Heteroplasmy of mitochondrial DNA in the ophiuroid Astrobrachion constrictum

We demonstrate the presence of mitochondrial heteroplasmy for the cytochrome oxidase I (COI) gene of the brittle star (Astrobrachion constrictum). One of the 117 individuals analyzed contained two distinct single-strand conformation polymorphism (SSCP) haplotypes differing by two substitutions; another showed sequence evidence for heteroplasmy. We used polymerase chain reaction (PCR) cloning, SSCP, and sequencing of a 480 bp region of the 5' end of COI to isolate and characterize these haplotypes. This is the first properly substantiated case of heteroplasmy in an echinoderm species and may have arisen from paternal leakage.     

Low resolution of mitochondrial COI barcodes for identifying species of the genus Larus (Charadriiformes: Laridae)

We evaluated whether cytochrome c oxidase subunit I (COI) barcodes that have been previously suggested for birds are useful for identifying species of the genus Larus, which are resident or migratory birds in Korea. We found 31 intra- or interspecific COI haplotypes from 12 of 13 Larus species in Korea. Haplotype analyses showed that the COI barcodes could not distinguish some interspecific haplotypes from 6 of 12 Larus species because there were no nucleotide substitutions among their COI haplotypes. The neighbor-joining tree formed shallow branches in the clades expected for L. saundersi, L. crassirostris, L. canus, L relictus, and L. ridbundus. In the nine Larus species, COI haplotypes were not grouped as distinct entities that were correctly assigned to their corresponding species, resulting in polytypic clades. These results indicate that the COI sequences need to be cautiously selected as a DNA barcode for identifying species of Korean Larus birds.     

Genetic differentiation of Helicoverpa armigera (Hübner) and H. assulta (Guenée) (Lepidoptera: Noctuidae) based on AFLP markers

Abstract Here we use amplified fragment length polymorphism (AFLP) to assess genetic differentiation of Helicoverpa armigera and H. assulta . The results indicated that both species-specific fingerprints and cluster analysis showed the ability of AFLP technique to discriminate the two sibling species; among a total 1963 AFLP markers amplified from nine primer combinations: 777 (39.6%) were H. armigera -specific, 602 (30.7%) were H. assulta -specific, and 584 (29.7%) were common bands. The mean number of H. armigera -specific bands was significantly more than that of H. assulta -specific bands for nine primer combinations ( P < 0.05); the intraspecific distance of H. armigera and H. assulta was 0.123 0 and 0.110 7 respectively, and the interspecific distance was 0.178 3. In addition, the percentage of polymorphic loci and estimated average heterozygosity were used to estimate genetic diversity of the two species. This study therefore demonstrates that AFLP analysis is a sensitive and reliable technique to study genetic differentiation and genetic relationships between species and provides sufficient molecular markers for future linkage map construction, location and eventual cloning of genes involved in traits differentiation.     

Two mitochondrial lineages in Korean freshwater Corbicula (Corbiculidae: bivalvia)

The Korean freshwater Corbicula was surveyed genetically by sequencing 614-bp homologous fragment of mitochondrial cytochrome oxidase I subunit. Among a total of 127 individuals collected from 12 Korean freshwater localities we found only two COI haplotypes and these differed by a total of 9 base substitutions. Although the sequence divergence between the two haplotypes is moderate (p = 1.47%), placing the two mitotypic sequences in the context of Asian mtDNA phylogeny reveals that Korean freshwater Corbicula is comprised of two independent freshwater mitochondrial lineages. These results are in serious disagreement with the long-standing conclusions of earlier conchology-based taxonomic work on Korean Corbicula in which a number of species names (a minimum of 10 nominal species) have been used. This indicates that morphological characteristics alone are poor criteria for species-level identification in this group. In addition, our COI dataset shows that there is an extremely low level of genetic variation in Korean freshwater populations, suggesting that these populations have passed through a severe population bottleneck that greatly reduced their genetic variability. Our data also provide new information on the biogeographic distribution of Korean freshwater Corbicula. When haplotypic frequencies were compared, it was evident that the two Korean freshwater mitochondrial lineages have achieved very different distribution ranges: the predominant lineage (FWKR1) is widely distributed in Korean freshwater systems, whereas the minor lineage (FWKR2) is restricted to a relatively narrow range.     

