Morphological and molecular characterization of Austrodiplostomum compactum metacercariae in the eyes and brains of fishes from the Ivaí River, Brazil / Caracterização morfológica e molecular de metacercárias de Austrodiplostomum compactum em olhos e cérebros de peixes do Rio Ivaí, Brasil

Abstract Austrodiplostomum spp. (Platyhelminthes: Digenea) are endoparasites with a broad geographic distribution in South America. During the larval stage, they parasitize the eyes, brains, muscles, gill, kidneys and swim bladder of a wide variety of fishes. The metacercariae of Austrodiplostomum spp. have several morphological characteristics during development, but are very similar among species, which makes it necessary to use molecular tools to contribute to the elucidation during the larval stage. The objective of this study was to perform morphological and molecular analyses of Austrodiplostomum sp. found in specimens of Hypostomus sourced from the Ivaí River in the state of Paraná, Brazil. Of the 93 analyzed specimens (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], and Hypostomus sp. 2 [n = 10]), 60 were parasitized. A total of 577 Austrodiplostomum sp. metacercariae was collected from the infected hosts; DNA from seven of these samples was extracted, amplified, and sequenced. The morphological data associated with the genetic distance values and the relationships observed in the COI gene tree, indicate that all metacercariae were A. compactum. This is the first record of A. compactum parasitizing H. hermanni, H. albopunctatus, Hypostomus sp. 1, and Hypostomus sp. 2 in the Ivaí River.

Resumo Austrodiplostomum spp. (Platyhelminthes: Digenea) são endoparasitos com uma ampla distribuição geográfica na América do Sul. Durante a fase larval, parasitam os olhos, cérebros, músculos, brânquias, rins e bexiga natatória de uma grande variedade de peixes. As metacercárias de Austrodiplostomum spp. apresentam várias características morfológicas durante o desenvolvimento, as quais são muito semelhantes entre as espécies, o que torna necessário o uso de ferramentas moleculares para contribuir para a elucidação durante a fase larval. O objetivo deste estudo foi realizar análises morfológicas e moleculares de Austrodiplostomum sp. encontradas em espécimes de Hypostomus provenientes do rio Ivaí, no Paraná, Brasil. Dos 93 espécimes analisados (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], e Hypostomus sp. 2 [n = 10]), 60 foram parasitados. Um total de 577 metacercárias de Austrodiplostomum foram coletadas dos hospedeiros infectados; o DNA de sete dessas amostras foi extraído, amplificado e sequenciado. Os dados morfológicos, associados aos valores de distância genética e as relações observadas na árvore gênica do COI, indicam que todas as metacercárias são A. compactum. Este é o primeiro registo de A. compactum parasitando H. hermanni, H. albopunctatus, Hypostomus sp. 1, e Hypostomus sp. 2 no rio Ivaí.

Molecular classification and comparative phylogeographic study of insectivorous bat species (Pipisitrellus coromandra) from Punjab, Pakistan / Classificação molecular e estudo filogeográfico comparativo de espécies de morcegos insetívoros (Pipistrellus coromandra) de Punjab, Paquistão

Molecular based identification of bat fauna in Pakistan has been relatively less explored. The current study was therefore planned to report for the first time the molecular classification of insectivorous bats (Pipistrellus coromandra) based on mitochondrion gene (COI) from Punjab, Pakistan. Specimens were collected from five different locations followed by DNA extraction with subsequent gene amplification and sequencing. All samples in the study had shown close identity matches with species (Pipistrellus coromandra) from India and (Pipistrellus tenuis) from Vietnam with percentage identity score of 96.11 and 95.58 respectively except one sequence which only revealed 86.78% identity match on Basic Local Alignment Search Tool (BLAST) and could only be assigned to genus level Pipistrellus sp. The results indicated negligible intra-population genetic distance among collected samples whereas the comparison with species from other countries had shown high intraspecific (P. coromandra) and interspecific (P. tenuis) mean genetic distances. The current study hence successfully proved the efficiency of COI gene as a molecular marker for species identification and in analyzing the patterns of genetic variation with species from other countries.

