Identificación molecular de Dípteros de importancia forense con el gen (COI Barcode), La Paz Bolivia / Molecular identification of Diptera of forensic importance with the gene (COI Barcode), La Paz Bolivia

Resumen Una de las dificultades encontradas es la correcta identificación de insectos asociados a la descomposición cadavérica, por la cual ha llevado a buscar y generar nuevas herramientas en biología molecular que facilitan la determinación de especímenes para la estimación del Intervalo Post-Mortem de una forma efectiva y certera a partir de estadios inmaduros; la colecta y taxonomía morfológica de Dípteros se realizaron en primera instancia y posteriormente se utilizó el sistema de Códigos de Barras (COI Barcode) para la identificación molecular de insectos problema por medio del gen mitocondrial COI en cualquier fase del ciclo biológico. Identificando tres especies de adultos con una probabilidad de correspondencia del 100%; los especímenes: Sarconesia versicolor de la Familia Calliphoridae y Fannia sp., no fueron hallados en las bases de datos mundiales del GenBank y del Boldsystems, siendo necesario su actualización realizando patrones de sucesión cronológica de fauna cadavérica en diferentes zonas geográficas, cuya práctica se aplicaría en las investigaciones criminales.

Abstract One of the difficulties encountered is the correct identification of insects associated with cadaveric decomposition, which has led to the search and generation of new tools in molecular biology that facilitate the determination of specificities for the modification of the Post-Mortem Interval in an effective and accurate from immature stages; the collection and morphological taxonomy of Diptera were made in the first instance and then the Bar Code System (COI Barcode) was used for the molecular identification of problem insects by means of the mitochondrial COI gene in any phase of the biological cycle. Identifying three species of adults with a probability of 100% correspondence; the specimens: Sarconesia versicolor of the Calliphoridae Family and Fannia sp., were not found in the world databases of the GenBank and the Boldsystems, being necessary to update them by chronological succession patterns of cadaveric fauna in different geographical areas, whose practice would be applied in criminal investigations.

ENZYMATIC AND ANTAGONIST ACTIVITY OF ENDOPHYTIC FUNGI FROM Sapindus saponaria L. (SAPINDACEAE) / Actividad enzimática y antagonista de los hongos endofíticos de Sapindus saponaria L. (Sapindaceae)

ABSTRACT Endophyte microorganisms have great biotechnological interest, with features applicable to different areas and are potentially useful in agriculture. The current study determines the biotechnological potential of endophytic fungi, isolated from leaves of Sapindus saponaria, to control phytopathogenic fungi and evaluate their enzyme production. Molecular taxonomy was performed by sequencing of the ITS1-5.8S-ITS2 ribosomal DNA region, identifying the genera Phomopsis, Sordariomycetes, Diaporthe, and Colletotrichum. In vitro antagonism against phytopathogens showed better results against Fusarium solani and provided inhibition indices between 41.8 % and 67.5 %. The endophytic strain SS81 (Diaporthe citri) presented the highest antagonism index against the pathogen. Against Glomerella sp. and Moniliophthora perniciosa, inhibition rates ranged between 18.7 % and 57.4 % and between 38.3 % and 64.8 %, respectively. Enzyme assays revealed that strain SS65 (Diaporthe sp.) produced 1.16 UI μmol/min of amylase; strain SS77 (Diaporthe sp.) produced 2.74 UI μmol/min of pectinase, and strain SS08 (Diaporthe sp.) produced 1.51 UI μmol/min of cellulase. Thus, the current study shows evidence the importance of isolated endophytes with phytoprotective properties of plants with medicinal properties as alternatives for biological control and natural sources of products with biotechnological interest.

