Identification of phlebotomine sand flies (Diptera: Psychodidae) from leishmaniasis endemic areas in southeastern Mexico using DNA barcoding

Leishmaniasis, a vector‐borne disease transmitted to humans through the bite of phlebotomine sand flies, is of public health significance in southeastern Mexico. Active and continuous monitoring of vectors is an important aspect of disease control for the prediction of potential outbreaks. Thus, the correct identification of vectors is paramount in this regard. In this study, we employed DNA barcoding as a tool for identifying phlebotomine sand flies collected in localized cutaneous leishmaniasis endemic areas of Quintana Roo, Mexico. Specimens were collected using CDC light and Shannon traps as part of the Mexican Ministry of Health surveillance program. DNA extraction was carried out using a nondestructive protocol, and morphological identification based on taxonomic keys was conducted on slide‐mounted specimens. Molecular taxonomic resolution using the 658‐bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was 100% congruent with the morphological identification. Seven species were identified: Lutzomyia cruciata (Coquillett 1907), Lutzomyia longipalpis (Lutz & Neiva 1912), Psathyromyia shannoni (Dyar 1929), Dampfomyia deleoni (Fairchild & Hertig 1947), Dampfomyia beltrani/steatopyga (Vargas & Díaz‐Nájera 1951), Bichromomyia olmeca olmeca (Vargas & Díaz‐Nájera, 1959), and Brumptomyia mesai (Sherlock 1962). Mean intraspecific divergence ranged from 0.12% to 1.22%, while interspecific distances ranged from 11.59% to 19.29%. Neighbor‐joining (NJ) analysis using the Kimura 2‐parameter model also showed specimens of the same species to be clustered together. The study provides the first cox1 sequences for three species of sand flies and indicates the utility of DNA barcoding for phlebotomine sand flies species identification in southeastern Mexico.     

Radiation of the Oriental phlebotomine sand flies (Diptera: Psychodidae)

Abstract The historical biogeography of phlebotomine sand fly taxa Hertigia, Warileya, Phlebotomus (Idiophlebotomus), P. (Spelaeophlebotomus), P. (Anaphlebotomus), and P. (Euphlebotomus) and the Phlebotomus (Euphlebotomus) argentipes species complex was investigated using phylogenetic inference from comparative genital morphology, distribution of ancestral taxa, fossil evidence and geological age. Idiophlebotomus and Euphlebotomus occur in the Oriental region with one species from northeast Australia, whereas Anaphlebotomus occurs both in the Afro‐tropical and Oriental regions. These disjunct distribution patterns across the Oriental region and the present day distribution are likely to be vicariance due to break of Gondwanaland. Fossil records, extant taxa distribution, phylogenetic analysis of the Old World Phlebotominae and paleogeography suggest that ancestors of Idiophlebotomus and Euphlebotomus originated apparently in the Cimmerian continent of northern margin of Gondwanaland in the early Permian (290 million years ago, MYA) and subsequently radiated in the Mesozoic by tectonic vicariance. The Phlebotomus argentipes species complex occurs in the South and South‐east Asian countries and transmits the protozoan parasite Leishmania donovani that causes visceral leishmaniasis (Kala‐azar) in India, Bangladesh and Nepal. The phylogeography of P. argentipes was caused through vicariance followed by dispersal events from 5O MYA (the Eocene) until the Recent era.     

Richness and diversity of sand flies (Diptera,Psychodidae) in an Atlantic rainforest reserve in southeastern Brazil

Our objective was to study and evaluate the richness and diversity of Phlebotominae fauna in the Duas Bocas Biological Reserve (DBBR) in the state of Espírito Santo, in southeastern Brazil. Sand fly collections were carried out during four consecutive nights each month between August 2007 and July 2008 at DBBR by using CDC automatic light traps and an illuminated Shannon trap. Specific richness (S) and Shannon diversity index (H) was calculated for each trap. We collected 18,868 sand flies belonging to 29 species and 13 genera. Nyssomyia yuilli yuilli was the most abundant species followed by Psychodopygus ayrozai, Ps. hirsutus, Psathyromyia pascalei, and Ps. matosi. We recorded Brumptomyia cardosoi, Br. troglodytes, and Ps. geniculatus for the first time in the state of Espírito Santo. We discuss the differences in diversity and richness of the sand flies in both traps and in relation to other Brazilian localities and biomes. We also discuss the possibility of wild transmission of Leishmania in the DBBR and the influence of the sand fly species in leishmaniasis transmission to the adjacent areas of the reserve.     