A identificação com base molecular da fauna de morcegos no Paquistão tem sido relativamente menos explorada. Portanto, o estudo atual foi planejado para relatar pela primeira vez a classificação molecular de morcegos insetívoros (Pipistrellus coromandra) com base no gene da mitocôndria (COI) de Punjab, Paquistão. As amostras foram coletadas em cinco locais diferentes, seguidas pela extração de DNA com subsequente amplificação e sequenciamento do gene. Todas as amostras no estudo mostraram coincidências de identidade próximas com espécies (Pipistrellus coromandra) da Índia e (Pipistrellus tenuis) do Vietnã, com pontuação de identidade percentual de 96,11 e 95,58, respectivamente, exceto uma sequência que revelou apenas 86,78% de correspondência de identidade na Ferramenta de Pesquisa de Alinhamento Local Básico (BLAST), a qual só poderia ser atribuída ao nível de gênero Pipistrellus sp. Os resultados indicaram distância genética intrapopulacional desprezível entre as amostras coletadas, enquanto a comparação com espécies de outros países mostrou altas distâncias genéticas intraespecíficas (P. coromandra) e interespecíficas (P. tenuis) médias. O presente estudo, portanto, comprovou com sucesso a eficiência do gene COI como marcador molecular para identificação de espécies e análise dos padrões de variação genética com espécies de outros países.

Molecular classification and comparative phylogeographic study of insectivorous bat species (Pipisitrellus coromandra) from Punjab, Pakistan / Classificação molecular e estudo filogeográfico comparativo de espécies de morcegos insetívoros (Pipistrellus coromandra) de Punjab, Paquistão

Molecular based identification of bat fauna in Pakistan has been relatively less explored. The current study was therefore planned to report for the first time the molecular classification of insectivorous bats (Pipistrellus coromandra) based on mitochondrion gene (COI) from Punjab, Pakistan. Specimens were collected from five different locations followed by DNA extraction with subsequent gene amplification and sequencing. All samples in the study had shown close identity matches with species (Pipistrellus coromandra) from India and (Pipistrellus tenuis) from Vietnam with percentage identity score of 96.11 and 95.58 respectively except one sequence which only revealed 86.78% identity match on Basic Local Alignment Search Tool (BLAST) and could only be assigned to genus level Pipistrellus sp. The results indicated negligible intra-population genetic distance among collected samples whereas the comparison with species from other countries had shown high intraspecific (P. coromandra) and interspecific (P. tenuis) mean genetic distances. The current study hence successfully proved the efficiency of COI gene as a molecular marker for species identification and in analyzing the patterns of genetic variation with species from other countries.

A identificação com base molecular da fauna de morcegos no Paquistão tem sido relativamente menos explorada. Portanto, o estudo atual foi planejado para relatar pela primeira vez a classificação molecular de morcegos insetívoros (Pipistrellus coromandra) com base no gene da mitocôndria (COI) de Punjab, Paquistão. As amostras foram coletadas em cinco locais diferentes, seguidas pela extração de DNA com subsequente amplificação e sequenciamento do gene. Todas as amostras no estudo mostraram coincidências de identidade próximas com espécies (Pipistrellus coromandra) da Índia e (Pipistrellus tenuis) do Vietnã, com pontuação de identidade percentual de 96,11 e 95,58, respectivamente, exceto uma sequência que revelou apenas 86,78% de correspondência de identidade na Ferramenta de Pesquisa de Alinhamento Local Básico (BLAST), a qual só poderia ser atribuída ao nível de gênero Pipistrellus sp. Os resultados indicaram distância genética intrapopulacional desprezível entre as amostras coletadas, enquanto a comparação com espécies de outros países mostrou altas distâncias genéticas intraespecíficas (P. coromandra) e interespecíficas (P. tenuis) médias. O presente estudo, portanto, comprovou com sucesso a eficiência do gene COI como marcador molecular para identificação de espécies e análise dos padrões de variação genética com espécies de outros países.

Molecular classification and comparative phylogeographic study of insectivorous bat species (Pipisitrellus coromandra) from Punjab, Pakistan

Abstract Molecular based identification of bat fauna in Pakistan has been relatively less explored. The current study was therefore planned to report for the first time the molecular classification of insectivorous bats (Pipistrellus coromandra) based on mitochondrion gene (COI) from Punjab, Pakistan. Specimens were collected from five different locations followed by DNA extraction with subsequent gene amplification and sequencing. All samples in the study had shown close identity matches with species (Pipistrellus coromandra) from India and (Pipistrellus tenuis) from Vietnam with percentage identity score of 96.11 and 95.58 respectively except one sequence which only revealed 86.78% identity match on Basic Local Alignment Search Tool (BLAST) and could only be assigned to genus level Pipistrellus sp. The results indicated negligible intra-population genetic distance among collected samples whereas the comparison with species from other countries had shown high intraspecific (P. coromandra) and interspecific (P. tenuis) mean genetic distances. The current study hence successfully proved the efficiency of COI gene as a molecular marker for species identification and in analyzing the patterns of genetic variation with species from other countries.

