Sex pheromones and reproductive isolation of three species in genus Adoxophyes.

We tested differences in female pheromone production and male response in three species of the genus Adoxophyes in Korea. Females of all three species produced mixtures of (Z)-9-tetradecenyl acetate (Z9-14:OAc) and (Z)-11-tetradecenyl acetate (Z11-14:OAc) as major components but in quite different ratios. The ratio of Z9-14:OAc and Z11-14:OAc in pheromone gland extracts was estimated to be ca. 100:200 for Adoxophyes honmai, 100:25 for Adoxophyes orana, and 100:4,000 for Adoxophyes sp. Field tests showed that males of each species were preferentially attracted to the two-component blends of Z9-14:OAc and Z11-14:OAc mimicking the blends found in pheromone gland extracts of conspecific females. The effects of minor components identified in gland extracts on trap catches varied with species. Addition of 10-methyldodecyl acetate (10me-12:OAc) or (E)-11-tetradecenyl acetate (E11-14:OAc) to the binary blend of Z9-14:OAc and Z11-14:OAc significantly increased captures of A. honmai males, whereas E11-14:OAc exhibited a strongly antagonistic effect on catches of Adoxophyes sp. males. Moreover, (Z)-9-tetradecen-1-ol (Z9-14:OH) or (Z)-11-tetradecen-1-ol (Z11-14:OH) added to the binary blends increased attraction of male A. orana but not A. honmai and Adoxophyes sp. males, suggesting that these minor components, in addition to the relative ratios of the two major components, play an important role in reproductive isolation between Adoxophyes species in the southern and midwestern Korea where these species occur sympatrically.

Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm, Helicoverpa assulta (Guenée).

Acetylcholinesterase (AChE) has been known to be the target of organophosphorous and carbamate insecticides. Only a single AChE, however, existed in insects and was involved in insecticide resistance, recently another AChE is reported in mosquitoes and aphids. We have cloned cDNAs encoding two ace genes, designated as Ha-ace1 and Ha-ace2 by a combined degenerate PCR and RACE strategy from adult heads of the oriental tobacco budworm, Helicoverpa assulta. The Ha-ace1 and Ha-ace2 genes encode 664 and 647 amino acids, respectively and have conserved motifs including a catalytic triad, a choline-binding site and an acyl pocket. Both Ha-AChEs were determined to be secretory proteins based on the existence of a signal peptide. The Ha-ace1 gene, the first reported ace1 in lepidopterans, belongs to the ace1 subfamily whereas the Ha-ace2 gene showed high similarity to those in the ace2 subfamily. Phylogenetic analysis showed that the Ha-ace1 gene was completely diverged from the Ha-ace2, suggesting that the Ha-ace genes are duplicated. Quantitative real time-PCR revealed that expression level of the Ha-ace1 gene was much higher than that of the Ha-ace2 in all body parts examined. The biochemical properties of purified proteins by affinity chromatography showed substrate specificity for acetylthiocholine iodide, and inhibitor specificity for BW284C51 and eserine and their peptide sequences partially identified by a MALDI-TOF mass spectrometer demonstrated that two Ha-AChEs were expressed in vivo.

Isolation and characterization of strain of Bacillus thuringiensis subsp. kenyae containing two novel cry1-type toxin genes.

To identify novel crystal proteins, Bacillus thuringiensis 2385-1 was isolated from Korean soil samples and characterized. The H-serotype of 2385-1 was identical to that of subsp. kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped. However, 2385-1 showed a much higher toxicity towards Plutella xylostella and Spodoptera exigua larvae than subsp. kenyae. In addition, the crystal protein profile and plasmid DNA pattern of 2385-1 differed from those of subsp. kenyae. To verify the crystal protein gene types of 2385-1, a PCR-RFLP analysis was performed, and the results revealed that 2385-1 contained two novel cry1-type crystal protein genes, cry1-5 and cry1-12, in addition to the cry1Ja1 gene. The deduced amino acid sequences of cry1-5 and cry1-12 showed a 97.9% and 75.7% sequence similarity with the CrylAb and Cry1Ja crystal proteins, respectively. Among the novel crystal proteins, Cry1-5 showed a high toxicity towards P. xylostella and S. exigua larvae. In conclusion, B. thuringiensis 2385-1 is a new isolate in terms of its gene types, and should be a promising source for an insecticide to control lepidopteran larvae.

Molecular characterization of pheromone biosynthesis activating neuropeptide from the diamondback moth, Plutella xylostella (L.).

Pheromone biosynthesis activating neuropeptide (PBAN) produced in the subesophageal ganglion stimulates pheromone production in the pheromone gland. A cDNA isolated from female adult heads of the diamondback moth (Plutella xylostella (L.)) encodes 193 amino acids including PBAN, designated as Plx-PBAN, and four other neuropeptides (NPs): diapause hormone (DH) homologue, alpha-NP, beta-NP and gamma-NP. All of the peptides are amidated in their C-termini and shared a conserved motif, FXPR(or K)L structure, as reported from other PBAN cDNAs. Plx-PBAN consists of 30 amino acids, the shortest PBAN so far reported. Plx-PBAN exhibited below 50% homology, compared with other known PBANs. The Plx-DH homologue is structurally different from DH of Bombyx mori. The length of Plx-beta-NP (16 amino acids) was the shortest and showed relatively low similarity, whereas gamma-NP (10 amino acids in length) was the longest among examined gamma-NPs. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in all examined body parts, with the highest expression level in the head of female adults. Analysis of RT-PCR products indicated the Plx-PBAN sequence was identical in all examined body parts of both sexes. Phylogenetic analysis revealed that the Plx-PBAN gene is distantly related to other PBANs, demonstrated by the relatively low similarity.

