A DNA ‘Barcode Blitz’: Rapid Digitization and Sequencing of a Natural History Collection

DNA barcoding protocols require the linkage of each sequence record to a voucher specimen that has, whenever possible, been authoritatively identified. Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera) species in the Australian National Insect Collection. Protocols were developed that enabled tissue samples, specimen data, and images to be assembled rapidly. Using these methods, a five-person team processed 41,650 specimens representing 12,699 species in 14 weeks. Subsequent molecular analysis took about six months, reflecting the need for multiple rounds of PCR as sequence recovery was impacted by age, body size, and collection protocols. Despite these variables and the fact that specimens averaged 30.4 years old, barcode records were obtained from 86% of the species. In fact, one or more barcode compliant sequences (>487 bp) were recovered from virtually all species represented by five or more individuals, even when the youngest was 50 years old. By assembling specimen images, distributional data, and DNA barcode sequences on a web-accessible informatics platform, this study has greatly advanced accessibility to information on thousands of species. Moreover, much of the specimen data became publically accessible within days of its acquisition, while most sequence results saw release within three months. As such, this study reveals the speed with which DNA barcode workflows can mobilize biodiversity data, often providing the first web-accessible information for a species. These results further suggest that existing collections can enable the rapid development of a comprehensive DNA barcode library for the most diverse compartment of terrestrial biodiversity – insects.     

Feasibility of using an alternative larval host and host plants to establish Cotesia flavipes (Hymenoptera: Braconidae) in the temperate Louisiana sugarcane ecosystem

Attempts to establish Cotesia flavipes Cameron (Hymenoptera: Braconidae) in Louisiana sugarcane fields to control the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae) have been unsuccessful. Experiments were conducted to investigate the feasibility of using an alternative larval host and host plants to overcome barriers preventing establishment. In addition, we evaluated C. flavipes' ability to search for D. saccharalis in sugarcane without above-ground internodes. Diatraea evanescens Dyar (Lepidoptera: Crambidae) was investigated as an alternative host for C. flavipes. Cotesia flavipes was reared for five generations on D. evanescens without any indication of diminishing fitness as measured by days to parasite pupation and average cocoon mass weight. However, there was a significant reduction in percent parasitism, cocoon mass weight, and percent emergence when C. flavipes parasitized D. evanescens as compared with D. saccharalis, resulting in a 75% reduction in the gross reproductive rate (R(0)). Greenhouse studies indicated little difference in parasitism of D. saccharalis on the weed hosts johnsongrass, Sorghum halepense (L.), and vaseygrass, Paspalum urvillei Steud. However, when planted as refuge plots, we found it difficult to establish infestations of D. saccharalis in either of these hosts, or in two energy sugarcanes. After 3 yr of infesting host plants and releasing parasitoids only one parasitized D. saccharalis larvae was recovered within the johnsongrass refuge. Diatraea evanescens readily established in vaseygrass; however, these larvae appear inaccessible to C. flavipes. In contrast, parasitism of D. saccharalis by C. flavipes infesting young sugarcane was 30%.     

Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens

Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants.     

Parasitism Behavior of Tetrastichus howardi (Hymenoptera: Eulophidae) on Larvae and Pupae of Sugarcane Borers

Journal of Insect Behavior - Control of the sugarcane borers, Diatraea saccharalis and Diatraea impersonatella (= D. flavipennella) (Lepidoptera: Crambidae), in Brazil, is based on mass release of...     

DNA barcodes for 1/1000 of the animal kingdom

This study reports DNA barcodes for more than 1300 Lepidoptera species from the eastern half of North America, establishing that 99.3 per cent of these species possess diagnostic barcode sequences. Intraspecific divergences averaged just 0.43 per cent among this assemblage, but most values were lower. The mean was elevated by deep barcode divergences (greater than 2%) in 5.1 per cent of the species, often involving the sympatric occurrence of two barcode clusters. A few of these cases have been analysed in detail, revealing species overlooked by the current taxonomic system. This study also provided a large-scale test of the extent of regional divergence in barcode sequences, indicating that geographical differentiation in the Lepidoptera of eastern North America is small, even when comparisons involve populations as much as 2800 km apart. The present results affirm that a highly effective system for the identification of Lepidoptera in this region can be built with few records per species because of the limited intra-specific variation. As most terrestrial and marine taxa are likely to possess a similar pattern of population structure, an effective DNA-based identification system can be developed with modest effort.     