Resumo A identificação com base molecular da fauna de morcegos no Paquistão tem sido relativamente menos explorada. Portanto, o estudo atual foi planejado para relatar pela primeira vez a classificação molecular de morcegos insetívoros (Pipistrellus coromandra) com base no gene da mitocôndria (COI) de Punjab, Paquistão. As amostras foram coletadas em cinco locais diferentes, seguidas pela extração de DNA com subsequente amplificação e sequenciamento do gene. Todas as amostras no estudo mostraram coincidências de identidade próximas com espécies (Pipistrellus coromandra) da Índia e (Pipistrellus tenuis) do Vietnã, com pontuação de identidade percentual de 96,11 e 95,58, respectivamente, exceto uma sequência que revelou apenas 86,78% de correspondência de identidade na Ferramenta de Pesquisa de Alinhamento Local Básico (BLAST), a qual só poderia ser atribuída ao nível de gênero Pipistrellus sp. Os resultados indicaram distância genética intrapopulacional desprezível entre as amostras coletadas, enquanto a comparação com espécies de outros países mostrou altas distâncias genéticas intraespecíficas (P. coromandra) e interespecíficas (P. tenuis) médias. O presente estudo, portanto, comprovou com sucesso a eficiência do gene COI como marcador molecular para identificação de espécies e análise dos padrões de variação genética com espécies de outros países.

Integrative taxonomy reveals a new species of pacu (Characiformes: Serrasalmidae: Myloplus) from the Brazilian Amazon

Pacus of the genus Myloplus represent a formidable taxonomic challenge, and particularly so for the case of M. asterias and M. rubripinnis, two widespread and common species that harbor considerable morphological diversity. Here we apply DNA barcoding and multiple species discovery methods to find candidate species in this complex group. We report on one well-supported lineage that is also morphologically and ecologically distinct. This lineage represents a new species that can be distinguished from congeners by the presence of dark chromatophores on lateral-line scales, which gives the appearance of a black lateral line. It can be further diagnosed by having 25-29 branched dorsal-fin rays (vs. 18-24), 89-114 perforated scales from the supracleithrum to the end of hypural plate (vs. 56-89), and 98-120 total lateral line scales (vs. 59-97). The new species is widely distributed in the Amazon basin, but seems to have a preference for black- and clearwater habitats. This ecological preference and black lateral line color pattern bears a striking similarity to the recently described silver dollar Metynnis melanogrammus.(AU)

Pacus do gênero Myloplus representam um desafio taxonômico formidável, e particularmente o caso de M. asterias e M. rubripinnis, duas espécies amplamente distribuídas e comuns que abrigam uma considerável diversidade morfológica. Aplicamos aqui a tecnologia do DNA barcoding e múltiplos métodos de descoberta de espécies para encontrar possíveis espécies novas nesse grupo complexo. Registramos uma linhagem bem suportada que também é distinta morfológica e ecologicamente. Essa linhagem representa uma nova espécie que pode ser distinguida das demais congêneres por apresentar cromatóforos escuros nas escamas da linha lateral que conferem uma aparência de linha lateral preta. Ela pode ser adicionalmente diagnosticada por ter 25-29 raios ramificados na nadadeira dorsal (vs. 18-24), 89-114 escamas perfuradas do supracleitro até o final da placa hipural (vs. 56-89) e 98-120 escamas totais na linha lateral (vs. 59-97). A nova espécie é amplamente distribuída na bacia Amazônica, mas aparentemente possui preferência por habitats de água preta e clara. A preferência ecológica e o padrão de colorido escuro da linha lateral consistem em semelhanças impressionantes com o silver dólar recém descrito Metynnis melanogrammus.(AU)

DNA barcoding of 4 species of cheyletid mites based on COI and 18S rRNA gene sequences / 中国血吸虫病防治杂志

