Occurrence of euchromatic B chromosomes in natural populations of Moenkhausia bonita and M. forestii (Pisces: Characidae)

Moenkhausia is a highly specious genus among the Characidae, composed of 96 valid species. Only twelve species have a known karyotype. Thus, here are presented the first cytogenetic data of two allopatric populations of Moenkhausia bonita and one of M. forestii, both belonging to the upper Paraná River basin (PR) with discussion on the evolutionary and cytotaxonomic aspects of the genus. The two species presented 2n = 50 chromosomes but different karyotype formulas and occurrence of 1-2 B chromosomes. These elements are small metacentrics in M. bonita and small acrocentrics in M. forestii. In both species, B chromosomes were euchromatic. Ag-NOR sites were found in pair 3 (metacentric), coinciding with fluorescent in situ hybridization (FISH) by the 18S rDNA probe in both species. However, the species differed in terms of the number and position of 5S rDNA sites. Heterochromatic blocks, mapped in M. bonita showed the least amount of heterochromatin in the terminal and pericentromeric regions, while the M. forestii karyotype revealed a greater amount of interstitial heterochromatic blocks. The karyotype distinctions between the two species, including the morphology of B chromosomes, may contribute as a reference in the taxonomic studies in this group.(AU)

Moenkhausia é um gênero altamente especioso dentre os Characidae, composto por 96 espécies válidas, mas apenas doze espécies têm seus cariótipos conhecidos. Portanto, são apresentados aqui os primeiros dados citogenéticos de duas populações alopátricas de Moenkhausia bonita e uma de M. forestii, ambas pertencentes à bacia do alto rio Paraná (PR), com uma ampla discussão sobre os aspectos evolutivos e citotaxonômicos do gênero. As duas espécies apresentaram 2n = 50 cromossomos, mas diferentes fórmulas cariotípicas e ocorrência de 1-2 cromossomos B. Esses elementos são pequenos metacêntricos em M. bonita e acrocêntricos pequenos em M. forestii. Em ambas as espécies, os cromossomos B apresentaram-se eucromáticos. Sítios Ag-NOR foram encontrados no par 3 (metacêntrico), coincidindo com a hibridização fluorescente in situ (FISH) pela sonda 18S rDNA em ambas as espécies. No entanto, as espécies diferiram em termos de número e posição dos sítios de 5S rDNA. Blocos heterocromáticos mapeados em M. bonita revelaram pequena quantidade de heterocromatina nas regiões terminal e pericentromérica, enquanto o cariótipo de M. forestii revelou uma maior quantidade de blocos heterocromáticos intersticiais. As distinções cariotípicas entre as duas espécies, incluindo a morfologia dos cromossomos B, podem contribuir como uma referência em estudos taxonômicos neste grupo.(AU)

Comparative cytogenetic analyses in Ancistrus species (Siluriformes: Loricariidae)

Ancistrus is a specious genus of armored catfishes that has been extensively used for cytogenetic studies in the last 17 years. A comparison of the extensive karyotypic plasticity within this genus is presented with new cytogenetic analysis for Ancistrus cf. multispinis and Ancistrus aguaboensis. This study aims to improve our understanding of chromosomal evolution associated with changes in the diploid number (2n) and the dispersion of ribosomal DNAs (rDNAs) within Ancistrus. Ancistrus cf. multispinis and A. aguaboensis exhibit 2n of 52 and 50 chromosomes, respectively. Given that A. cf. multispinis shares a 2n = 52 also found in Pterygoplichthyini, the sister group for Ancistrini, a Robertsonian (Rb) fusion event is proposed for the 2n reduction in A. aguaboensis. 5S rDNAs pseudogenes sites have already been associated with Rb fusion in Ancistrus and our analysis suggests that the 2n reduction in A. aguaboensis was triggered by double strand breaks (DSBs) and chromosomal rearrangements at 5S rDNA sites. The presence of evolutionary breakpoint regions (EBRs) into rDNA cluster is proposed to explain part of the Rb fusion in Ancistrus. Cytogenetic data presented extends the diversity already documented in Ancistrus to further understand the role of chromosomal rearrangements in the diversification of Ancistrini.(AU)