Buffalo species identification and delineation using genetic barcoding markers

Enrichment of barcode databases with mitochondrial cytochrome c oxidase subunit I (COI) barcode sequences in different animal taxa has become important for identification of animal source in food samples to prevent commercial fraud. In this study, COI barcode sequence in seventy one river buffalo samples were determined, analyzed and deposited in Genbank barcode database and barcode of life database (BOLD) to contribute for construction of public reference library for COI barcode sequence in river buffalo. Moreover COI barcode sequence was used to identify the closely related buffalo groups: river buffalo, swamp buffalo, lowland anoa and African buffalo. Results indicated the success of the COI barcode in the identification of each of the tested groups. Whereas a suggested sequence of other mitochondrial segment representing two successive transfer RNA (tRNA) genes; tRNA-Threonine (MT-TT) and tRNA-Proline (MT-TP) was failed to be used as a barcode marker for differentiation between the tested buffalo groups.     

Pyrosequencing for mini-barcoding of fresh and old museum specimens

DNA barcoding is an effective approach for species identification and for discovery of new and/or cryptic species. Sanger sequencing technology is the method of choice for obtaining standard 650 bp cytochrome c oxidase subunit I (COI) barcodes. However, DNA degradation/fragmentation makes it difficult to obtain a full-length barcode from old specimens. Mini-barcodes of 130 bp from the standard barcode region have been shown to be effective for accurate identification in many animal groups and may be readily obtained from museum samples. Here we demonstrate the application of an alternative sequencing technology, the four-enzymes single-specimen pyrosequencing, in rapid, cost-effective mini-barcode analysis. We were able to generate sequences of up to 100 bp from mini-barcode fragments of COI in 135 fresh and 50 old Lepidoptera specimens (ranging from 53-97 year-old). The sequences obtained using pyrosequencing were of high quality and we were able to robustly match all the tested pyro-sequenced samples to their respective Sanger-sequenced standard barcode sequences, where available. Simplicity of the protocol and instrumentation coupled with higher speed and lower cost per sequence than Sanger sequencing makes this approach potentially useful in efforts to link standard barcode sequences from unidentified specimens to known museum specimens with only short DNA fragments.     

DNA barcode goes two-dimensions: DNA QR code web server

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.     

Evaluation of lectin-expressing transgenic sugarcane against stalkborers (Lepidoptera: Pyralidae): effects on life history parameters

The impact of snowdrop lectin (Galanthus nivalis agglutinin, GNA) expressed in transgenic sugarcane on life history parameters of Mexican rice borer [Eoreuma loftini (Dyar)] and sugarcane borer [Diatraea saccharalis (F.)] (both Lepidoptera: Pyralidae) was evaluated. In the laboratory, lyophilized sugarcane leaf sheath tissue was incorporated in a meridic diet resulting in a GNA concentration of 0.47% of total protein, and used for insect bioassays over two successive generations. Deleterious effects of GNA were not observed on survival, weight, and developmental periods of larvae and pupae, nor on adult fecundity and egg viability of D. saccharalis. Moreover, in the first generation, addition of transgenic sugarcane tissue to the diet enhanced larval growth in D. saccharalis resulting in higher larval and pupal weight compared with diet with nontransgenic sugarcane, but this effect was not observed in the second generation. In contrast, larval survival, percent adult emergence, and female fecundity of E. loftini were significantly reduced when fed transgenic sugarcane diet compared with nontransgenic sugarcane diet. In addition, a substantial reduction of female pupal weight of E. loftini was observed in the second generation. For both species, the only consistent effect of GNA in both generations was a reduction in adult female longevity. Life table parameters showed that GNA at the level found in the transgenic diet negatively affected development and reproduction of E. loftini, whereas it had a nil to positive effect on development and reproduction of D. saccharalis.     