Objective To analyze the sequences of mitochondrial cytochrome C oxidase subunit I gene (COI) and 18S ribosomal RNA gene (18S rRNA), so as to identify the feasible DNA barcodes for 4 species of cheyletid mites and improve the DNA barcoding database for cheyletid mites. Methods Cheyletid mite samples were collected from small-scale flour mills in Fuyang, Wuhu and Tongling cities of Anhui Province from May 2018 to July 2019, extracted and morphologically identified. Then, genomic DNA was extracted from a single cheyletid mite, and the COI and 18S rRNA gene sequences were obtained by PCR amplification, cloning and sequencing. The obtained sequences were aligned using the BLAST software. Multiple sequence alignment was done using the software ClustalX version 1.83 using the known gene sequences from cheyletid mites. The genetic distance was calculated using the software MEGA X, and the phylogenetic tree was created using the maximum likelihood method. Results The DNA barcoding results of Cheyletus malaccensis, C. carnifex and Cheletomorpha lepidopterorum were consistent with the morphological identification, while no sequences pertaining to Eucheyletia reticulate were retrieved in the GenBank database. The proportions of A + T were 69.6% and 55.1% in the COI and 18S rRNA sequences of 4 cheyletid mites species, respectively, and the numbers of base substitutions were 137 and 46, respectively. There were 154 to 321 and 58 to 99 inter-species variation loci in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, respectively, and the intra-species genetic distance was all 0.020 or less in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, with inter-species genetic distance of 0.235 to 0.583 and 0.078 to 0.114, respectively. Phylogenetic analysis based on COI and 18S rRNA genes showed that all four species of cheyletid mites were clustered into a branch with a 100% supportive rate, which was consistent with the morphological identification. Conclusion Mitochondrial COI gene is superior to 18S rRNA gene as DNA barcodes for 4 species of cheyletid mites, which is more suitable to be used to investigate the phylogenetic relationship of at genus and species levels.

Population genetics of invasive Pomacea spp. in Hangzhou City, Zhejiang Province / 中国血吸虫病防治杂志

Objective To investigate the distribution and identify the genetic genetics of invasive Pomacea species in Xihu District, Hangzhou City, so as to understand the spread tendency of Pomacea species. Methods The specimens of Pomacea species were collected from five sites in water systems (lakes, rivers and wetlands) and its costal lands in Xihu District, Hangzhou City in 2017 for morphological identification. Total DNA was isolated from the foot tissues of adult snails for amplification of the COI gene, and haplotype diversity and nucleic acid diversity analyses were performed. In addition, a phylogenetic tree was created based on the haplotype captured from GenBank and those from this study to investigate the phylogenetic relationships. Results Pomacea specimens, which were preliminarily characterized as Pomacea, were found in ponds, rivers and wetlands in Xihu District of Hangzhou City. A total of 16 sequences were captured from the DNA samples of Pomacea specimens, which belonged to 3 haplotypes, including Hap1, Hap2 and Hap3. A high frequency was seen in Hap1 and Hap3, and a low frequency was found in Hap2. The Pomacea specimens collected from the 5 sites in Xihu Districts included P. canaliculata and P. maculate. The Pomacea specimens with a Hap1 had a close genetic relationship with the P. canaliculata from Argentina, Guangdong Province and Hong Kong Special Administrative Region of China, and the Pomacea specimens with a Hap2 had a close genetic relationship with the P. canaliculata from Argentina, Japan and Guangzhou City of Guangdong Province, China, while the Pomacea specimens with a Hap2 had a close genetic relationship with the P. maculate from Argentina and Brazil. Conclusions P. canaliculata and P. maculata are present in Xihu District of Hangzhou City. P. maculata may spread to Xihu District through multiple introductions or water flow.

Molecular identification of Tricula spp. and the parasitized trematode cercariae in schistosomiasis-endemic areas of Yunnan Province / 中国血吸虫病防治杂志

Objective To characterize a species of the genus Tricula and parasitized trematodes in schistosomiasis-endemic areas of Yunnan Province using a molecular analysis, so as to understand their taxonomic positions. Methods Tricula spp. and Oncomelania snails were collected from Xiangyun County, Yunnan Province, and cercaria parasitizing snails were observed using crushing followed by microscopy. Cercaria parasitizing Tricula snails at various morphologies were sampled using a shedding method. Genomic DNA was extracted from snail soft tissues and cercariae, and the 16S rRNA, COI, 28S rDNA genes in snails and the ND1 and 28S rDNA genes in cercariae were amplified using a PCR assay and sequenced. The species of Tricula snails and their parasitized trematodes was characterized using sequence alignment and phylogenetic analysis. Results Among 382 Tricula snails detected, there were three types of trematode cercariae found, including the non-forked (20.94%, 80/382), double-forked (3.40%, 13/382) and swallow shapes (7.07%, 27/382). Sequence and phylogenetic analyses showed that the 16S rRNA, COI and 28S rDNA gene sequences of this species of Tricula had high homology to those in Delavaya dianchiensis, and were clustered in a branch. Sequencing analysis of the ND1 and 28S rDNA genes revealed that the non-forked cercariae belonged to the family Pleu- rogenidae, the swallow-shaped cercariae belonged to the family Opecoelidae, and the double-forked cercariae belonged to another species of the genus Schistosoma that was different from S. sinensium and S. ovuncatum. Conclusion The species and taxonomy of Triculla spp. and their parasitized trematodes are preliminarily determined in schistosomiasis-endemic areas of Yunnan Province; however, further studies are required to investigate the more definite taxonomy and pathogenicity.