RESUMEN Los microorganismos endofíticos tienen gran interés biotecnológico, con características aplicables a diferentes áreas y potencialmente útiles en la agricultura. El presente estudio determinó el potencial biotecnológico de los hongos endofíticos, aislados de las hojas de Sapindus saponaria, en el control de hongos fitopatógenos y evaluación de su producción de enzimática. La taxonomía molecular fue realizada por la secuencia de la región ITS1-5.8S-ITS2 del ADN ribosomal, identificando los géneros Phomopsis, Sordariomycetes, Diaporthe y Colletotrichum. El antagonismo in vitro contra fitopatógenos mostró mejores resultados contra Fusarium solani y proporcionó índices de inhibición de entre el 41,8 % y el 67,5 %. El linaje endofítico SS81 (Diaporthe citri) presentó el mayor índice de antagonismo contra los patógenos. Contra Glomerella sp. y Moniliophthora perniciosa, las tasas de inhibición variaron entre el 18,7 % y el 57,4 % y entre el 38,3 % y el 64,8 %, respectivamente. El ensayo enzimático reveló que el linaje SS65 (Diaporthe sp.) produjo 1,16 UI μmol / min de amilasa; el linaje SS77 (Diaporthe sp.) produjo 2,74 UI μmol / min de pectinasa; y el linaje SS08 (Diaporthe sp.) produjo 1,51 UI μmol / min de celulasa. Así, el presente estudio evidencia la importancia de los endófitos aislados con propiedades fitoprotectoras como alternativas para el control biológico y como fuentes naturales de productos con interés biotecnológico.

Taxonomía molecular de Anopheles del Ecuador mediante ADN mitocondrial (Citocromo c Oxidasa I) y optimización por Parsimonia Máxima / Molecular Taxonomy of Anopheles from Ecuador, using mitochondrial DNA (Cytochrome c Oxidase I) and Maximum Parsimony optimization

La identificación de especies de Anopheles es compleja debido a la presencia de varios complejos de especies o especies cripticas cuyos ejemplares son de difícil distinción morfológica. Se corroboró o se corrigió la identificación de especies de Anopheles spp. capturadas en Ecuador, utilizando 393 secuencias de ADN mitocondrial, Citocromo Oxidasa I (COI) de 36 especies de Anofelinos neotropicales depositadas en GenBank y mediante la construcción de árboles filogenéticos usando parsimonia máxima. Se analizaron las identificaciones moleculares de cinco especies de Anopheles mediante los criterios de monofília y topología del árbol, secuencias provenientes de la localidad tipo y la divergencia genética intra/inter especies. Las topologías de los árboles resultantes corroboran la identificación de secuencias/especies de Anopheles (Anopheles) pseudopunctipennis Theobald, An. (Ano.) eiseni Coquillett y An. (Nyssorhynchus) albimanus Wiedemann; se corrige la identificación molecular de An. (Nys.) rangeli Gabaldón, Cova-García y López (no oswaldoi) y permanece en duda la identificación correcta de las secuencias relacionadas el grupo Punctimacula, hasta tener disponibles un mayor número de secuencias COI de especies relacionadas. La matriz de ADN-COI de 241 secuencias/haplotiposx36 especies construida para estos análisis resultará de utilidad para la identificación molecular de especies de Anofelinos de Ecuador y del Neotrópico con base en la porción de 520 pb entre 2.272 a 2.792pb de COI.

The Anopheles species identification is complicated by the presence of several complexes of species and species whose specimens are difficult morphological distinction. We use 393 mitochondrial DNA sequences, Cytochrome Oxidase I (COI) of 36 Neotropical species of Anophelinae deposited in GenBank and by constructing phylogenetic trees using maximum parsimony the objective was to corroborate or correct the molecular identification of five sequences/species of Anopheles collected in Ecuador and also availables in Genbank. We identified the taxonomic units, based on monophyly, phylogenetic species definition, tree topology, ocurrence of sequences from the type locality and genetic intra/inter divergence of clades. The topologies of the resulting trees corroborate the identification of sequences of Anopheles (Anopheles) pseudopunctipennis Theobald, An. (Ano.) eiseni Coquillett y An. (Nyssorhynchus) albimanus Wiedemann, while the molecular identification of An. (Nys.) rangeli Gabaldón, Cova-García y López (not “oswaldoi”) is corrected and remains in doubt the correct identification of related sequences for sequences belonging to Punctimacula group, until have available a greater number of COI sequences from related species. However according to the criteria of type locality, these are not punctimacula in sensu stricto. Finally, the matrix of alignment DNA-COI haplotypes sequences of 241x36 species built for these analyzes will be useful for molecular identification of Anophelinae species from Ecuador and the Neotropics based on the portion of 520 bp COI (between 2,272- 2,792 bp).