Comparative efficacy of three suction traps for collecting phlebotomine sand flies (Diptera: Psychodidae) in open habitats

The efficacy of three suction traps for trapping phlebotomine sand flies (Diptera: Psychodidae) was compared. Traps were baited with Co₂ and used without any light source. CO₂‐baited CDC traps were evaluated either in their standard downdraft orientation or inverted (iCDC traps). Mosquito Magnet‐X (MMX) counterflow geometry traps were tested in the updraft orientation only. Both updraft traps (iCDC and MMX) were deployed with their opening ～10 cm from the ground while the opening of the downdraft (CDC) trap was ～40 cm above ground. Comparisons were conducted in two arid locations where different sand fly species prevail. In the Jordan Valley, 3,367 sand flies were caught, 2,370 of which were females. The predominant species was Phlebotomus (Phlebotomus) papatasi, Scopoli 1786 (>99%). The updraft‐type traps iCDC and MMX caught an average of 118 and 67.1 sand flies per trap night, respectively. The CDC trap caught 32.9 sand flies on average per night, significantly less than the iCDC traps. In the Judean desert, traps were arranged in a 3×3 Latin square design. A total of 565 sand flies were caught, 345 of which were females. The predominant species was P. (Paraphlebotomus) sergenti Parrot 1917 (87%). The updraft traps iCDC and MMX caught an average of 25.6 and 17.9 sand flies per trap per night, respectively. The CDC trap caught 7.8 sand flies on average per night, significantly less than the iCDC traps. The female to male ratio was 1.7 on average for all trap types. In conclusion, updraft traps deployed with their opening close to the ground are clearly more effective for trapping sand flies than downdraft CDC traps in open habitats.     

A summary of the evidence for the change in European distribution of phlebotomine sand flies (Diptera: Psychodidae) of public health importance

The phlebotomine sand flies (Diptera: Psychodidae, Phlebotominae) are vectors of several infectious pathogens. The presence of a sand fly vector is considered to be a risk factor for the emergence of leishmaniasis in temperate Europe. Hence, the occurrence of phlebotomine sand flies and any changes in their distribution is important in determining the potential change in distribution of leishmaniasis in Europe. Therefore, published evidence for a changing distribution of the important phlebotomine sand fly vectors of leishmaniasis and phlebovirus infection in Europe is reviewed. This paper presents evidence of an increasing risk of establishment by sand fly species, especially for the Atlantic Coast and inland parts of Germany, Switzerland, and Austria. In addition to detection in potentially appropriate areas, the findings show areas of potential future establishment of the species. The most important and urgent necessity within the community of entomologists working on phlebotomines is the need to record the extremes of distribution of each species and obtain data on their regional presence/absence along with increased sharing of the data throughout European projects.     

Catalogue of the type material of Phlebotominae (Diptera,Psychodidae) deposited in the Instituto Evandro Chagas,Brazil

The available type material of Phlebotominae (Diptera, Psychodidae) deposited in the “Coleção de Flebotomíneos” of the Instituto Evandro Chagas (ColFleb IEC) is now presented in an annotated catalogue comprising a total of 121 type specimens belonging to 12 species as follow: Nyssomyia richardwardi (2 female paratypes), Nyssomyia shawi (9 male and 25 female paratypes), Nyssomyia umbratilis (female holotype and 1 female paratype), Nyssomyia yuilli yuilli (1 male and 1 female paratypes), Pintomyia gruta (1 male and 2 female paratypes), Psychodopygus lainsoni (2 male syntypes), Psychodopygus leonidasdeanei (male holotype, female “allotype” and 45 female paratypes), Psychodopygus llanosmartinsi (2 female paratypes), Psychodopygus wellcomei (1 male and 4 female “syntypes”), Trichophoromyia readyi (male holotype, female “allotype” and 1 male paratype), Trichophoromyia adelsonsouzai (male holotype, 13 male 5 female paratypes), and Trichophoromyia brachipyga (1 male paratype).     