Authentication of commercially available frozen shrimp meats using DNA barcoding

Shrimps and prawns are quite nutritious and provide quality protein, omega-3 fatty acids, certain antioxidants,vitamins, and minerals like iodine. Therefore, they are healthy food for human consumption. In order to findout fish fraud in value-added products, in this study, six frozen shrimp species packed and sold by differentcommercial companies were purchased, correctly identified using DNA barcoding of mt-COI gene, and thesequences were authenticated properly with the GenBank. The genomic DNA isolated from frozen shrimpsamples showed greater than 10 kb. Their amplified products showed 564–770 bp of mt-COI gene. The BLASTresults for the labeled frozen shrimp sample Litopenaeus vannamei showed maximum similarity (99%–100%)with the same species sequences available with NCBI database. Five unlabeled sample sequences (two samplestaken from two independent packets, and three samples taken from one mixed shrimp packet) showed maximumsimilarity with the sequences available in the NCBI database and exactly identified as Fenneropenaeusmerguiensis, Penaeus monodon, Fenneropenaeus indicus, Penaeus semisulcatus, and P. monodon, respectively.These six sequences showed more variable amino acid sites than the identical and similar amino acid residueswithin themselves. The AT biases (61.5%–67.51%, P. semisulcatus and P. monodon taken from unlabeledmixed packet) and GC biases (32.9%–38.6%, P. monodon and P. semisulcatus) varied among these six speciessequences, which indicated less NUMTs gene. The interspecies divergence rate calculated for these six speciesranged between 0.434 and 3.401 (L. vennamei vs. F. indicus, and F. merguiensis vs. P. monodon, taken fromunlabeled independent packet). Thus, it is recommended that DNA barcoding of mt-COI gene can be used toidentify any unlabeled or labeled shrimp/prawn/seafood available in the market to avoid fish fraud

Application of DNA barcoding to identification of rodents in Zhejiang Province / 预防医学

Objective@# To apply DNA barcoding to identifying the rodents in Zhejiang Province. @*Methods @#Rodents were captured from Jiashan,Longyou,Yunhe and Ninghai counties in Zhejiang Province. The DNA was extracted from ears of rodent samples,and was amplified and sequenced with mitochondrial cytochrome C oxidase subunit I（COI）genes. The obtained sequences were compared with the related sequences in GenBank,and neighbour-joining evolutionary tree was constructed. Then the results by DNA barcoding and by morphological identification were compared. @*Results @#A total of 22 COI gene samples were amplified. The evolutionary tree constructed by 18 samples was consistent with the morphological identification results and 4 samples were different：Suncus murinus should be Crocidura lasiura,infant rats of Rattus losea and Rattus tanezumi was re-identified as Rattus rattus,infant rats of Microtus fortis（sample number：NH-1）needs further identification. @*Conclusion @#DNA barcoding can effectively correct the errors of morphological identification,thus combining the two methods could improve the accuracy of rodent identification.

Application progress on mitochondrial DNA markers in identification of animal medicinal materials / 中草药

Animal medicinal materials are important parts of traditional Chinese medicine (TCM) with a long history and remarkable efficacy. Due to the destruction of wild animal resources and increasingly market demands, the adulterations of animals medicinal materials have become more common in medicine market and bring security risks for clinical application. In recent years, mitochondrial DNA is widely used in the field of animal population genetics, phylogeography, and phylogenetic development due to its maternally inherited features and abundant genetic diversity, and has achieved fruitful results in the field of molecular identification, which provides technical support for quality control of animal medicinal materials. This paper summarizes the application value and research status of COI, Cyt b, and 12S rRNA in the identification of animal medicinal materials, and gives a brief discussion on the follow-up development of mtDNA marker technique in the identification of animal medicinal materials in combination with previous result, which provides technical support for reasonable utilization of medicinal animal resources.