Hybridization between Helicoverpa armigera and Helicoverpa assulta (Lepidoptera: Noctuidae): development and morphological characterization of F1 hybrids

Reciprocal hybridizations between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) were studied. The cross between females of H. armigera and males of H. assulta yielded only fertile males and sterile individuals lacking an aedeagus, valva or ostium bursae. A total of 492 larvae of the F1 generation were obtained and 374 of these completed larval development and pupated. Only 203 pupae were morphologically normal males, the remaining 171 pupae were malformed. Larvae and pupae that gave rise to morphologically abnormal adults exhibited longer development times. Sterility was not only associated with malformed external sex organs, but also a range of abnormalities of the internal reproductive system: (i) loss of internal reproductive organs, (ii) with one to three copies of an undeveloped bursa copulatrix; or (iii) with one or two undeveloped testes. Normal male hybrid adults showed higher flight activity in comparison with males of both species. In contrast, the cross between females of H. assulta and males of H. armigera yielded morphologically normal offspring (80 males and 83 females). The interaction of the Z-chromosome from H. assulta with autosomes from H. armigera might result in morphological abnormalities found in hybrids and backcrosses, and maternal-zygotic incompatibilities might contribute to sex bias attributed to hybrid inviability.     

Mechanisms of premating isolation between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae)

Helicoverpa armigera and Helicoverpa assulta are sympatric sibling species, and in the laboratory they can interbreed and produce viable offspring. To assess the contributions of temporal barriers and sexual barriers to premating isolation, we investigated both the temporal rhythms of calling behavior and pheromone titers of H. armigera and H. assulta females and the behavioral responses of males to conspecific and heterospecific calling females in a wind tunnel. Both H. armigera and H. assulta females called throughout the scotophase, and there was more calling during the second half of the scotophase than during the first half. Maximal pheromone titer and maximal calling activity in H. armigera synchronously occurred at the sixth hour into the scotophase, whereas, in H. assulta, the maximal pheromone titer occurred 2 h before the peak of calling. Pheromone blend ratios of the two species were opposite and, within each species, changes in the ratio within the scotophase and at different ages were relatively small. Males of both H. armigera and H. assulta responded strongly to their conspecific calling females in the wind tunnel and completed the whole courtship sequence. In contrast, they did not land and had no copulation attempts in response to heterospecific calling females. These results show that the two species do not have obvious temporal differences in calling behavior and pheromone production, and the specificity of sex pheromone blend emitted by females plays a key role in their premating isolation. In addition, we summarized the potential isolation mechanisms of H. armigera and H. assulta.     

Cryptic divergence and strong population structure in the colonial invertebrate Pycnoclavella communis (Ascidiacea) inferred from molecular data

We studied sequence variation in the mitochondrial gene cytochrome c oxidase subunit I (COI) for 135 individuals from eight Mediterranean populations of the colonial ascidian Pycnoclavella communis across most of its presently known range of distribution in the Mediterranean. Three haplotypes from Atlantic locations were also included in the study. Phylogenetic, phylogeographic and population genetic analyses were used to unravel the genetic variability within and between populations. The study revealed 32 haplotypes for COI, 29 of them grouped within two Mediterranean lineages of P. communis (mean nucleotide divergence between lineages was 8.55%). Phylogenetic and network analyses suggest the possible existence of cryptic species corresponding to these two lineages. Population genetic analyses were restricted to the five populations belonging to the main genetic lineage, and for these localities we compared the information gleaned from COI sequence data and from eight microsatellite loci. A high genetic divergence between populations was substantiated using both kinds of markers (COI, global Fst=0.343; microsatellite loci, global Fst=0.362). There were high numbers of private haplotypes (COI) and alleles (microsatellites) in the populations studied. Restricted gene flow and inbreeding occur in the present range of distribution of the species. Microsatellite loci showed a strong incidence of failed amplifications, which we attribute to the marked intraspecies variability that hampered the application of these highly specific markers. Our results show important genetic variability at all levels studied, from within populations to between basins, possibly coupled to speciation processes. This variability is attributable to restricted gene flow among populations due to short-distance dispersal of the larvae.     