Isolation and characterization of microsatellite loci in the sand fly Phlebotomus papatasi (Diptera: Psychodidae)

Phlebotomus papatasi is a proven vector of Leishmania major which is one of the causative agents of cutaneous leishmaniasis in the Old World. Although it has a wide geographical range, its population structure is not yet well understood. In an effort to better understand the population dynamics of this vector, we developed a panel of di‐ and trinucleotide microsatellite markers, using a magnetic bead hybridization enrichment protocol. These microsatellite loci showed three to seven alleles with an expected heterozygosity range between 0.702 and 0.876. The level of polymorphisms found in this study suggests that these microsatellite loci can be used for population analysis of P. papatasi.     

Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae: Phlebotominae)

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium–potassium iodide (Os–KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis.     

Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.     

Species composition and nocturnal activity of phlebotomine sand flies (Diptera: Psychodidae) inhabiting a limestone cave in Thailand

We investigated the nocturnal activity of cave‐dwelling sand flies at different time intervals and determined their species composition and seasonal variation. Sand flies were captured on one night each month using CDC light traps from 18:00–06:00 with the collecting bag being changed every two h between February, 2010 and January, 2011. A total of 18,709 individuals, including 10,740 males and 7,969 females, was collected. The overall ratio between male and female specimens was 1:0.74. The collected specimens included 14 species from four genera, Chinius, Idiophlebotomus, Phlebotomus, and Sergentomyia. Sergentomyia phadangensis was the most abundant species (comprising 31.9% of the collected individuals), followed by Se. anodontis (22.8%) and Ph. mascomai (18.2%). The highest number of specimens was collected in July (15.6%), followed by May (15.5%) with the peak of collection recorded at the time interval of 00:01–02:00, followed by 22:01–00:00. However, there were no significant differences observed among time intervals of sand fly collections (p=0.154). Observations of the nocturnal activity of male and female sand flies throughout the night suggest that phlebotomine sand flies show the greatest activity level after midnight.     

DNA条形码技术在毒品原植物大麻鉴定中的应用

准确鉴定毒品原植物大麻的种属及品种具有重要的理论和实践意义。为了探讨DNA条形码技术用于毒品原植物大麻种属鉴定及品种鉴定的可行性,该研究以60份大麻原植物(分别采自内蒙、黑龙江、陕西延安、陕西榆林4个地区的栽培大麻雌雄各6株及新疆玛纳斯地区的野生大麻雌雄各6株)为材料,通过从其叶片中提取的DNA为模版,利用核糖体DNA基因间隔区的通用引物ITS2和叶绿体DNA的通用引物psbAtrnH进行PCR扩增,对扩增片段进行双向测序,将测序结果进行人工矫正和比对。结果显示:所有大麻样本的ITS2扩增片段序列没有变异完全一致,但psbA-trnH扩增片段变异较大共检测出8种cpDNA单倍型,用MEGE5.1软件计算种间遗传距离,并构建NJ系统聚类树可以有效把这五个地区的大麻样本区别开来,因此证明DNA条形码技术在毒品原植物大麻的种属鉴定方面具有可行性,但其用于大麻的种属鉴定的准确性、可靠性及在其来源地鉴定及品种鉴定中的可能性还有待进一步深入地研究。     

Malformations of the genitalia in male Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae)

Phlebotomus papatasi ( Scopoli, 1786 ) (Diptera: Psychodidae) is a major vector of Leishmania major (Kinetoplastida: Trypanosomatidae), a causative agent of zoonotic cutaneous leishmaniasis. Morphological characters of sand fly genitalia are key indicators for species identification. Various anomalies affecting male genitalia have been previously described. We take advantage of a large sand flies survey conducted in 32 stations in Central and Southern Morocco to systematically quantify the prevalence and spatial distribution of malformations affecting the genitalia of P. papatasi. Among 597 examined males, 122 were abnormal (20.4%). Malformations were widespread and largely concerned the number of spines in the lateral lobes and in the styles. Asymmetrical anomalies in lateral lobes were common. Correspondence analysis of our results highlighted the symmetrical anomalies observed in the lateral lobes, and abnormal styles of the male genitalia were found to be associated with environmental disturbances since they were prevalent in sewage dumps.     

