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.     

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.     

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.     

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.     

Acceptability and suitability of the European Ostrinia nubilalis Hübner for Macrocentrus cingulum Brischke from Asia and Europe

We examined whether Macrocentrus cingulum (Hymenoptera: Braconidae) of Asian origin could serve as a biological control agent of the maize pest Ostrinia nubilalis (Lepidoptera: Crambidae) in Europe. M. cingulum is already present in Europe, where it does not parasitize O. nubilalis but Ostrinia scapulalis, a related species feeding on wild dicotyledons. In contrast, M. cingulum have been imported from Europe and Asia into North America (where O. nubilalis had been accidentally introduced from Europe), and does parasitize O. nubilalis there. We conducted laboratory infestations to assess host acceptability (parasitoid’s propensity to oviposit) and suitability (parasitoid’s ability to develop) of European O. nubilalis for M. cingulum of European and Asian origin, and of Ostrinia furnacalis (their original host) for Asian M. cingulum. Asian M. cingulum parasitized European O. nubilalis as readily as O. furnacalis, and developed equally well in terms of: % female cocoons, time to first emergence from the cocoon, total number of adult offspring, % female offspring and adult longevity. Adult female parasitoids were significantly larger when emerging from O. nubilalis, mixed-sex and male cocoons were significantly more and less frequent, respectively. The acceptability of O. nubilalis was significantly lower for European than for Asian M. cingulum, and its suitability was zero. Asian M. cingulum appears a potential candidate for introduction as a biological control agent of a major maize pest, European O. nubilalis, provided environmental impact studies, economic analyses, and foreseeable interactions with other biological control agents such as the egg parasitoid Trichogramma brassicae (Hymenoptera: Trichogrammatidae) are satisfying.     

Fine structure of endocrine hindgut cells of a lepidopteran,Ostrinia nubilalis (Hübn.)

Summary Cells of the hindgut of the European corn borer, Ostrinia nubilalis, have two functions, namely, ion transport and secretion of a hormone called proctodone. In this species, proctodone is an essential requisite to the prepupal molt in conjunction with brain hormone synthesis. Apical infoldings and their associated mitochondria form mitochondrial pumps for ion transport from the gut lumen to the hemocoel. The endocrine function of the cells is evidenced by rhythmical formation and discharge of inclusion bodies every 8 hours. These measure from 800 Å to 3 . in diameter and vary in composition from bodies with whorled, myeloid content to inclusions with densely granular matrix. During the discharge phase, granular material appears in the basal infoldings and accumulates in large quantities underneath the basal lamina and in the hemocoelic clefts adjacent to the active cells.This study constitutes publication No. 358 from the Oregon Regional Primate Research Center, supported in part by postdoctoral training fellowship 1-T1AM-5521-02 and Grant FR 00163 from the National Institutes of Health.     

Physiological host specificity: a model using the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and microsporidia of row crop and other stalk-boring hosts

We investigated vertical and horizontal transmission as means by which entomopathogenic microsporidia may be isolated in their hosts. Ostrinia nubilalis larvae were challenged with microsporidia isolated from other stalk-boring and row crop Lepidoptera and were susceptible to seven species. Two species were horizontally transmitted. A Nosema sp. from Eoreuma loftini was transmitted among O. nubilalis larvae but not among larvae of the E. loftini host. This species was also vertically transmitted to the offspring of infected O. nubilalis females. An rDNA sequence showed the E. loftini isolate to be Nosema pyrausta, a naturally occurring species in O. nubilalis. Our results suggest that both horizontal and vertical transmission provide physiological barriers to host switching in the microsporidia, thus restricting the natural host range.     