Genetic basis of sex pheromone blend difference between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae)

The two closely related moth species, Helicoverpa armigera and H. assulta, are sympatric in China. Both species use a mixture of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) as their sex pheromones but in widely different ratios. Hybridization and backcrossing experiments between H. armigera and H. assulta were conducted and sex pheromone compositions of the parent species, their F(1) hybrids and backcrosses were compared to study the genetic basis of the production of their sex pheromone blend composition. Results show that the difference in sex pheromone blend ratios of these Helicoverpa species is mainly controlled by an autosomal locus with two alleles, with the allele from H. armigera being almost completely dominant over that derived from H. assulta.     

Genomic structure of the luciferase gene and phylogenetic analysis in the Hotaria-group fireflies

The luminescent fireflies have species specific flash patterns, being recognized as sexual communication. The luciferase gene is the sole enzyme responsible for bioluminescence. We describe here the complete nucleotide sequence and the exon-intron structure of the luciferase gene of the Hotaria-group fireflies, H. unmunsana, H. papariensis and H. tsushimana. The luciferase gene of the Hotaria-group firefly including the known H. parvula spans 1950 bp and consisted of six introns and seven exons coding for 548 amino acid residues, suggesting highly conserved structure among the Hotaria-group fireflies. Although only one luciferase gene was cloned from H. papariensis, each of the two sequences of the gene was found in H. unmunsana (U1 and Uc) and H. tsushimana (T1 and T2). The amino acid sequence divergence among H. unmunsana, H. papariensis, and H. tsushimana only ranged from zero to three amino acid residues, but H. parvula differed by 10-11 amino acid residues from the other Hotaria-group fireflies, suggesting a divergent relationship of this species. Phylogenetic analysis using the deduced amino acid sequences of the luciferase gene resulted in a monophyletic group in the Hotaria excluding H. parvula, suggesting a close relationship among H. unmunsana, H. papariensis and H. tsushimana. Additionally, we also analyzed the mitochondrial cytochrome oxidase I (COI) gene of the Hotaria-group fireflies. The deduced amino acid sequence of the COI gene of H. unmunsana was identical to that of H. papariensis and H. tsushimana, but different by three positions from H. parvula. In terms of nucleotide sequences of the COI gene, intraspecific sequence divergence was sometimes larger than interspecies level, and phylogenetic analysis placed the three species into monophyletic groups unresolved among them, but excluded H. parvula. In conclusion, our results suggest that H. unmunsana, H. papariensis and H. tsushimana are very closely related or might be an identical species, at least based on the luciferase and COI genes.     

Comparative study on the responses of maxillary sensilla styloconica of cotton bollwormHelicoverpa armigera and Oriental tobacco budwormH. assulta larvae to phytochemicals

Using the electro-physiological technique, the sensory mechanisms of maxillary sensilla styloconica to stimulants and deterrents were explored on two closely related species, the generalist Helicoverpa armigera and the specialist H. assulta. The results showed that: (i) in both species, cells sensitive to sucrose and azadirachtin were mainly in the lateral sensillum styloconicum, and those to inositol were in the medial sensillum styloconicum; (ii) sensitivity of medial sensillum styloconicum in H. assulta to inositol was higher than that in H. armigera; (iii) among 6 tested deterrents, only azadirachtin evoked high impulse discharge from the lateral sensillum styloconicum in both insects; (iv) the deterrents could disturb stimulants evoking impulse discharge from maxillary sensilla styloconica of both species in different degrees: To sucrose evoking impulses on lateral sensillum styloconicum, for H. armigera capsaicin had a strong inhibition and gossypol had a weak inhibition, while for H. assulta tann     