Genetic characteristics of Spermophilus in Shaanxi Province, China / 中国人兽共患病学报

We studied the genetic characteristics of Spermophilus in Shaanxi Province,China.The COI,Cyt-b gene were sequenced and the results were compared with those of dauricus from Inner Mongolia Keyouzhong Banner and Zhengxiangbai Banner,and S.alaschanicus from Haiyuan County of Ningxia.And genetic distance was analyzed and Neighbor-Joining tree was built.Results showed that the genetic distance of COI gene sequences between Spermophilus from Dingbian County in Shaanxi and S.alaschanicus in Ningxia was ≤0.5％,and the genetic distance was ranged from 7.9％ to 9.3％ with Citellus dauricus from Inner Mongolia.The genetic distance of Cyt-b gene between Spermophilus from Dingbian County in Shaanxi and S.alaschanicus in Ningxia was ≤2.2％,and ranged from 8.9％ to 11.2％ with Citellus dauricus from Inner Mongolia.The Neighbor-Joining tree of COI,Cyt-b gene showed two major clusters.One of them were clustered by Spermophilus from Dingbian County in Shaanxi and S.alaschanicus in Ningxia,and another one was Citellus dauricus from Inner Mongolia.The Neighbor-Joining tree of COI gene showed that all samples from Shaanxi Province clustered in a group.In conclusion,the Spermophilus in Shaanxi Province were S.alaschanicus.

Epidemiology and COI gene heredity evolutionary analysis of Gasterophilus third instars / 中国兽医学报

In order to investigate the species and categorization of Gasterophilus in Ili horse.We analysised the COI gene of the identified Gasterophilus dominant species and constructed NJ phylogenetic tree in the study.The results showed that infection rate was 100％ in total of 16 775 the third phase Gasterophilus instar larvae.Four Gasterophilus species were identified,and showed serious mix infections.Dominant species were Gasterophilus nasalis,its relative dominance were 53.17％,and prefer to live in the cardia,others to irregular live in the pylorus of the horses.COI gene homology of GasterophiIus nasalis,Gasterophilus intestinalis,Gasterophilus pecorum,Gasterophilus haemorrhoidalis (GenBank Accession No.:GU265752.1,KR230402.1,KU578262.1,KT946620.1) were 99％,99％,99％ and 100％ respectively.Phylogenetic analysis results showed that the data were clustered with the Gasterophilus app.which publshed on the GenBank.G.intestinalis and G.haemorrhoidalis cluster together first,and then cluster with G.nasalis,at last all three kinds of Gasterophilus cluster with G.pecorum.When the COI gene is the target,in-group and out-group of the Gasterophilus can forms an independent evolutionary branch.This study provides useful parameters for the classification of Gasterophilus.

Identification of two Bithynia species from Heng County,Guangxi Zhuang Autonomous Region,China by using morphological and DNA barcoding methods / 中国血吸虫病防治杂志

Objective To distinguish two Bithynia species,Bithynia fuchsiana and Bithynia robusta collected from Heng County,Guangxi Zhuang Autonomous Region,by using morphological and DNA barcoding methods. Methods The adult B. fuchsiana and B. robusta were collected from the biotope such as rivers,ditches and ponds in Heng County of Guangxi Zhuang Autonomous Region,China. The two species specimens were identified by measuring shell morphological parameters,compar?ing the characters of the male reproductive system,and using the COI gene barcoding technique and building phylogenetic tree. Results B. fuchsiana and B. robusta were similar morphologically in the shell appearance;they had the similar snail height, snail width,shape and male reproductive structure. The DNA sequence analysis showed that the COI gene of the two Bithynia species had low sequence divergence with 11 variation sites among 22 sequences. The length of the COI gene segment was 517 bp and no insertion sites and deletion loci after sequence edited. All individuals of the two species gathered to one clade in the phylogenetic tree based on COI gene. Conclusion According to the evidence of morphology and COI gene coding sequence,B. fuchsiana and B. robusta from Heng County,Guangxi Zhuang Autonomous Region,are likely to be the same species.

Population genetics of Chrysoperla externa (Neuroptera: Chrysopidae) and implications for biological control / Genética de populações de Chrysoperla externa (Neuroptera: Chrysopidae) e implicações para o controle biológico

Abstract Green lacewings are insects with great potential to be use in the biological control of agricultural pests, but relatively few studies have attempted to understand the genetic structure of these agents, especially those of predatory insects. The purpose of this study was to characterize genetically populations of C. externa using sequences of subunit I of the cytochrome oxidase, a mitochondrial gene, and examine the population structure of this species in sampled areas in São Paulo state. The results indicate high genetic diversity but no genetic structure, detected by AMOVA analysis, and high levels of haplotype sharing in the network. These genetic patterns could be a consequence of environmental homogeneity provided by agroecosystem (citrus orchard), allowing gene flow among populations. Probably there is a unique population in the area sampled that could be used as a population (genetic) source for mass-reared and posterior release in these farms.