Ancistrus é um gênero rico em espécies de peixes conhecidos como cascudos e tem sido alvo de estudos citogenéticos nos últimos 17 anos. Uma comparação da plasticidade presente no gênero é apresentada com novas análises citogenéticas para Ancistrus cf. multispinis e Ancistrus aguaboensis. Este estudo visa melhorar nossa compreensão da evolução cromossômica associada as alterações do número diploide (2n) e a dispersão de DNAs ribossômicos (rDNAs) em Ancistrus. Ancistrus cf. multispinis e A. aguaboensis apresentaram 2n de 52 e 50 cromossomos, respectivamente. Visto que A. cf. multispinis compartilha 2n = 52 também encontrado em Pterygoplichthyini, o grupo irmão para Ancistrini, um evento de fusão Robertsoniana (Rb) é proposto para a redução do 2n em A. aguaboensis. Sítios de pseudogenes de rDNA 5S já foram associados a eventos de fusão Rb em Ancistrus e nossas análises sugerem que a redução do 2n em A. aguaboensis foi desencadeada por quebras na dupla fita e rearranjos cromossômicos em sítios de rDNA 5S. A presença de evolutionary breakpoint regions (EBRs) em clusters de rDNA foi proposta para explicar parte da fusão Rb em Ancistrus. Os dados citogenéticos apresentados ampliam a diversidade já documentada em Ancistrus visando melhor entender o papel dos rearranjos cromossômicos na diversificação de Ancistrini.(AU)

Neoechinorhynchus macrospinosus (Acanthocephala: Neoechinorhynchidae) in Rabbit fish Siganus rivulatus (Siganidae): morphology and phylogeny / Neoechinorhynchus macrospinosus (Acanthocephala: Neoechinorhynchidae) em peixes-coelho Siganus rivulatus (Siganidae): morfologia e filogenia

Siganids are the most important marine fish distributed along the African coast. Therefore, the current study aimed to investigate parasite fauna infects one of the most important mariculture fish species in the Red Sea, the Rabbit fish Siganus rivulatus. One acanthocephalan species has been isolated from the posterior region of fish intestine, belonging to the Neoechinorhynchidae family, and named as Neoechinorhynchus macrospinosus Amin & Nahhas, 1994 based on its morphological and morphometric features. In order to determine the accurate taxonomic position of this acanthocephalan species, molecular phylogenetic analysis was carried out based on the partial sequences of 18S rDNA gene region. The obtained data revealed that this species was associated with a close identity ˃71% for other species belonging to the Neoechinorhynchidae family. In addition, the recovered species deeply embedded in the Neoechinorhynchus genus, closely related to the previously described Neoechinorhynchus sp., N. mexicoensis, and N. golvani with identity percent of 95.14, 93.59, 93.59%, respectively. Therefore, the present study provide a better understanding about the taxonomic status of N. macrospinosus based on 18S rDNA that can be useful for achieving a proper assessment of biodiversity.

Os siganídeos são os peixes marinhos mais importantes distribuídos ao longo da costa africana. Portanto, o presente estudo teve como objetivo investigar a fauna de parasitas infectando uma das espécies mais importantes de peixes para maricultura no Mar Vermelho, o peixe-coelho Siganus rivulatus. Uma espécie de acantocéfalo foi isolada da região posterior do intestino de peixes pertencentes à família Neoechinorhynchidae, e denominadas Neoechinorhynchus macrospinosus Amin & Nahhas, 1994, com base em suas características morfológicas e morfométricas. A fim de determinar a posição taxonômica precisa dessa espécie de acantocéfalo, a análise filogenética molecular foi realizada com base nas sequências parciais da região do gene 18S rDNA e revelou que essa espécie estava associada a uma identidade próxima de até 71% para outras espécies pertencentes a família Neoechinorhynchidae e profundamente enraizada no gênero Neoechinorhynchus, intimamente relacionada a Neoechinorhynchus sp., N. mexicoensis, and N. golvani descrito anteriormente com percentual de identidade de 95,14, 93,59, 93,59%, respectivamente. Portanto, o presente estudo fornece uma melhor compreensão sobre o status taxonômico de N. macrospinosus com base no 18S rDNA que pode ser útil para obter uma avaliação adequada da biodiversidade.

[RETRACTED ARTICLE] Neoechinorhynchus macrospinosus (Acanthocephala: Neoechinorhynchidae) in Rabbit fish Siganus rivulatus (Siganidae): morphology and phylogeny / [ARTIGO RETRATADO] Neoechinorhynchus macrospinosus (Acanthocephala: Neoechinorhynchidae) em peixes-coelho Siganus rivulatus (Siganidae): morfologia e filogenia