棉铃虫不同寄主种群遗传分化的RAPD分析

应用RAPD技术对生活于辣椒,烟草和番茄3种不同寄主上的棉铃虫（Helicover pa armigera)种群,并以烟草烟青虫（H.assulta)为外群进行了种群遗传分化的研究,研究结果表明：4个处理内个体间最大的遗传距离均小于处理间最小的遗传距离,烟青虫的遗传距离为0．177－0．346,棉铃虫的辣椒种群为0．289－0．404,烟草种群为0．396－0．505,番茄种群为0．329－0．382,采用UPDGA法进行聚类,并构建系统发育树。各处理成聚的遗传距离为：烟青虫是0．334,棉铃虫的辣椒种群是0．372,烟草种群是0．463,番茄种群是0．360。各处理均首先成聚,棉铃虫与烟青虫成聚的遗传距离（0．703）,稍大于棉铃虫各种群的成聚值（0．639）。上述结果表明,棉铃虫不同寄主种群已经存在明显的遗传分化。     

棉铃虫和烟青虫性信息素腺体脂肪醇和乙酸酯转化的比较研究

棉铃虫Helicoverpa armigera和烟青虫H. assulta属于可同域发生的近缘种昆虫,通过产生比例相反的两种性信息素化合物——顺9-十六碳烯醛和顺11-十六碳烯醛维持种间生殖隔离。本研究应用外源不饱和脂肪醇及乙酸酯在棉铃虫和烟青虫性信息素腺体进行在体转化,利用气相色谱法分析转化产物,从酶学角度探讨了上述两近缘种昆虫性信息素腺体组分差异的形成原因。实验结果表明,两种昆虫信息素腺体表皮伯醇氧化酶对外源顺9-十六碳烯醇、顺11-十六碳烯醇和反10-十六碳烯醇无催化专一性,说明末端氧化过程对于醛类性信息素组分特定比例的形成不起作用。棉铃虫性信息素腺体组织具有较高的乙酸酯酶活性,可水解外源乙酸酯,但烟青虫性信息素腺体乙酸酯酶活性很低。这些发现对于进一步了解两种昆虫的生殖隔离机制有重要参考价值。     

Molecular identification of three Ompok species using mitochondrial COI gene

A DNA-based barcode identification system that is applicable to all animal species will provide a simple, universal tool for the identification of fish species. The barcode system is based on sequence diversity in subunit 1 cytochrome c oxidase (COI) gene. Identification and characterization of fish species based on morphological characters are sometimes found to be erroneous and environmentally affected. There are no studies on the genus Ompok in India at molecular level and species identification of the Ompok is usually carried out through morphological features. A total of 106 samples from three species Ompok pabda, O. pabo and O. bimaculatus were collected from eight sampling sites of seven Indian rivers. One hundred and six sequences were generated from COI region of three Ompok species and 21 haplotypes were observed. The sequence analysis of COI gene revealed three genetically distinct Ompok species and exhibited identical phylogenetic resolution among them. The partial COI gene sequence can be used as a diagnostic molecular marker for identification and resolution of taxonomic ambiguity of Ompok species.     

Comparative study of sex pheromone composition and biosynthesis in Helicoverpa armigera, H. assulta and their hybrid

Two Helicoverpa species, H. armigera and H. assulta use (Z)-11-hexadecenal and (Z)-9-hexadecenal as their sex attractant pheromone components but in opposite ratios. Since both female and male interspecific hybrids produced by female H. assulta and male H. armigera have been obtained in our laboratory, we can make a comparative study of sex pheromone composition and biosynthesis in the two species and their hybrid. With GC and GC-MS analyses using single gland extracts, the ratio of (Z)-9-hexadecenal to (Z)-11-hexadecenal was determined as 2.1:100 in H. armigera, and 1739:100 in H. assulta. The hybrid has a ratio of 4.0: 100, which is closer to that of H. armigera, but significantly different from H. armigera. We investigated pheromone biosynthesis with labeling experiments, using various fatty acid precursors in H. armigera, H. assulta and the hybrid. In H. armigera, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation; (Z)-9-hexadecenal results from delta11 desaturation of stearic acid, followed by one cycle of chain shortening, reduction and terminal oxidation. delta11 desaturase is the unique desaturase for the production of the two pheromone components. In our Chinese strain of H. assulta, palmitic acid is used as the substrate to form both the major pheromone component, (Z)-9-hexadecenal and the minor one, (Z)-11-hexadecenal. Our data suggest that delta9 desaturase is the major desaturase, and delta11 desaturase is responsible for the minor component in H. assulta, which is consistent with previous work. However, the weak chain shortening acting on (Z)-9 and (Z)-11-octadecenoic acid, which is present in the pheromone glands, does occur in this species to produce (Z)-7 and (Z)-9-hexadecenoic acid. In the hybrid, the major pheromone component, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation. The direct fatty acid precursor of the minor component, (Z)-9-hexadecenoic acid is mainly produced by delta9 desaturation of palmitic acid, but also by delta11 desaturation of stearic acid and one cycle of chain shortening. The greater relative amounts of (Z)-9-hexadecenal in the hybrid are due to the fact that both palmitic and stearic acids are used as substrates, whereas only stearic acid is used as substrate in H. armigera. The evolutionary relationships between the desaturases in several Helicoverpa species are also discussed in this paper.     