Molecular Characterization of Various Trichomonad Species Isolated from Humans and Related Mammals in Indonesia

Trichomonad species inhabit a variety of vertebrate hosts; however, their potential zoonotic transmission has not been clearly addressed, especially with regard to human infection. Twenty-one strains of trichomonads isolated from humans (5 isolates), pigs (6 isolates), rodents (6 isolates), a water buffalo (1 isolate), a cow (1 isolate), a goat (1 isolate), and a dog (1 isolate) were collected in Indonesia and molecularly characterized. The DNA sequences of the partial 18S small subunit ribosomal RNA (rRNA) gene or 5.8S rRNA gene locus with its flanking regions (internal transcribed spacer region, ITS1 and ITS2) were identified in various trichomonads; Simplicimonas sp., Hexamastix mitis, and Hypotrichomonas sp. from rodents, and Tetratrichomonas sp. and Trichomonas sp. from pigs. All of these species were not detected in humans, whereas Pentatrichomonas hominis was identified in humans, pigs, the dog, the water buffalo, the cow, and the goat. Even when using the high-resolution gene locus of the ITS regions, all P. hominis strains were genetically identical; thus zoonotic transmission between humans and these closely related mammals may be occurring in the area investigated. The detection of Simplicimonas sp. in rodents (Rattus exulans) and P. hominis in water buffalo in this study revealed newly recognized host adaptations and suggested the existence of remaining unrevealed ranges of hosts in the trichomonad species.

Whole-cell antigenicity data support sequence-based kinetoplastid taxonomy

Early immunological data, obtained by immunodiffusion and immunoelectrophoresis, on the whole-cell antigenicity of kinetoplastid protozoa were retrieved and used to construct a dendrogram of antigenic distances. Remarkably, they supported the same taxonomic conclusions as analyses based on DNA and protein sequence data.

Morphological and molecular characterisation of Heliconema hainanensis sp. nov. (Spirurina: Physalopteridae) from congers in the South China Sea, with a key to the species of Heliconema

Heliconema hainanensis sp. nov. collected from Uroconger lepturus (Richardson) (Anguilliformes: Congridae), Muraenesox cinereus (Forsskål) and Congresox talabonoides (Bleeker) (Anguilliformes: Muraenesocidae) in the South China Sea was described using light and scanning electron microscopy. The new species differs from its congeners by the following morphology: pseudolabia, the number and arrangement of caudal papillae (4 pairs of pedunculate precloacal papillae arranged in 2 groups of 2 and 2 pairs and 6 pairs of pedunculate postcloacal papillae arranged in 4 groups of 1, 2, 1 and 2 pairs), the length of spicules [left spicule 0.51-0.69 mm, right spicule 0.20-0.27 mm, spicule (right:left) ratio 1:2.20-2.69] and the morphology of the female tail tip. In addition, specimens of the new species collected from the three different hosts and specimens of an unidentified species of Heliconema collected from U. lepturus were characterised using molecular methods by sequencing the internal transcribed spacer (ITS) of ribosomal DNA. Analyses and comparison of the ITS sequence of H. hainanensis sp. nov. with Heliconema sp. support the validity of the new species based on morphological observations. An identification key to the species of Heliconema is also provided.

Phytochemical and biological research of Fritillaria medicine resources / 中国天然药物

The genus Fritillaria is a botanical source for various pharmaceutically active components, which have been commonly used in traditional Chinese medicine for thousands of years. Increasing interest in Fritillaria medicinal resources has led to additional discoveries of steroidal alkaloids, saponins, terpenoids, glycosides and many other compounds in various Fritillaria species, and to investigations on their chemotaxonomy, molecular phylogeny and pharmacology. In continuation of studies on Fritillaria pharmacophylogeny, the phytochemistry, chemotaxonomy, molecular biology and phylogeny of Fritillaria and their relevance to drug efficacy is reviewed. Literature searching is used to characterize the global scientific effort in the flexible technologies being applied. The interrelationship within Chinese Bei Mu species and between Chinese species, and species distributed outside of China, is clarified by the molecular phylogenetic inferences based on nuclear and chloroplast DNA sequences. The incongruence between chemotaxonomy and molecular phylogeny is revealed and discussed. It is essential to study more species for both the sustainable utilization of Fritillaria medicinal resources and for finding novel compounds with potential clinical utility. Systems biology and omics technologies will play an increasingly important role in future pharmaceutical research involving the bioactive compounds of Fritillaria.