Abstract Siganids are the most important marine fish distributed along the African coast. Therefore, the current study aimed to investigate parasite fauna infects one of the most important mariculture fish species in the Red Sea, the Rabbit fish Siganus rivulatus. One acanthocephalan species has been isolated from the posterior region of fish intestine, belonging to the Neoechinorhynchidae family, and named as Neoechinorhynchus macrospinosus Amin & Nahhas, 1994 based on its morphological and morphometric features. In order to determine the accurate taxonomic position of this acanthocephalan species, molecular phylogenetic analysis was carried out based on the partial sequences of 18S rDNA gene region. The obtained data revealed that this species was associated with a close identity ˃71% for other species belonging to the Neoechinorhynchidae family. In addition, the recovered species deeply embedded in the Neoechinorhynchus genus, closely related to the previously described Neoechinorhynchus sp., N. mexicoensis, and N. golvani with identity percent of 95.14, 93.59, 93.59%, respectively. Therefore, the present study provide a better understanding about the taxonomic status of N. macrospinosus based on 18S rDNA that can be useful for achieving a proper assessment of biodiversity.

Resumo Os siganídeos são os peixes marinhos mais importantes distribuídos ao longo da costa africana. Portanto, o presente estudo teve como objetivo investigar a fauna de parasitas infectando uma das espécies mais importantes de peixes para maricultura no Mar Vermelho, o peixe-coelho Siganus rivulatus. Uma espécie de acantocéfalo foi isolada da região posterior do intestino de peixes pertencentes à família Neoechinorhynchidae, e denominadas Neoechinorhynchus macrospinosus Amin & Nahhas, 1994, com base em suas características morfológicas e morfométricas. A fim de determinar a posição taxonômica precisa dessa espécie de acantocéfalo, a análise filogenética molecular foi realizada com base nas sequências parciais da região do gene 18S rDNA e revelou que essa espécie estava associada a uma identidade próxima de até 71% para outras espécies pertencentes a família Neoechinorhynchidae e profundamente enraizada no gênero Neoechinorhynchus, intimamente relacionada a Neoechinorhynchus sp., N. mexicoensis, and N. golvani descrito anteriormente com percentual de identidade de 95,14, 93,59, 93,59%, respectivamente. Portanto, o presente estudo fornece uma melhor compreensão sobre o status taxonômico de N. macrospinosus com base no 18S rDNA que pode ser útil para obter uma avaliação adequada da biodiversidade.

Application of 18S rDNA Clone Library to Detect Diatom Population Diversity in Dianchi / 法医学杂志

Objective To detect the diatom population diversity in Dianchi by constructing a 18S rDNA clone library. Methods DNA from diatoms in 6 water samples of Dianchi was amplified with diatom 18S rDNA specific primer.The 18S rDNA clone library was constructed, and clones were randomly selected for sequence. Sequence alignment was performed by BLAST. The diatom population distribution in Dianchi was analyzed and the phylogenetic tree of diatom 18S rDNA in Dianchi waters was established with the MEGA v7.0.14 software. Results Two hundred and forty clones were sequenced, with 167 diatom sequences obtained, including 11 diatom species such as Stephanodiscus, Diatoma, and Melosira. There were certain differences in diatom population distribution among the 6 samples. Conclusion The population distribution of diatom species in Dianchi shows unique features and the sequence analysis of diatom 18S rDNA has a certain reference value to the inference of forensic drowning sites.

IDENTIFICACIÓN MOLECULAR Y REGISTRO DE Aphelenchoides spp. EN CULTIVOS COMERCIALES DE Hydrangea EN ANTIOQUIA, COLOMBIA / MOLECULAR IDENTIFICATION AND REPORT OF Aphelenchoides spp. IN COMMERCIAL CROPS OF Hydrangea IN ANTIOQUIA, COLOMBIA

RESUMEN El cultivo comercial de hortensias para flor de exportación ocupa un renglón importante en el sector económico del oriente antioqueño, por ser fuente de empleo y de desarrollo en la zona. La hortensia (Hydrangea macrophylla) es afectada por numerosos organismos fitopatógenos, entre ellos, nematodos del género Aphelenchoides, los que ocasionan, en el follaje, lesiones necróticas angulares, malformación de flor, enanismo y un daño indirecto en la tasa fotosintética, demeritando los parámetros de calidad para exportación. El objetivo de este estudio fue identificar, molecularmente, las especies del nematodo Aphelenchoides asociadas al cultivo de hortensias de color, en los municipios de Medellín (Santa Elena), La Ceja y Rionegro, siendo este el primer reporte para Colombia, de las especies de este género. Para la ejecución del estudio, se realizaron 10 muestreos en cultivos comerciales, distribuidos entre los tres municipios mencionados. Los nematodos extraídos, se sometieron a pruebas basadas en el análisis de ADN, haciendo uso del marcador ribosomal 18S. Los análisis filogenéticos practicados mostraron la presencia de la especie Aphelenchoides ritzemabosi en cultivos de hortensias, del corregimiento de Santa Elena y, de A. fragarie, en los municipios de La Ceja y Rionegro.

