongly suggest that geotaxis evolved as a singular instance of behavioural resistance in a major agricultural pest.     

Comparison on DNA patterns of different ecotypes of European corn borer (Ostrinia nubilalis Hübner) in Hungary

For a molecular genetic study on Hungarian populations of European corn borer L5 stage larvae were collected from 14 places of three different regions of the country (uni- and bivoltine ecotypes). Additionally, the study included larvae from Egypt, too (multivoltine ecotype). Molecular examinations of European corn borer larvae using the study of mitochondrial cytochrome b (cyt b) revealed that by single strand conformation polimorphism (SSCP) the populations found in Hungary represented the same haplotype. Even the Egyptian sample showed no genetic divergence. Some minor deviatons were found in the case of a sample from Székkutas, but that did not prove the genetic divergence of the bivoltine ecotype either, since the other samples of South-East Hungary did not display this kind of genetic variation. On the basis of our investigations it can be said that the univoltine and bivoltine generations, have uniform genetic complements.     

Binding specificity of OBPs in the yellow peach moth Conogethes punctiferalis (Guenée)

Odorant-binding proteins (OBPs) are translators of the external chemical signals, which are critical for maintaining insect life. However, few OBPs were reported in the yellow peach moth (YPM), Conogethes punctiferalis (Guenée). In the current study, five OBPs (CpunOBP1, CpunOBP2, CpunOBP7, CpunPBP2 and CpunPBP4) were expressed and purified from the antennae of the YPM. The results showed that the proteins encoded by five CpunOBPs had six conserved cysteine residues, which were typical structural features of classic OBPs. Moreover, the fluorescence competitive binding assays indicated that the binding affinity of five CpunOBPs to the selected YPM female sex pheromones, host plant volatiles and Penicillium-inoculated apple volatiles was obviously different. The binding affinities of CpunOBP1 and CpunOBP2 with β-ionone were the strongest. CpunOBP7 could bind with 12 host plant volatiles but was unable to interact with any one of the three tested female sex pheromones. CpunPBP2 and CpunPBP4 exhibited the highest binding affinity to female sex pheromone trans-10-hexadecenal among 30 tested compounds. In conclusion, these results suggest the functional differentiation of the CpunOBPs in recognizing sex pheromones, host plant volatiles and fungus-infected host plant volatiles, which will provide new insights into selecting target proteins for YPM biocontrol.