DNA barcoding of tropical black flies (Diptera: Simuliidae) of Thailand

The ecological and medical importance of black flies drives the need for rapid and reliable identification of these minute, structurally uniform insects. We assessed the efficiency of DNA barcoding for species identification of tropical black flies. A total of 351 cytochrome c oxidase subunit 1 sequences were obtained from 41 species in six subgenera of the genus Simulium in Thailand. Despite high intraspecific genetic divergence (mean = 2.00%, maximum = 9.27%), DNA barcodes provided 96% correct identification. Barcodes also differentiated cytoforms of selected species complexes, albeit with varying levels of success. Perfect differentiation was achieved for two cytoforms of Simulium feuerborni, and 91% correct identification was obtained for the Simulium angulistylum complex. Low success (33%), however, was obtained for the Simulium siamense complex. The differential efficiency of DNA barcodes to discriminate cytoforms was attributed to different levels of genetic structure and demographic histories of the taxa. DNA barcode trees were largely congruent with phylogenies based on previous molecular, chromosomal and morphological analyses, but revealed inconsistencies that will require further evaluation.     

The genus Apsiphortica from China with DNA barcoding information (Diptera: Drosophilidae)

Two new species of the genus Apsiphortica are described from China: A. orthophallos n. sp. and A. sinuatipenis n. sp. Species delimitations are improved by integrating morphological and DNA barcoding information. The intra- and interspecific pairwise p-distances (proportional distance) are summarized for five Apsiphortica species from China. Furthermore, nucleotide sites with fixed status in the alignment of the COI sequences (639 nucleotide sites in length) are used as “pure” molecular diagnostic characters to delineate the five species. A key to all the Chinese species of the genus Apsiphortica is provided.     

Species identification of North American guinea worms (Nematoda: Dracunculus) with DNA barcoding

Dracunculus insignis is a nematode parasite that infects the subcutaneous tissues of mammals such as raccoon (Procyon lotor), mink (Neovison vison) and fisher (Martes pennanti). D. lutrae, a morphologically similar species, has only been recovered from the otter (Lontra canadensis). Species identification of these two North American guinea worms has only been achieved by morphology of males and host identity. As a result, where only female specimens are present, accurate identifications are not possible. To date, specimens recovered from otter have been assumed to be D. lutrae, while those from all other hosts are assumed to be D. insignis. This study uses DNA barcoding to differentiate between these two North American dracunculoids. Our results show that D. insignis is a 'true' generalist, showing little sequence divergence regardless of host association, although our studies did validate its occurrence in a new host - the otter. Interestingly, specimens of the host specialist, D. lutrae, showed some sequence divergence, although it was low. The finding of D. insignis in otter substantiates the need to supplement morphology-based methods in providing species identifications for certain dracunculoids.     

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well‐supported clusters. Intraspecific Kimura 2‐parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra‐ and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.     

DNA barcoding for the identification of smoked fish products

DNA barcoding was applied to the identification of smoked products from fish in 10 families in four orders and allowed identification to the species level, even among closely related species in the same genus. Barcoding is likely to become a standard tool for identification of fish specimens and products.     

苹果园鳞翅目夜蛾科DNA条形码鉴定

为了检验DNA条形码在鳞翅目夜蛾科蛾类鉴定中的可行性,本文对采自北京昌平苹果园内的夜蛾科14种71头蛾类标本分别提取了DNA,并扩增了线粒体cox1及核基因28S,利用系统发育树、遗传距离、阈值等方法进行了鉴定和比较分析。同时,检验了目前BOLD系统的鉴定成功率。实验表明,基于cox1基因和BOLD系统的鉴定成功率达到了100%,而基于28S则很低,为64.8%。用不同方法构建的系统发育树,鉴定结果均相同。93%的种内遗传距离小于1%,94%的种间遗传距离为大于3%,种内种间的遗传距离形成明显的3%阈值现象。     

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.     

Modification of Disney trap for capture of sand flies (Diptera: Psychodidae: Phlebotominae)

This paper describes the modifications made to the original model of the Disney trap, with a view to easier handling of the same, greater practicability in the collection of sand flies, protection of the animal bait and durability of the trap in the field.