Resumo Crisopídeos são insetos com grande potencial para uso em controle biológico de pragas agrícolas, mas relativamente poucos estudos têm tentado compreender a estrutura genética destes agentes, especialmente no caso de insetos predadores. O objetivo deste trabalho foi caracterizar geneticamente populações de C. externa utilizando sequências da subunidade I do gene mitocondrial citocromo oxidase e avaliar a estruturação populacional desta espécie em áreas amostras no estado de São Paulo. Os resultados indicaram elevada diversidade genética e nenhuma estruturação genética, detectada pela AMOVA, além de elevado compartilhamento na rede haplotípica. Este padrão genético poderia ser uma consequência da homogeneidade ambiental favorecida pelos agroecossistemas (citricultura), permitindo fluxo gênico entre as populações. Provavelmente há uma única população, do ponto de vista genético, na área amostrada que poderia ser utilizada em criações massais e em liberações nas fazendas desta região.

DNA barcode of parodontidae species from the La Plata river basin - applying new data to clarify taxonomic problems

In the past years, DNA barcoding has emerged as a quick, accurate and efficient tool to identify species. Considering the difficulty in identifying some Parodontidae species from the La Plata basin and the absence of molecular data for the group, we aimed to test the effectiveness of DNA barcoding and discuss the importance of using different approaches to solve taxonomic problems. Eight species were analyzed with partial sequences of Cytochrome c oxidase I. The mean intraspecific K2P genetic distance was 0.04% compared to 4.2% for mean interspecific K2P genetic distance. The analyses of distance showed two pairs of species with K2P genetic divergence lower than 2%, but enough to separate these species. Apareiodon sp. and A. ibitiensis, considered as the same species by some authors, showed 4.2% genetic divergence, reinforcing their are different species. Samples of A. affinis from the Uruguay and Paraguay rivers presented 0.3% genetic divergence, indicating a close relationship between them. However, these samples diverged 6.1% from the samples of the upper Paraná River, indicating that the latter represents a potentially new species. The results showed the effectiveness of the DNA barcoding method in identifying the analyzed species, which, together with the morphological and cytogenetic available data, help species identification.

Nos últimos anos o DNA barcoding surgiu como uma ferramenta rápida, precisa e eficiente para identificar espécies. Considerando a dificuldade na identificação de algumas espécies de Parodontidae da bacia do rio da Prata e da ausência de dados moleculares para o grupo, testamos a eficácia do código de barras de DNA e discutimos a importância do uso de diferentes abordagens para resolver problemas taxonômicos. Oito espécies foram analisadas com sequencias parciais do gene citocromo c oxidase I. A distância genética média K2P intraespecífica foi de 0,04% comparado com 4,2% para distância genética média K2P interespecífica. As análises de distância mostraram dois pares de espécies com divergência genética K2P inferior a 2%, mas o suficiente para separar estas espécies. Apareiodon sp. e A. ibitiensis, consideradas a mesma espécie por alguns autores, mostraram 4,2% de divergência genética, confirmando serem espécies diferentes. Amostras de A. affinis dos rios Uruguai e Paraguai apresentaram 0,3% de divergência genética, indicando um maior grau de relação entre elas, no entanto, esses exemplares divergiram em 6,1% em relação aos exemplares do alto rio Paraná, o que indica que estes últimos representam uma espécie potencialmente nova. Os resultados mostraram a eficácia do método de DNA barcoding na identificação das espécies analisadas, os quais, em conjunto com os dados morfológicos e citogenéticos disponíveis auxiliam na identificação inequívoca das espécies.

DNA barcoding of common medicinal snakes in China / 中国药学杂志

OBJECTIVE: To establish a DNA barcode database of common medicinal snakes in China for species identification and phylogenetic analysis. METHODS: The COI barcode sequences of 44 samples from 23 snake species were amplified and sequenced. The aligned sequences, 658 base pairs in length, were analyzed for divergence using the Kimura-2-parameter distance model with MEGA5.0. RESULTS: The genetic distance and NJ tree demonstrated that there were 277 variable sites in species, the intraspe-cific K2P distance ranged from 0-0.0409, the interspecific genetic distance ranged from 0.085 0-0.2568, and there was significant difference between the interspecific genetic distance and intraspecific genetic distance. The minimum interspecific distance was 0.0850 between Lycodon synaptor and Lycodon ruhstrati. The maximum interspecific distance was 0.2568 between Elaphe radiate and Naja atra. The snakes examined in this study were clustered in three major clades corresponding to their three families. Each species formed a monophyletic clad. CONCLUSION: DNA barcoding based on the COI sequences is accurate and reliable and could be applied to identify the common medicinal snakes in our study. The study provides new information for the phylogenetic relationships of snake species.