Two Different Bacillus thuringiensis Delta-Endotoxin Receptors in the Midgut Brush Border Membrane of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae)

Binding of three Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Ostrinia nubilalis larvae was characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections. Our results demonstrate that two independent ICP receptors are present in the brush border of O. nubilalis gut epithelium. From competition binding experiments performed with I-labeled and native ICPs it was concluded that CryIA(b) and CryIA(c) are recognized by the same receptor. An 11-fold-higher binding affinity of CryIA(b) for this receptor correlated with a 10-fold-higher toxicity of this ICP compared with CryIA(c). The CryIB toxin did not compete for the binding site of CryIA(b) and CryIA(c). Immunological detection of ingested B. thuringiensis ICPs on gut sections of O. nubilalis larvae revealed binding only along the epithelial brush border membrane. CryID and CryIE, two ICPs that are not toxic to O. nubilalis, were not bound to the apical microvilli of gut epithelial cells. In vitro binding experiments performed with native and biotinylated ICPs on tissue sections confirmed the correlation between ICP binding and toxicity. Moreover, by performing heterologous competition experiments with biotinylated and native ICPs, it was confirmed that the CryIB receptor is different from the receptor for CryIA(b) and CryIA(c). Retention of activated crystal proteins by the peritrophic membrane was not correlated with toxicity. Furthermore, it was demonstrated that CryIA(b), CryIA(c), and CryIB toxins interact in vitro with the epithelial microvilli of Malpighian tubules. In addition, CryIA(c) toxin also adheres to the basement membrane of the midgut epithelium.     

Diapause in the European corn borer,Pyrausta nubilalis (Hübn.)

Diapause in mature larvae of the European corn borer was found to be associated with three readily discernible characteristics: (1) arrested gonadal development; (2) failure to pupate shortly after cessation of larval feeding; (3) reduction of oxygen consumption to about 25 per cent that of non-diapause mature borer larvae. Diapause was found to be induced by photoperiods of from 9 to 15·5 hr of light per 24-hr period. Within this range of photoperiods, incidence of diapause was inversely proportional to the rearing temperature employed, except that with 10·5–13·5 hr photoperiods all larvae entered diapause without regard to the ambient temperature. At moderate rearing temperature (23–25°C), the mean threshold photoperiod was 15·4 hr of light per 24 hr. The diapause threshold for photoperiods under 13 hr was not determined. The physiological changes associated with diapause were found to be largely reversible up to at least the early part of the fifth larval instar. No critical growth stage for diapause determination was detected.     

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite loci from the European corn borer, Ostrinia nubilalis (Hübner) (Insecta: Lepidoptera: Crambidae)

Few useful microsatellites are available for population studies of the European corn borer, Ostrinia nubilalis (Hübner). An enrichment strategy was used to develop microsatellite markers for O. nubilalis, and over 500 positive clones were isolated. Seventy-five contained unique microsatellites, 10 of which were polymorphic with discernable polymerase chain reaction products. The 10 loci were surveyed for variability in 72 wild individuals from central Iowa. Five loci showed no deviation from Hardy-Weinberg proportions, and all were successfully cross-amplified in the related Asian corn borer, Ostrinia furnacalis. These loci represent a significant addition to microsatellites appropriate for population studies of O. nubilalis.     

Identification and characterization of genes abnormally expressed in wing-deficient mutant (flügellos) of the silkworm,Bombyx mori

The wing-deficient mutant, flügellos (fl), of the silkworm lacks four wings in the pupa and the adult, due to aberrant wing morphogenesis during metamorphosis. To elucidate the mechanisms of wing-specific deficiencies in the fl mutant, we used mRNA differential display and identified five genes abnormally expressed in the fl wing discs. Northern blot and RT-PCR analyses revealed that four genes were overexpressed, but the fifth one was not transcribed in the fl wing discs. The expression level of ribosome-associated protein p40 in the fl wing discs was elevated approximately 10 times compared to the wild-type (WT) discs. Another overexpressed gene CB10 encodes a novel wing-specific protein with a putative zinc-finger motif. Overexpression of two components of extracellular matrix, cuticle protein 18 (BMCP18) and a fibrillin-like protein AD10, may result in the abnormal wing morphogenesis in the fl mutant. In contrast, a novel member of multifunctional Ca2+-binding protein annexins, designated as annexin b13 (Anx b13), was expressed dominantly in the wing discs of WT but completely repressed in the fl tissues. Strong expression of Anx b13 in wing discs during the fourth and fifth instar indicates that ANX B13 plays an important role in wing morphogenesis.     