ABSTRACT Hydrangea macrophylla commercial crops have an important economic significance for the eastern region of the department of Antioquia, Colombia, due to its impact on employment and local development. This flowering plant can be attacked by several phytopathogens among them nematodes of the genus Aphelenchoides; which can produce necrotic lesions on the plant leaves as well decreased photosynthetic rate, dwarfism, and flower malformation. Additionally, this pathogen is considered a regulated pest and limits the international commercialization of the flower. The aim of this study was to molecularly identify the Aphelenchoides species associated with infected H. macrophylla plants in crops the municipalities of Medellín, La Ceja, and Rionegro. This is the first report of the species of this phytopathogen for Colombia. The sampling activities were performed in ten commercial crops locate at the three municipalities. The isolated nematodes were subject to DNA-based tests were the 18S rDNA gene was amplified, sequenced and analyzed using phylogenetic methods. The obtained results showed the infection of Aphelenchoides ritzemabosi within the Santa Elena (Medellin) area in crops and A. fragarie within the municipalities of La Ceja, and Rionegro.

Contributions to the systematic of Pimelodidae (Osteichthyes, Siluriformes): basic and molecular cytogenetics on seven species of Pimelodus from three Brazilian hydrographic systems

Pimelodidae harbors several species and is widely distributed throughout the Neotropical region. Pimelodus is the genus with the largest number of species, however it is a polyphyletic group. Cytogenetic analyzes of the valid species still covers less than half of them. Herein, seven Pimelodus species from three Brazilian hydrographic systems were analyzed through basic (Giemsa, AgNORs and C banding) and molecular (5S and 18S rDNA-FISH) cytogenetic methods. All species had 2n=56 chromosomes with different karyotype formulas observed among the species. AgNORs were corresponding to 18S rDNA and localized on long arm of one chromosome pair in all species. Heterochromatin distribution follows the pattern commonly verified in the family and allows to identify each one of the studied species. 5S rDNA marker was interspecifically variable in number and position of cistrons. Pimelodus ortmanni had B chromosomes varying intra and inter-individually. We performed a discussion on our own and available cytogenetic data for Pimelodidae, and the associating of them with available phylogeny enable us identifying features that distinguish subgroups within Pimelodidae, such as NORs location (terminal/long arm for species belonging to "Iheringichthys-Parapimelodus" and "Pimelodus maculatus" subclades) and location of 5S rDNA sites (pericentromeric/interstitial/ long arm for species belonging to Pimelodus group).(AU)

Pimelodidae abriga várias espécies e é amplamente distribuída ao longo da região Neotropical. Pimelodus é o gênero com o maior número de espécies, porém é um grupo polifilético. Análises citogenéticas foram realizadas em menos da metade das espécies válidas. Aqui, sete espécies de Pimelodus de três sistemas hidrográficos brasileiros foram estudadas através das técnicas citogenéticas básicas (Giemsa, AgRONs e banda C) e moleculares (FISH-DNAr 5S e 18S). Todas as espécies apresentaram 2n=56 cromossomos, sendo observadas variações na fórmula cariotípica entre algumas espécies. As AgRONs correspondentes ao DNAr 18S foram localizadas no braço longo de um par de cromossomos em todas as espécies. A heterocromatina segue o padrão comumente observado na família e permite identificar cada uma das espécies estudadas. O DNAr 5S apresentou variação interespecífica em número e na posição dos cístrons. Cromossomos B foram evidenciados em P. ortmanni com variação intra e interindividual. Nós discutimos os nossos resultados com os dados citogenéticos válidos para Pimelodidae, e a associação desses dados com a filogenia válida nos permitiu identificar características que distinguem subgrupos dentro de Pimelodidae, tais como a localização das RONs (terminal/braço longo para espécies pertencentes aos subclados "Iheringichthys-Parapimelodus" e "Pimelodus maculatus") e localização dos sítios de DNAr 5S (pericentromérico/intersticial no braço longo para espécies pertencentes ao grupo Pimelodus).(AU)

New morphological data and molecular diagnostic of Henneguya friderici (Myxozoa: Myxobolidae), a parasite of Leporinus friderici (Osteichthyes: Anostomidae) from southeastern Brazil / Novos dados morfológicos e diagnóstico molecular de Henneguya friderici (Myxozoa: Myxobolidae), parasito de Leporinus friderici (Osteichthyes: Anostomidae) do sudeste do Brasil