烟夜蛾性肽受体基因的克隆与表达模式分析

【目的】克隆烟夜蛾Helicoverpa assulta (Guenée)性肽受体基因并分析其表达模式, 为深入研究性肽与交配后反应的关系奠定基础。【方法】采用RT-PCR方法, 从烟夜蛾雌蛾性信息素腺体中得到性肽受体基因cDNA全序列。利用荧光定量PCR方法, 分析该基因的表达模式。【结果】序列分析结果显示, 烟夜蛾性肽受体基因cDNA全长2 048 bp, 命名为HassSPR（GenBank登录号: AFH53182.1）。该基因的开放阅读框长1 275 bp, 编码424个氨基酸残基, 序列中含有7个跨膜域结构, 预测分子量和等电点分别为48.6 kDa和9.25。序列比对分析表明, HassSPR与近缘种棉铃虫H. armigera和其他蛾类性肽受体的氨基酸序列一致性分别达98.35%和超过84%, 与已经报道的其他昆虫的性肽受体的氨基酸序列一致性也在64%以上。不同组织表达分析表明, HassSPR在测定的1日龄雌蛾不同组织中均有表达, 以在脑中的表达量最高。时序表达分析表明, 在羽化前1 天至羽化后6日龄雌蛾的信息素腺体中均有表达, 以3日龄表达量最高。雌蛾交配后, HassSPR在性信息素腺体和脑中的表达量显著上调, 而在交配囊和卵巢中的表达量显著下调。【结论】从烟夜蛾雌蛾性信息素腺体中克隆得到性肽受体基因HassSPR, 其表达模式提示该基因的表达水平与雌蛾的生殖生理和生殖行为有关。     

Nucleotide sequence variation in mitochondrial COI gene among 147 silkworm (Bombyx mori) strains from Japanese, Chinese, European and moltinism classes

We characterized the nucleotide sequences of PCR-amplified mitochondrial COI fragments of 147 silkworm (Bombyx mori) strains that have been maintained in the National Institute of Agrobiological Sciences. Coding sequences (714 bp) of the 147 COI fragments were classified into eight haplotypes based on nucleotide differences at eight segregating sites. No length variation was identified in this region. The 5'-noncoding region showed different features, wherein changes in the number of Ts in the T-stretch, together with two base substitutions, were observed. As a result, the 147 COI noncoding sequences were classified into six haplotypes. Combining the coding and noncoding regions, we identified 14 haplotypes. One of the 14 haplotypes, Hap1A was exclusively abundant in the Japanese native strain class, while this haplotype was less frequent in the other three native strain classes. This finding suggests that the Japanese strain class underwent significant genetic differentiation from the Chinese, European, and moltinism classes, when the each class is regarded as a population. Comparison of the nucleotide sequences to those of B. mandarina (which inhabits Japan) revealed changes that are significantly larger than those within either B. mori or B. mandarina. Furthermore, we detected no common haplotypes between them, which suggests the concept of suppressed gene flow between the two species.     