Genetic and morphological characterisation of a new species of the genus Hysterothylacium (Nematoda) from Paralichthys isosceles Jordan, 1890 (Pisces: Teleostei) of the Neotropical Region, state of Rio de Janeiro, Brazil

Taking into account the difficulties of taxonomic identification of larval anisakid nematodes based on morphological characters, genetic analyses were performed, together with those usually applied, in order to identify anisakid larvae found in the flounder Paralichthys isosceles from the littoral of the state of Rio de Janeiro, Brazil. The analysis of 1,820 larvae revealed a new species, similar to Hysterothylacium MD, Hysterothylacium 2, Hysterothylacium KB and Hysterothylacium sp regarding the absence of the larval tooth, an excretory pore situated below the nerve ring level, and slender lateral alae. Moreover, the new species differs from Hysterothylacium fortalezae and Hysterothylacium reliquens with regard to the number and size of spines present on the tail end and from Hysterothylacium patagonicus by the absence of interlabia. The maximum parsimony and neighbour joining tree topologies based on the 18S ribosomal DNA gene, complete internal transcribed spacer region and cytochrome oxidase 2 (COII) gene demonstrated that the Brazilian larvae belong to Raphidascarididae and represent a unique genetic entity, confirmed as a new Hysterothylacium species. Furthermore, the new species presents COII genetic signatures and shares polymorphisms with Raphidascarididae members. This is the first description of a new anisakid species from Brazil through the integration of morphological and molecular taxonomy data.

DNA Barcoding of Fish, Insects, and Shellfish in Korea

DNA barcoding has been widely used in species identification and biodiversity research. A short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence serves as a DNA bio-barcode. We collected DNA barcodes, based on COI sequences from 156 species (529 sequences) of fish, insects, and shellfish. We present results on phylogenetic relationships to assess biodiversity the in the Korean peninsula. Average GC% contents of the 68 fish species (46.9%), the 59 shellfish species (38.0%), and the 29 insect species (33.2%) are reported. Using the Kimura 2 parameter in all possible pairwise comparisons, the average interspecific distances were compared with the average intraspecific distances in fish (3.22 vs. 0.41), insects (2.06 vs. 0.25), and shellfish (3.58 vs. 0.14). Our results confirm that distance-based DNA barcoding provides sufficient information to identify and delineate fish, insect, and shellfish species by means of all possible pairwise comparisons. These results also confirm that the development of an effective molecular barcode identification system is possible. All DNA barcode sequences collected from our study will be useful for the interpretation of species-level identification and community-level patterns in fish, insects, and shellfish in Korea, although at the species level, the rate of correct identification in a diversified environment might be low.

Molecular and Morphological Characterization of Endophytic Heterobasidion araucariae from Roots of Capsicum annuum L. in Korea

A species of Heterobasidion was encountered during a diversity study of endophytic fungi from healthy root tissues of chili pepper (Capsicum annuum L.) in Korea. The fungal species (CNU081069) was identified as Heterobasidion araucariae based on phylogenetic analyses of the internal transcribed spacer and translation elongation factor gene sequences. Morphological descriptions of the endophytic isolate matched well with the previous references and supported the molecular identification. The fungus Heterobasidion araucariae CNU081069 is new to Korea.

Acercamiento a los estudios actuales sobre el filo Tardigrada y su importancia en la medicina: [revisión] / Current studies on phylum Tardigrada and its importance in medicine: [review]

La siguiente revisión permite obtener una visión global de los diferentes estudios que se han llevado a cabo en la actualidad respecto a los tardígrados, principalmente a nivel molecular y genético. Los documentos consultados permiten un acercamiento a la anatomía del Tardígrado y al proceso de criptobiosis, así como a la taxonomía tradicional. Posteriormente, se abordan la genética y la biología molecular del Tardígrado, y se recopilan algunos de los resultados obtenidos respecto a su genoma, teniendo en cuenta genes o fragmentos secuenciados reportados en GenBank y estudios cromosómicos, con referencia a los métodos de extracción de ADN, taxonomía molecular, construcción de bibliotecas de ADNc y evolución-filogenia...