Abstract The myxozoan Henneguya friderici is a parasite of the gills, intestine, kidney and liver of Leporinus friderici, a characiform fish belonging to the family Anostomidae. Forty-two specimens of L. friderici that had been caught in the Mogi Guaçú River, state of São Paulo, were studied. Elongated white plasmodia were found in the gill filaments of 10 host specimens (24%). The mature spores had an ellipsoidal body with polar capsules of equal size and caudal length greater than body length. This study also described 18S rDNA sequencing of H. friderici infecting the gill filaments. This produced a sequence of 1050 bp that demonstrated significant genetic differences with previously described species of Henneguya. Similarity analysis using sequences from species that clustered closest to those produced by this study showed that the species with greatest genetic similarity to H. friderici was H. leporinicola, with 94% similarity.

Resumo O myxozoa Henneguya friderici é um parasito encontrado nas brânquias, fígado, intestino e rins de Leporinus friderici, (Characiformes: Anastomidae). Foram capturados e examinados quarenta e dois espécimes de L. friderici oriundos do Rio Mogi Guaçú, estado de São Paulo. Cistos alongados e brancos foram encontrados nos filamentos branquiais de 10 (24%) hospedeiros. Os esporos maduros apresentaram o corpo alongado com as cápsulas polares em tamanhos iguais e o comprimento caudal maior do que o comprimento corporal. Com isso, o presente trabalho, descreve o sequenciamento de 1050 pb do gene 18S rDNA de H. friderici infectando os filamentos branquiais, o que demonstrou diferenças genéticas significativas em comparação com espécies previamente descritas de Henneguya. A análise de similaridade utilizando as sequencias de espécies que se agruparam mais próximas às produzidas por este estudo mostrou que a espécie com maior semelhança genética com H. friderici foi H. leporinicola, com 94% de similaridade.

Identification and characterization of a taxol-producing endophytic fungus from Taxus media / 生物工程学报

To enrich the resource pool of endophytic fungi from plants which produce taxol, a taxol-producing endophytic fungus TMS-26 was isolated from the stem of Taxus Media. The result of high performance liquid chromatography (HPLC) showed that TMS-26 extract exhibited similar chromatographic peaks and retention time (4.545 min) with authentic taxol. Then mass spectrometry (MS) analysis further confirmed that TMS-26 extracts contained the same mass peaks with authentic taxol ((M+Na)+=876). These indicated that the isolated endophytic fungus TMS-26 can produce taxol. According to the morphological characteristics, the molecular analysis of 18S rDNA and internal transcribed spacer nuclear rDNA gene sequence, the fungus was identified as Aspergillus fumigatus TMS-26.

Allopatric chromosomal variation in Nematocharax venustus Weitzman, Menezes & Britski, 1986 (Actinopterygii: Characiformes) based on mapping of repetitive sequences

Characiformes is the most cytogenetically studied group of freshwater Actinopterygii, but karyotypical data of several taxa remain unknown. This is the case of Nematocharax , regarded as a monotypic genus and characterized by marked sexual dimorphism. Therefore, we provide the first cytogenetic report of allopatric populations of Nematocharax venustus based on distinct methods of chromosomal banding and fluorescence in situ hybridization (FISH) with repetitive DNA probes (18S and 5S rDNA). The karyotype macrostructure was conserved in all specimens and populations, independently on sex, since they shared a diploid number (2n) of 50 chromosomes divided into 8m+26sm+14st+2a. The heterochromatin was mainly distributed at pericentromeric regions and base-specific fluorochrome staining revealed a single pair bearing GC-rich sites, coincident with nucleolar organizer regions (NORs). On the other hand, interpopulation variation in both number and position of repetitive sequences was observed, particularly in relation to 5S rDNA. Apparently, the short life cycles and restricted dispersal of small characins, such as N. venustus , might have favored the divergence of repetitive DNA among populations, indicating that this species might encompass populations with distinct evolutionary histories, which has important implications for conservation measures.