Molecular identification of Adoxophyes honmai (Yasuda) (Lepidoptera: Tortricidae) based on mitochondrial COI gene sequences.

Molecular identification techniques are used where morphological characters are not useful for distinguishing species that resemble each other closely. The example studied here is the Adoxophyes species complex, in which A. orana (Fischer von Rösslerstamm) is officially the only known Korean species in the genus Adoxophyes (Lepidoptera: Tortricidae). However there have been suspicions that at least two types of A. orana exist in Korea based on the distribution and range of the host, with A. orana attacking apples and peaches, and another Adoxophyes sp. attacking tea and pears. The latter is presumed to be A. honmai (Yasuda), but the two have remained confused because of their extreme morphological similarity, despite several Asian studies of pheromonal and morphological characteristics. To confirm the occurrence of an Adoxophyes species other than A. orana in Korea, we compared 940 bp of the mitochondrial cytochrome oxidase I (COI) gene from 16 samples of Adoxophyes and found that there is a second Adoxophyes species different from A. orana. Comparison of the different sequences to that of Japanese A. honmai confirmed that they belong to the latter. From the sequence difference between the two Korean species, we were able to develop new PCR primer sets that distinguish them. This molecular identification technique with no enzyme digestion or sequencing step is a convenient and rapid way of differentiating between species that are hard to distinguish morphologically.

Pheromone biosynthetic pathways in the moths Helicoverpa zea and Helicoverpa assulta.

Sex pheromones of many Lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathway. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via delta 11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by delta 11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by delta 9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the delta 9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the delta 11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes.

Identification of a new member of PBAN family and immunoreactivity in the central nervous system from Adoxophyes sp. (Lepidoptera: Tortricidae).

Production of sex pheromones, Z9-14:OAc and Z11-14:OAc, of the smaller tea tortrix, Adoxophyes sp. was stimulated by injection of the female or male head extracts as well as synthetic pheromone biosynthesis activating neuropeptide (PBAN) into decapitated females. The amount of pheromone produced reached a maximum level 3 h after injection of synthetic PBAN into females. A cDNA isolated from brain-suboesophageal ganglion complex (Br-SEG) of A. sp. females contained an ORF of 576 nucleotides encoding 192 amino acids. Based on endoproteolytic sites, it can be predicted to be cleaved into five putative peptide domains including PBAN and four other neuropeptides. Ado-PBAN consisting of 31-amino acids is the shortest PBAN so far reported. Four other putative PBAN-encoding gene neuropeptides (PGN) are predicted with PGN-24, PGN-7, PGN-20, and PGN-8 amino acids. All of the peptides are amidated in their C-termini with a FXPR(or I, K)L structure, except for PGN-8 (TVKLTPRLamide). PBAN-like immunoreactive material was observed in Br, SEG and ventral nerve cord (VNC) of the female adult. In the brain, 5-7 pairs of neurons containing PBAN-like immunoreactivity were found in each protocerebral hemisphere. Three groups of cell clusters found in the SEG corresponded to the mandibular, maxillary and labial neurons as in other moths. PBAN-like immunoreactive neurons in the VNC were found in thoracic (three pairs) and abdominal ganglia (two pairs). As compared to other moths, a relatively low similarity of peptide sequences deduced from Ado-PBAN gene and a different expression pattern of PBAN-like immunoreactivity could indicate phylogenetical distance from the other species.

Cloning, mutagenesis, and expression of the acetylcholinesterase gene from a strain of Musca domestica; the change from a drug-resistant to a sensitive enzyme.

Insect acetylcholinesterase (AChE) is known to be a primary target of organophosphorus and carbamate insecticides. However chronic exposure to these chemicals has led to resistance to applied insecticides, due usually to mutation of the AChE gene. Analysis of the AChE gene (hm) of Musca domestica (the housefly), which is cloned in this report, reveals the relationship between mutation and insecticide resistance. The 2,076 bp hm encodes a mature protein of 612 amino acids (67 kDa), and an 80 residue signal peptide. Unlike the enzyme of 'sensitive' strains, the AChE used in this study was resistant to the organophosphorus insecticide, trichlorphon. DNA sequencing showed that this AChE is identical to that of the sensitive strains with the exception of three amino acids Met-82, Ala-262, and Tyr-327. Site-directed mutagenesis of the Ala-262 and Tyr-327 residues largely restored sensitivity to the insecticide, suggesting that these two residues are the key structural elements controlling sensitivity. In addition to these residues, Glu-234 and Ala-236 in the conserved sequence FGESAG are thought to play a role in modulating sensitivity to organophosphorus insecticides.

Expression of a recombinant Cry1Ac crystal protein fused with a green fluorescent protein in Bacillus thuringiensis subsp. kurstaki Cry-B.

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis Cry(-)B strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the cry1Ac gene (pProAc-GFP). The B. thuringiensis Cry(-)B strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immunoblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single species, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis Cry(-)B strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuringiensis.

Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.).

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.