Occurrence of the Indo-Pacific freshwater prawn Macrobrachium equidens Dana 1852 (Decapoda, Palaemonidae) on the coast of Brazilian Amazonia, with notes on its reproductive biology

The freshwater prawn Macrobrachium equidens, which is native species of the Indo-Pacific Region, was recorded for the first time on the Amazon coast of Brazil. This species was found to inhabit the same environment as two native Macrobrachium species, M. amazonicum and M. acanthurus, and is morphologically very similar to the latter. The identification of the species was confirmed by the genetic analysis of sequences of the mitochondrial Cytochrome Oxidase (COI) gene. A detailed description of the morphological features and reproductive biology of M. equidens in this new environment is presented.

O camarão de água doce Macrobrachium equidens, nativo da região do Indo-Pacífico, foi registrada pela primeira vez na costa da Amazônia Brasileira. Esta espécie foi encontrada habitando o mesmo ambiente que duas espécies nativas do gênero Macrobrachium: M. amazonicum e M. acanthurus, e é morfologicamente muito similar à última. A identificação dessa espécie foi confirmada pela análise da seqüência genética do gene mitocondrial Citocromo Oxidase (COI). Uma descrição detalhada das características morfológicas e biologia reprodutiva de M. equidens neste novo ambiente é apresentada.

A new species of Tetragonopterus Cuvier, 1816 (Characiformes: Characidae: Tetragonopterinae) from the rio Jari, Amapá, northern Brazil

Uma espécie nova de Tetragonopterus é descrita do rio Jari, um tributário da margem esquerda do rio Amazonas, na divisa entre os Estados do Amapá e Pará, norte do Brasil. Esta é morfologicamente diferenciada das outras espécies do gênero (T. argenteus, T. chalceus e T. rarus combinação nova) pelo formato losangular da mancha no pedúnculo caudal contra uma mancha arredondada a quadrangular nas demais espécies. Sequências parciais do gene mitocondrial citocromo oxidase C subunidade I de representantes de todas as espécies válidas de Tetragonopterus, incluindo esta espécie nova, foram analisadas. Os resultados obtidos revelaram uma significante distância genética entre esta espécie nova e as demais do gênero. É apresentada uma discussão sobre a nova combinação, Tetragonopterus rarus.

A new species of Tetragonopterus is described from the rio Jari, a tributary to the left margin of rio Amazonas, at the border between Amapá and Pará States, northern Brazil. It is morphologically diagnosed from the other species of the genus (T. argenteus, T. chalceus, and T. rarus new combination) by the lozenge-shaped spot on the caudal peduncle vs. rounded to square spot on the other species. Partial sequences of the mitochondrial gene Cytochrome Oxidase C subunit I, from representatives of all valid species of Tetragonopterus, including this new species, were analyzed. The obtained results revealed a significant genetic distance between the new species and its congeners. A discussion on the new combination, Tetragonopterus rarus, is also provided.

Molecular identification, phylogeny and geographic distribution of Brazilian mangrove oysters (Crassostrea)

Oysters (Ostreidae) manifest a high degree of phenotypic plasticity, whereby morphology is of limited value for species identification and taxonomy. By using molecular data, the aim was to genetically characterize the species of Crassostrea occurring along the Brazilian coast, and phylogenetically relate these to other Crassostrea from different parts of the world. Sequencing of the partial cytochrome oxidase c subunit I gene (COI), revealed a total of three species of Crassostrea at 16 locations along the Brazilian coast. C. gasar was found from Curuçá (Pará state) to Santos (São Paulo state), and C. rhizophorae from Fortim (Ceará state) to Florianópolis (Santa Catarina state), although small individuals of the latter species were also found at Ajuruteua beach (municipality of Bragança, Pará state). An unidentified Crassostrea species was found only on Canela Island, Bragança. Crassostrea gasar and C. rhizophorae grouped with C. virginica, thereby forming a monophyletic Atlantic group, whereas Crassostrea sp. from Canela Island was shown to be more similar to Indo-Pacific oysters, and either arrived in the Atlantic Ocean before the convergence of the Isthmus of Panama or was accidentally brought to Brazil by ship.