Barcoding Beetles: A Regional Survey of 1872 Species Reveals High Identification Success and Unusually Deep Interspecific Divergences

With 400 K described species, beetles (Insecta: Coleoptera) represent the most diverse order in the animal kingdom. Although the study of their diversity currently represents a major challenge, DNA barcodes may provide a functional, standardized tool for their identification. To evaluate this possibility, we performed the first comprehensive test of the effectiveness of DNA barcodes as a tool for beetle identification by sequencing the COI barcode region from 1872 North European species. We examined intraspecific divergences, identification success and the effects of sample size on variation observed within and between species. A high proportion (98.3%) of these species possessed distinctive barcode sequence arrays. Moreover, the sequence divergences between nearest neighbor species were considerably higher than those reported for the only other insect order, Lepidoptera, which has seen intensive analysis (11.99% vs up to 5.80% mean NN divergence). Although maximum intraspecific divergence increased and average divergence between nearest neighbors decreased with increasing sampling effort, these trends rarely hampered identification by DNA barcodes due to deep sequence divergences between most species. The Barcode Index Number system in BOLD coincided strongly with known species boundaries with perfect matches between species and BINs in 92.1% of all cases. In addition, DNA barcode analysis revealed the likely occurrence of about 20 overlooked species. The current results indicate that DNA barcodes distinguish species of beetles remarkably well, establishing their potential to provide an effective identification tool for this order and to accelerate the discovery of new beetle species.     

Purification and characterization of three beta-glycosidases from midgut of the sugar cane borer,Diatraea saccharalis

Three beta-glycosidases, named betaGly1, betaGly2 and betaGly3, were isolated from midgut tissues of the sugar cane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae). The three enzymes have similar Mr (58,000; 61,000; 61,000), pI (7.5, 7.4, and 7.4) and optimum pH (6.7, 6.3, and 7.2) and were resolved by hydrophobic chromatography. The beta-glycosidases prefer beta-glucosides to beta-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose beta-1,3 linkages better than beta-1, 4- or beta-1,6 linkages. betaGly1 and 2 were completely purified, whereas betaGly3 was isolated with a contaminant peptide that has no activity upon beta-glycosides.By using competing substrates, it was shown that betaGly 1 and 3 have one active site, whereas betaGly2 has two, one hydrolyzing natural and the other synthetic substrates. betaGly2 is the only D. saccharalis beta-glycosidase that can efficiently hydrolyse prunasin, the glycoside remaining after glucose removal from the plant glycoside amygdalin and that liberates the cyanogenic mandelonitrile. As shown elsewhere, betaGly2 activity is reduced when D. saccharalis is reared in amygdalin containing diets. From the results, we propose that the physiological role of betaGly 1 and 3 is the digestion of oligo- and disaccharides derived from hemicelluloses and of betaGly2 is glycolipid hydrolysis.Free energy relationships showed that D. saccharalis betaGly3 and Tenebrio molitor (Coleoptera) betaGly1 have active sites that bind similarly the transition states formed with different substrates. The same is also true for the active sites of D. saccharalis betaGly1 and T. molitor betaGly2. This suggests that active sites of similar enzymes are probably homologous, displaying nearly identical bonds between active site amino acids and substrate moieties.     

Histochemical and ultrastructural evidence of lipid secretion by the silk gland of the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae)

The silk gland in Lepidoptera larvae is responsible for the silk production used for shelter or cocoon construction. The secretion of fibroin and sericin by the different silk gland regions are well established. There are few attempts to detect lipid components in the insect silk secretion, although the presence of such element may contribute to the resistance of the shelter to wet environment. This study characterizes the glandular region and detects the presence of lipid components in the secretion of the silk gland of Diatraea saccharalis(Fabricius). The silk gland was submitted to histochemical procedure for lipid detection or conventionally prepared for ultrastructural analyses. Lipid droplets were histochemically detected in both the apical cytoplasm of cell of the anterior region and in the lumen among the microvilli. Ultrastructural analyses of the anterior region showed lipid material, visualized as myelin-like structures within the vesicular Golgi complex and in the apical secretory globules, mixed up with the sericin; similar material was observed into the lumen, adjacent to the microvilli. Lipids were not detected in the cells neither in the lumen of the posterior region. Our results suggest that the silk produced by D. saccharalis has a minor lipid content that is secreted by the anterior region together with the sericin.     