The following review allows a general vision of different studies that have been performed on Tardigrades, mainly those related to molecular biology and genetics. In general, this document shows some aspects on Tardigrade´s anatomy, cryptobiosis processes and the methodology of traditional taxonomy. Then we follow with some of the findings reported on the genetics and molecular biology of Tardigrades, taking into account sequenced genes or fragments reported in GenBank and chromosomal studies, likewise, with reference to DNA extraction methods, molecular taxonomy, cDNA library construction and phylogenyevolution...

Descripción del ARN de transferencia mitocondrial para Serina (UCN) de Lutzomyia columbiana (Diptera, Psychodidae) / Description of the mitochondrial serine transfer RNA (UCN) of Lutzomyia columbiana (Diptera, Psychodidae)

Lutzomyia columbiana es un flebotomíneo considerado como vector sospechoso de Leishmania mexicana y Leishmania braziliensis en Colombia. Este insecto pertenece al grupo verrucarum, que incluye algunos taxones isomórficos, lo que ha estimulado la búsqueda de marcadores moleculares que permitan, además de diferenciar las especies, estudiar sus relaciones de parentesco. En este artículo se describe por primera vez la estructura putativa del ARN de transferencia mitocondrial para serina que reconoce el codón UCN (ARNtSer) de Lu. columbiana. El ADN genómico fue extraído, amplificado y secuenciado a partir de seis especímenes colectados con cebo humano. La estructura secundaria del ARNtSer fue inferida con el programa tRNAscan-SE 1.21. El gen ARNts consistió de 67 pares de bases (pb), encontrándose un solo haplotipo en los seis individuos secuenciados. El ARNtSer de Lu. columbiana mostró 7 apareamientos intracatenarios en el brazo aceptor del aminoácido, 3 en el brazo dihidrouridina (DHU), 5 en el brazo del anticodón y 5 en el brazo ribotimidina-pseudouridina-citosina (T›C). El tamaño de las lupas correspondió a 5 nucleótidos en la DHU, 7 en la anticodón, 4 en la variable y 7 en la T›C. Lu. columbiana se distingue del resto de especies de Lutzomyia y Phlebotomus secuenciadas a la fecha por la presencia de una guanina en la posición nucleotídica 64, que produce un apareamiento no canónico tipo uracilo-guanina en el brazo aceptor. Se necesitan más estudios para confirmar la utilidad del ARNtSer como marcador molecular para la discriminación de especies de flebotomíneos.

The sand fly Lutzomyia columbiana is considered a suspected vector of Leishmania mexicana and Leishmania braziliensis in Colombia. Lu. columbiana belongs to the Lutzomyia verrucarum species group, which included some sibling species. This has motivated the search for molecular markers to distinguish these taxa. In this paper, we described for the first time the putative secondary structure of the mitochondrial serine transfer RNA that recognizes the codon UCN of Lu. columbiana (tRNA Ser). DNA was extracted, amplified and sequenced from six individuals collected in human biting activity. The secondary structure of the tRNA Ser was inferred using the program tRNAscan-SE 1.21. The tRNA Ser gene length was 67 pair of bases (pb), and a single haplotype was detected among the six specimens sequenced. In the inferred secondary structure of the tRNA Ser of Lu. columbiana, the acceptor arm consisted of 7 bp, the dihydrouridine (DHU) arm of 3 pb, the anticodon arm of 5 pb, and the ribothymidine-pseudouridine-cytosine (T›C) arm of 5 pb. Similarity, the estimated size of the loops was 5 nucleotides in the DHU, 7 in the anticodon, 4 in the variable, and 7 in the T›C. Lu. columbiana differs from other Lutzomyia and Phlebotomus species sequenced to date by the presence of guanine in the nucleotide position 64, which induce a non-canonical base pair conformation type uracil-guanine in the acceptor arm. More studies are necessary to confirm the usefulness of the tRNA Ser as a suitable molecular tool for sand fly species identification.