Characiformes é o grupo de Actinopterygii de água doce mais estudado citogeneticamente, porém dados cariotípicos de vários taxa permanecem desconhecidos. Este é o caso de Nematocharax , considerado um gênero monotípico e caracterizado pelo acentuado dimorfismo sexual. Em vista disso, nós fornecemos a primeira descrição citogenética de populações alopátricas de Nematocharax venustus , baseada em métodos distintos de bandamento cromossômico e hibridação fluorescente in situ (FISH) com sondas de DNA repetitivo (DNAr 18S e 5S). A macroestrutura cariotípica mostrou-se conservada em todos os espécimes e populações, independentemente do sexo, uma vez que compartilharam um número diploide (2n) de 50 cromossomos dividido em 8m+26sm+14st+2a. A heterocromatina distribuiu-se principalmente nas regiões pericentroméricas e a coloração com fluorocromos base-específicos revelou um único par portador de sítios GC-ricos, coincidentes com as regiões organizadoras de nucléolo (RONs). Por outro lado, foi observada uma variação interpopulacional no número e na posição das sequências repetitivas, especialmente em relação ao DNAr 5S. Aparentemente, ciclos de vida curtos e dispersão restrita dos pequenos caracídeos, tal como N. venustus , podem ter favorecido a divergência do DNA repetitivo entre as populações, indicando que essa espécie pode englobar populações com distintas histórias evolutivas, o que tem implicações importantes para medidas de conservação.

B chromosome and NORs polymorphism in Callichthys callichthys (Linnaeus, 1758) (Siluriformes: Callichthyidae) from upper Paraná River, Brazil

B chromosomes are extra chromosomes from the normal chromosomal set, found in different organisms, highlighting their presence on the group of fishes. Callichthys callichthys from the upper Paraná River has a diploid number of 56 chromosomes (26 m-sm + 30 st-a) for both sexes, with the presence of a sporadically acrocentric B chromosome. Moreover, one individual presented a diploid number of 57 chromosomes, with the presence of a morphologically ill-defined acrocentric B chromosome in all analyzed cells. The physical mapping of 5S and 18S rDNA shows multiple 5S rDNA sites and only one pair of chromosomes with 18S sites in C. callichthys, except for two individuals. These two individuals presented a third chromosome bearing NORs (Ag-staining and 18S rDNA) where 5S and 18S rDNA genes are syntenic, differing only in position. The dispersion of the 18S rDNA genes from the main st-a chromosome pair 25 to one of the chromosomes from the m-sm pair 4 would have originated two variant individuals, one of which with the ill-defined acrocentric B chromosome. Mechanisms to justify the suggested hypothesis about this B chromosome origin are discussed in the present study...

Cromossomos B são cromossomos extras ao conjunto cromossômico normal, encontrado em diferentes organismos, com destaque para sua presença no grupo de peixes. Callichthys callichthys do alto rio Paraná tem um número diploide de 56 cromossomos (26 m-sm + 30 st-a) para ambos os sexos, com a presença esporádica de um cromossomo B acrocêntrico. Além do mais, um indivíduo apresentou número diploide de 57 cromossomos, com a presença de um cromossomo B acrocêntrico morfologicamente mal definido em todas as células analisadas. O mapeamento físico do DNAr 5S e 18S mostrou múltiplos sítios de DNAr 5S e apenas um par de cromossomos com sítio para o DNAr 18S em C. callichthys, com exceção para dois indivíduos. Estes dois indivíduos apresentaram um terceiro cromossomo portador das RONs (Ag-RONs e 18S rDNA), onde os genes DNAr 5S e 18S são sintênicos, diferindo apenas na posição. A dispersão dos genes DNAr 18S do par de cromossomos principal st-a 25 para um dos cromossomos do par m-sm 4 teria originado dois indivíduos variantes, um dos quais com cromossomo B acrocêntrico mal definido. Mecanismos para justificar a hipótese sugerida sobre a origem deste cromossomo B são discutidos no presente estudo...

Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

PRIMER REPORTE DE Cryptosporidium parvum EN TERNEROS HOLSTEIN (Bos taurus) DE MANIZALES, CALDAS, COLOMBIA / FIRST REPORT OF Cryptosporidium parvum IN HOLSTEIN CALVES (Bos taurus) FROM MANIZALES, CALDAS, COLOMBIA

El objetivo de este estudio fue identificar especies o genotipos del protozoario parásito Cryptosporidium presentes en heces colectadas de terneros Holstein del municipio de Manizales, Departamento de Caldas, Colombia. El ADN fue extraído a 80 muestras de materia fecal, de las cuales 11 fueron diagnosticadas positivas para Cryptosporidium spp., mediante reacción en cadena de la polimerasa (PCR). El análisis PCR-RFLP del locus 18S ADNr, identificó la presencia de Cryptosporidium parvum en todas las muestras positivas analizadas. Este hallazgo sugiere que el ganado puede ser una fuente potencial de infección por Cryptosporidium en humanos y se constituye en el primer reporte publicado de C. parvum en bovinos de Manizales, Caldas.