Flight dynamics analysis of the European corn borer (Ostrinia nubilalis (Hübner)) populations in Hungary from the second part of the twentieth century until the present

The data originating from six different points of the Hungarian Light Trap Network (1960–2005) were processed for comprehensive examination of the European corn borer's (ECB) (Ostrinia nubilalis Hübner) flight dynamics. The annual flight diagrams were visually assayed. The annual alteration tendency of the quotients of the total trapped individual numbers (Σ), the relative individual number per day (1RIN) and the flight peak (FQ) were examined by means of linear regression analysis. The period and time of the alterations with comparative assay of five consecutive years series of data with Student's t-probe were revealed. Eventually I investigated the effects of annual average temperature on the examined flight parameters with the help of variance analysis. The change of ecology and flight dynamics of the ECB started in the first and extended to the second part of twentieth century is now continuing. This statement has been confirmed by the increasing number of trapped individuals (regression quotient calculated from Σ, etc.: Velence-Sukoró: 65.58; Tarhos: 131). This phenomenon can be best characterised by the appearance and spreading of the second late summer flight peak (average regression quotient calculated from FQ, etc.: 0.082). According to my experiment the gradually increasing temperature and climate play a part in the background of the national flight changes [significant connection between the annual average temperatures of Hungary and Σ (P = 0.021), - 1RIN (P = 0.043)]. The spreading of the bivoltine ecotype can be predicted from these results and the literary data of Hungary.     

Cloning,expression and immunocytochemical localization of a general odorant-binding protein gene from Helicoverpa armigera (Hübner)

A cDNA clone coding for general odorant-binding protein2 was isolated from the antenna of Helicoverpa armigera by RT-PCR and (5'/3')-RACE technique. Results of sequencing and structural analyses showed that the full-length of GOBP2Harm was 636 bp, possessing 162 amino acid residues and a signal peptide of 21 amino acids. Its predicted molecular weight and isoelectric point were 18.2 kDa and 5.21, respectively. This deduced amino acid sequence shared some common structural features with odorant-binding proteins from several moth species, including the six conserved cysteine motif, typical of insect's OBPs. Northern blot showed that GOBP2Harm is specifically expressed in the antenna of Helicoverpa armigera at similar levels in both sexes. In order to obtain sufficient GOBP2 for further determining its biochemical and physiological properties, a bacterical expression vector of GOBP2 was constructed and successfully expressed. The protein was obtained mainly as insoluble inclusion bodies, that, however, could be solubilized and refolded. The rGOBP2 was purified by affinity chromatography and gel filtration. The rGOBP2 was shown to cross-react with an anti-GOBP antiserum from Antheraea polyphemus. Finally, polyclonal antibodies against GOBP2Harm were used to mark the distribution of the protein in olfactory sensilla and were tested by immuno-electron microscopy. In the male, GOBP2Harm is mainly expressed in sensilla basiconica, while in the female, it is equally expressed in sensilla basiconica and in sensilla trichodea.     

Molecular cloning and bacterial expression of pheromone binding protein in the antennae of Helicoverpa armigera (Hübner)

A cDNA clone coding for pheromone binding protein was isolated from the antennae of Helicoverpa armigera by RT-PCR and (5'/3')-RACE technique. The full-length of H. armigera pheromone binding protein (HarmPBP) was 952 bp, possessing 162 amino acid residues including a signal peptide of 20 amino acids. Its predicted molecular weight and isoelectric point were 18.26 kDa and 5.23, respectively. This deduced amino acid sequence shared some common structural features with odorant-binding proteins from several moth species, including the six conserved cysteine motif, a typical characteristic of insect's odorant-binding proteins. Northern blot showed that HarmPBP is specifically expressed in the antennae of Helicoverpa armigera and more abundantly expressed in male than female. During the antennal development, HarmPBP is first expressed about 4 days prior to adult eclosion and rises to a plateau 2 days prior to adult eclosion. In order to obtain sufficient PBP for further determining its biochemical and physiological properties, a bacterical expression vector of PBP was constructed and successfully expressed in Escherichia coli. The recombinant PBP was shown to cross-react with an anti-PBP antiserum from Antheraea polyphemus. Polyclonal antibodies against HarmPBP were used to mark the distribution of the protein in olfactory sensilla. Very strong labeling was observed in the sensillum lymph of the hair lumen and of the sensillum-lymph cavity. In the male, HarmPBP is expressed in sensilla trichodea and not in sensilla basiconica, while in the female, it is expressed both in sensilla basiconica and sensilla trichodea.