小蔗螟Cytb基因序列的分析研究(鳞翅目:螟蛾总科:草螟亚科)

扩增并测定了小蔗螟线粒体细胞色素b(Cyt b)基因,其序列全长1146 bp,利用分子生物学软件分析了包括小蔗螟在内的4目(鳞翅目、双翅目、鞘翅目、缨尾目)共19条Cyt b基因序列.分析结果显示:鳞翅目昆虫AT含量明显低于其它昆虫,其中小蔗螟AT含量为75.1%;AT含量与A或T碱基使用的偏倚没有明显的相关性;碱基替换主要发生在密码子第三位,并且颠换率远大于转换率;密码子使用频率与基因碱基组成AT偏倚有相关性;相比于核苷酸数据,氨基酸所显示的遗传距离更为准确;使用Cyt b基因构建的系统进化树支持昆虫的单源进化学说.     

菌物DNA条形码技术原理与操作

DNA条形码技术是通过对1个较短目的基因的DNA序列进行分析从而进行物种鉴定的方法,它通过对1个或多个相关基因进行大范围的扫描,进而鉴定未知物种或者发现新种。当传统的分类学受到阻碍时,这种技术可以发挥其优势。相对于其他生物,菌物的生活史独特而复杂,这就使得对其进行的形态学鉴定要受到菌物自身生长发育时期的限制。国内外科学家对寻找适合于大多数菌物的标准DNA条码进行过探索,但还没有找到满足全部特征的基因片段。文中对DNA条形码技术的概念、原理依据、操作步骤和优缺点方面进行了介绍,并对DNA条形码技术在我国菌物研究方面的应用前景进行了展望。     

Sugarcane borer (Lepidoptera: Crambidae) management threshold assessment on four sugarcane cultivars

This research assesses the potential for using different economic injury thresholds in management of a key insect pest on susceptible and resistant commercially produced cultivars of sugarcane (Saccharum spp. hybrids). In a 2-yr sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), study involving four commercially produced sugarcane cultivars and four insecticide treatment thresholds, 'LCP 85-384' and 'HoCP 91-555' were the most susceptible based on percentage of bored internodes compared with the more resistant 'HoCP 85-845' and 'CP 70-321'. In 2001, the 10% infested stalks threshold was not as effective as the 5% early season-10% late season and 5% full season for HoCP 91-555. Based on D. saccharalis injury under natural infestation conditions, susceptible cultivars seem to require a lower infestation threshold than the more resistant cultivars to achieve adequate injury reduction. Among yield components, only the theoretical recoverable sugar per stalk was significantly increased by applying insecticides. With the resistant HoCP 85-845, differences were not detected for percentage of bored internodes among treated versus untreated management regimes. The resistant HoCP 85-845 had higher levels of fiber in our study; however, no clear pattern on resistance mechanisms was established, because the resistant cultivar CP 70-321 had comparatively low levels of fiber. The development of cultivar-specific thresholds is expected to lower the amount of insecticide used for D. saccharalis management in the sugarcane industry, reduce selection pressure, and delay the development of insecticide resistance.     