The objective of this study was to identify species or genotypes of Cryptosporidium parasite present in feces collected from Holstein calves in Manizales city, Caldas Department, Colombia. DNA was extracted from 80 fecal samples, which 11 were diagnosed positive for Cryptosporidium spp., by the Polymerase Chain Reaction method. PCR-RFLP analysis of 18S rDNA locus identified the presence of Cryptosporidium parvum in all samples tested positive. This finding suggests that cattle may be a potential source of human infection by Cryptosporidium, and it becomes the first published report of C. parvum in cattle in Manizales, Caldas.

Diversity of Culturable Soil Micro-fungi along Altitudinal Gradients of Eastern Himalayas

Very few studies have addressed the phylogenetic diversity of fungi from Northeast India under the Eastern Himalayan range. In the present study, an attempt has been made to study the phylogenetic diversity of culturable soil fungi along the altitudinal gradients of eastern Himalayas. Soil samples from 24 m above sea level to 2,000 m above sea level altitudes of North-East India were collected to investigate soil micro-fungal community structure and diversity. Molecular characterization of the isolates was done by PCR amplification of 18S rDNA using universal primers. Phylogenetic analysis using BLAST revealed variation in the distribution and richness of different fungal biodiversity over a wide range of altitudes. A total of 107 isolates were characterized belonging to the phyla Ascomycota and Zygomycota, corresponding to seven orders (Eurotiales, Hypocreales, Calosphaeriales, Capnodiales, Pleosporales, Mucorales, and Mortierellales) and Incertae sedis. The characterized isolates were analysed for richness, evenness and diversity indices. Fungal diversity had significant correlation with soil physico-chemical parameters and the altitude. Eurotiales and Hypocreales were most diverse and abundant group of fungi along the entire altitudinal stretch. Species of Penicillium (D = 1.44) and Aspergillus (D = 1.288) were found to have highest diversity index followed by Talaromyces (D = 1.26) and Fusarium (D = 1.26). Fungal distribution showed negative correlation with altitude and soil moisture content. Soil temperature, pH, humidity and ambient temperature showed positive correlation with fungal distribution.

Cytogenetic and molecular markers reveal the complexity of the genus Piabina Reinhardt, 1867 (Characiformes: Characidae)

Cytogenetic and molecular analyses were carried out in fish representative of the genus Piabina. This study specifically involved the species P. argentea and P. anhembi collected from areas of the Paranapanema and Tietê River basins, Brazil. Our findings suggest that fish classified as Piabina argentea in the Paranapanema and Tietê Rivers may represent more than one species. The samples analyzed differed by cytogenetic particularities and molecular analyses using partial sequences of the genes COI and CytB as genetic markers revealed three distinct groups of P. argentea with genetic distances sufficient to support the conclusion that the three samples analyzed are three distinct taxonomic units.

Foram realizadas análises citogenéticas e moleculares em representantes do gênero Piabina. O estudo envolveu especificamente as espécies P. argentea e P. anhembi coletadas nas áreas das bacias hidrográficas dos rios Paranapanema e Tietê (Brasil). Os dados sugerem que a espécie P. argentea coletada nas bacias dos rios Paranapanema e Tietê podem representar mais de uma espécie. As amostras analisadas diferem por particularidades citogenéticas e nas análises moleculares utilizando-se sequências parciais dos genes COI e CytB, revelando três grupos distintos de P. argentea com distâncias genéticas suficientes para sustentar a conclusão de que as três amostras analisadas são unidades taxonômicas distintas.

Karyotypic conservatism in samples of Characidium cf. zebra (Teleostei, Characiformes, Crenuchidae): physical mapping of ribosomal genes and natural triploidy

Basic and molecular cytogenetic analyses were performed in specimens of Characidium cf. zebra from five collection sites located throughout the Tietê, Paranapanema and Paraguay river basins. The diploid number in specimens from all samples was 2n = 50 with a karyotype composed of 32 metacentric and 18 submetacentric chromosomes in both males and females. Constitutive heterochromatin was present at the centromeric regions of all chromosomes and pair 23, had additional interstitial heterochromatic blocks on its long arms. The nucleolar organizer regions (NORs) were located on the long arms of pair 23, while the 5S rDNA sites were detected in different chromosomes among the studied samples. One specimen from the Alambari river was a natural triploid and had two extra chromosomes, resulting in 2n = 77. The remarkable karyotypic similarity among the specimens of C. cf. zebra suggests a close evolutionary relationship. On the other hand, the distinct patterns of 5S rDNA distribution may be the result of gene flow constraints during their evolutionary history.

Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region

We present filaria-nested polymerase chain reaction (PCR), which is based on amplification of first internal transcribed spacer rDNA to distinguish three parasitic filarial species (Onchocerca volvulus, Mansonella ozzardiand Mansonella perstans) that can be found in the Amazon Region. Nested PCR-based identifications yielded the same results as those utilizing morphological characters. Nested PCR is highly sensitive and specific and it detects low-level infections in both humans and vectors. No cross-amplifications were observed with various other blood parasites and no false-positive results were obtained with the nested PCR. The method works efficiently with whole-blood, blood-spot and skin biopsy samples. Our method may thus be suitable for assessing the efficacy of filaria control programmes in Amazonia by recording parasite infections in both the human host and the vector. By specifically differentiating the major sympatric species of filaria, this technique could also enhance epidemiological research in the region.

Morphological and molecular identification of Cyclospora-like organism from dogs / 中国人兽共患病学报

The morphological and molecular identifications of Cyclospora-like oocysts of dogs were underwent in the present study, in which the morphological characteristics of the Cyclospora-like oocysts firstly found in the stool samples of dogs, such as shape, size, acid-fast staining,sporulation and auto-fluorescene, were observed. According to the published sequence of the rDNA gene of Cyclospora in GenBank, 3 primers were designed and were used to amplify part of the 18S rDNA gene of dog-associated Cyclospora-like organism by nested PCR.The amplicons were purified and cloned into vector pMD19T. Then, the positive clones screened were sequenced and subjected to sequence homology and phylogenetic analysis. It was found that the morphological charactertistics of the Cyclospora-like oocysts in dogs were similar to that of the human Cyclospoa oocysts and the size of-the amplified fragment of 18S rDNA was proved to be 715 bp, that was identical to that of the target fragment. Based on the results of sequence homology and phylogenetic analysis, the dog-associated Cyclospora-like organism was identified as the Cyclospora species.

Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA3) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO3 staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.

Cytogenetic data on Astyanax jacuhiensis (Characidae) in the lago Guaíba and tributaries, Brazil

Cytogenetic analyses were performed in Astyanax jacuhiensis from lago Guaíba, Brazil. The diploid number was 50, with a karyotype composed of 8m+30sm+4st+8a chromosomes, FN = 92. The AgNORs were observed in 2 to 5 chromosomes, with intra- and interindividual variation. The sm pair 8 observed always carried NORs on the short arms, presenting size heteromorphism between homologous. Fluorescence in situ hybridization (FISH) with an 18S rDNA probe only confirmed the location of ribosomal cistrons in the sm pair 8, and heteromorphism of these regions between the homologous chromosomes. C-banding revealed the occurrence of weak C-positive heterochromatin in the pericentromeric regions of several chromosomes, in addition to more evident bands interstitially located on some chromosome pairs and in the terminal region of the short arms in pair 8. C-banding plus CMA3 revealed light fluorescent signals in different chromosomes of the karyotype, with a strong terminal site in pair 8, indicating the occurrence of several GC-rich heterochromatic regions in this species. Our results provide the first description of the Astyanax jacuhiensis karyotype, showing karyotype similarities when compared to various populations of A. altiparanae and A. bimaculatus, indicating that chromosomal features are very similar for these three species.

Análises citogenéticas foram realizadas em Astyanax jacuhiensis do lago Guaíba, Brasil. O número diplóide foi 50, sendo o cariótipo composto por 8m+30sm+4st+8a cromossomos, NF = 92. As regiões organizadoras de nucléolos (AgNORs) foram observadas em 2 a 5 cromossomos, evidenciando uma variação intra e interindividual nesta espécie. O par sm 8 foi constantemente detectado com NORs nos braços curtos, mostrando um heteromorfismo de tamanho entre os homólogos. Entretanto, a hibridação in situ fluorescente (FISH) com sonda de DNAr 18S, localizou cístrons ribossômicos apenas no par 8, confirmando o heteromorfismo de tamanho entre os homólogos. O bandamento C revelou a presença de bandas discretas de heterocromatina na região pericentromérica da maioria dos cromossomos, além de algumas bandas mais evidentes intersticiais, bem como na região terminal dos braços curtos do par 8. A associação de BC+CMA3 evidenciou marcações fluorescentes mais discretas em diferentes cromossomos e uma forte marcação terminal no par 8, confirmando vários sítios de heterocromatina GC-rica nessa espécie. Nossos resultados fornecem a primeira descrição do cariótipo de Astyanax jacuhiensis, apresentando semelhanças em relação ao cariótipo de diferentes populações de A. altiparanae e A. bimaculatus, indicando que as características cromossômicas são muito semelhantes para estas três espécies.