支持向量机和邻接法在夜蛾科昆虫条码研究中的应用

【背景】鳞翅目夜蛾科昆虫种类繁多,目前已经超过3．5万种,绝大多数是农林生产的主要害虫。由于多数近缘属种形态相似,难以鉴定,给农林害虫的防治工作带了很大的困难。DNA条形码技术是一种快速、准确鉴定物种的方法。支持向量机作为一种新的机器学习方法,自1995年被提出以来已经在数据分类和高维模式识别等领域取得不错的效果。【方法】将北京妙峰山采集的58种夜蛾101个样品的COI序列分成3套数据集,分别通过邻接法和支持向量机对其进行验证。【结果】通过对DNA条形码物种鉴定结果的验证表明,邻接法优于支持向量机。但DNA条形码在鉴定夜蛾科的一些近缘种上,效果不佳,如棉铃虫和烟青虫。【结论与意义】DNA条形码作为一种新兴的物种鉴定方法,在分类学上具有很高的应用价值。通过邻接法和支持向量机的比较,虽然支持向量机的成功率低于邻接法,但是其在DNA条形码中的应用是对数据问询方式的一种探索。     

Molecular markers to discriminate among four pest species of Helicoverpa (Lepidoptera: Noctuidae)

The four significant pest species in the Helicoverpa genus (H. armigera, H. assulta, H. punctigera and H. zea) are morphologically similar and can only be reliably distinguished through dissection of adult genitalia. Two partial regions of the mitochondrial DNA (mtDNA), the cytochrome oxidase subunit I (COI) and the cytochrome b (Cyt b) genes were amplified by PCR and digested with restriction endonucleases. The restriction patterns, generated by the endonucleases BstZ17I and HphI, demonstrated reliable differentiation of the four Helicoverpa pest species. This technique is fast, reliable and effective at distinguishing specimens irrespective of their life stages and offers support to conventional taxonomic differentiation based on morphological characters.     

Metabolic detoxification of capsaicin by UDP-glycosyltransferase in three Helicoverpa species

Capsaicin β-glucoside was isolated from the feces of Helicoverpa armigera, Helicoverpa assulta, and Helicoverpa zea that fed on capsaicin-supplemented artificial diet. The chemical structure was identified by NMR spectroscopic analysis as well as by enzymatic hydrolysis. The excretion rates of the glucoside were different among the three species; those in the two generalists, H. armigera and H. zea, were higher than in a specialist, H. assulta. UDP-glycosyltransferases (UGT) enzyme activities measured from the whole larval homogenate of the three species with capsaicin and UDP-glucose as substrates were also higher in the two generalists. Compared among five different larval tissues (labial glands, testes from male larvae, midgut, the Malpighian tubules (MT), and fat body) from the three species, the formation of the capsaicin glucoside by one or more UGT is high in the fat body of all the three species as expected, as well as in H. assulta MT. Optimization of the enzyme assay method is also described in detail. Although the lower excretion rate of the unaltered capsaicin in H. assulta indicates higher metabolic capacity toward capsacin than in the other two generalists, the glucosylation per se seems to be insufficient to explain the decrease in capsaicin in the specialist, suggesting that H. assulta might have another important mechanism to deal with capsaicin more specifically.     

DNA barcoding of the endemic New Zealand leafroller moth genera, Ctenopseustis and Planotortrix

Molecular techniques such as DNA barcoding have become popular in assisting species identification especially for cryptic species complexes. We have analysed data from a 468-bp region of the mitochondrial cytochrome oxidase subunit I (COI) gene from 200 specimens of 12 species of endemic New Zealand leafroller moths (Tortricidae) from the genera Planotortrix and Ctenopseustis to assess whether the DNA barcoding region can distinguish these species. Among the 200 sequences analysed, 72 haplotypes were recovered, with each genus forming a separate major clade. Maximum likelihood phylogenetic methods were used to test whether species fell into reciprocally monophyletic clades. The optimal phylogeny showed that four species within the genus Ctenopseustis (C. obliquana, C. herana, C. filicis and C. fraterna) and three within Planotortrix (P. octo, P. excessana and P. avicenniae) are polyphyletic. Shimodaira-Hasegawa tests rejected a null hypothesis of monophyly for the species C. obliquana, C. herana, P. octo and P. excessana. Comparisons of within and between species levels of sequence divergence for the same set of seven species showed cases where maximum levels of within-species divergence were greater than some levels of between-species divergence. DNA barcoding using this region of the COI gene is able to distinguish the two genera and some species within each genus; however, many species cannot be identified using this method. Finally, we discuss the possible reasons for this polyphyly, including incomplete lineage sorting, introgression, horizontal gene transfer and incorrect taxonomy.