Comparative susceptibility of two different hosts to genotypic variants of the Anticarsia gemmatalis nuclear polyhedrosis virus

The susceptibility of third instar larvae of Anticarsia gemmatalis (Lepidoptera: Noctuidae) and Diatraea saccharalis (Lepidoptera: Pyralidae) to ten distinct plaque purified genotypic variants of a selected isolate of the Anticarsia gemmatalis multiple-embedded nuclear polyhedrosis virus (AgMNPV), was compared. Despite the fact that this isolate, AgMNPV-Ds20, represents a wild strain of the AgMNPV selected for higher virulence to D. saccharalis, an alternate host, most of the variants are much more virulent to the original host Anticarsia than to Diatraea. Bioassays have shown an over one hundred-fold variation in LD₅₀ values ranging from 1700 polyhedron inclusion bodies (PIBs) to more than 200 000 PIBs/larva. The PIB production in infected larvae increased with the pathogenicity of the variant to the host, showing an average ten-fold reduction in Diatraea when compared to Anticarsia for the same variant. The virus particle yield ranged from 6×10⁷ to more than 10⁹ PIBs/g of infected larvae in Diatraea and from 8×10⁸ to more than 10¹⁰ PIBs/g of infected Anticarsia larvae. The data show a clear difference of the pathogenicity of the genotypic variants of AgMNPV in vivo both between the original and alternate host and between the individual variants for the same host. These differences found in vivo indicate that monitoring of shifts in variant frequency of wild and laboratory-propagated viral isolates in these highly heterogeneous populations would help ensure the efficacy of biological control programs.     

中国蜘蛛抱蛋属植物DNA条形码研究

利用植物DNA条形码候选序列mat K、psb A-trn H、psb K-psb I和rbc L对蜘蛛抱蛋属(Aspidistra)植物的19种104批样品进行扩增和测序,并采用相似性搜索算法(BLAST)对各序列的鉴定效率进行评价,得出蜘蛛抱蛋属物种鉴定的最佳序列。结果显示,psb K-psb I的物种鉴定成功率为88.7%,在单一序列中成功率最高。通过多序列组合鉴定效率的比较,发现组合序列的鉴定成功率明显高于单一序列,其中mat K+(psb K-psb I)组合的鉴定成功率高达100%,基于该序列组合构建蜘蛛抱蛋属植物的系统发育树,结果显示同一物种的样品聚集度较好,多表现为单系。研究结果表明mat K+(psb K-psb I)序列组合可作为蜘蛛抱蛋植物种鉴定的最佳条形码序列。     

Morphometry of the midgut epithelium of Diatraea saccharalis Fabricius, 1794 (Lepidoptera) parasitized by Cotesia flavipes Cameron, 1891 (Hymenoptera)

The morphometric study of the midgut in Diatraea saccharalis (Lepidoptera) larvae parasitized by the Cotesia flavipes (Hymenoptera) showed that there was significant increase in the columnar, goblet and regenerative cells and their nuclei; the midgut lumen diameter and the epithelial height were also increased in the parasitized larvae. The multivariate analysis showed that parasitism affected the columnar cell only in the posterior region, and the goblet cells along the midgut length (anterior and posterior regions).     

Testing DNA Barcode Performance in 1000 Species of European Lepidoptera: Large Geographic Distances Have Small Genetic Impacts

This study examines the performance of DNA barcodes (mt cytochrome c oxidase 1 gene) in the identification of 1004 species of Lepidoptera shared by two localities (Finland, Austria) that are 1600 km apart. Maximum intraspecific distances for the pooled data were less than 2% for 880 species (87.6%), while deeper divergence was detected in 124 species. Despite such variation, the overall DNA barcode library possessed diagnostic COI sequences for 98.8% of the taxa. Because a reference library based on Finnish specimens was highly effective in identifying specimens from Austria, we conclude that barcode libraries based on regional sampling can often be effective for a much larger area. Moreover, dispersal ability (poor, good) and distribution patterns (disjunct, fragmented, continuous, migratory) had little impact on levels of intraspecific geographic divergence. Furthermore, the present study revealed that, despite the intensity of past taxonomic work on European Lepidoptera, nearly 20% of the species shared by Austria and Finland require further work to clarify their status. Particularly discordant BIN (Barcode Index Number) cases should be checked to ascertain possible explanatory factors such as incorrect taxonomy, hybridization, introgression, and Wolbachia infections.