Morphological and molecular diagnosis of anisakid nematode larvae from cutlassfish (Trichiurus lepturus) off the coast of Rio de Janeiro, Brazil

Submitted by Sandra Infurna ([email protected]) on 2016-09-13T16:45:12Z No. of bitstreams: 1 luizfelipe_cunha_etal_IOC_2012.pdf: 1411336 bytes, checksum: 540887704c1393ac7d93054eb573b850 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-09-13T16:55:13Z (GMT) No. of bitstreams: 1 luizfelipe_cunha_etal_IOC_2012.pdf: 1411336 bytes, checksum: 540887704c1393ac7d93054eb573b850 (MD5)Made available in DSpace on 2016-09-13T16:55:13Z (GMT). No. of bitstreams: 1 luizfelipe_cunha_etal_IOC_2012.pdf: 1411336 bytes, checksum: 540887704c1393ac7d93054eb573b850 (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Avaliação e Promoção e Saúde Ambiental. Rio de Janeiro, RJ, Brasil / Universidade Federal Fluminense. Laboratório de Biologia do Nécton e Ecologia Pesqueira, Biologia Marinha. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Avaliação e Promoção e Saúde Ambiental. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Avaliação e Promoção e Saúde Ambiental. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense. Laboratório de Biologia do Nécton e Ecologia Pesqueira, Biologia Marinha. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Avaliação e Promoção e Saúde Ambiental. Rio de Janeiro, RJ, Brasil.Anisakid nematode larvae from Trichiurus lepturus off coast of Rio de Janeiro were studied using light, laser confocal and scanning electron microscopy, in addition to a molecular approach. Mitochondrial cytochrome c-oxidase subunit 2 (mtDNA cox-2), partial 28S (LSU) and internal transcribed spacers (ITS-1, 5.8S, ITS-2) of ribosomal DNA were amplified using the polymerase chain reaction and sequenced to evaluate the phylogenetic relationships between the nematode taxa. The morphological and genetic profiles confirmed that, of the 1,030 larvae collected from the 64 fish examined, 398 were analysed, of which 361 were Hysterothylacium sp. and 37 were Anisakis typica. Larvae of Hysterothylacium sp. were not identified to the species level due to the absence of similar sequences for adult parasites; however, the ITS sequence clustered in the phylogenetic tree with sequences of H. deardorffoverstreetorum, whereas an mtDNA cox-2 and LSU concatenated phylogenetic analysis demonstrated the presence of two clades, both of them under the same name as the larval H. deardorffoverstreetorum. Data on the occurrence of parasites during the winter and summer months were compared using the t-test. The greatest prevalence and intensity of infection were recorded for larval Hysterothylacium, with a prevalence of 51.56% and an intensity of up to 55 parasites per fish. The larval Anisakis exhibit a higher abundance and intensity of infection in the winter months, and those of Hysterothylacium during the summer. However, the t-test indicated no significant differences between the abundance and intensity of infection recorded during the months of collection for either of these larval nematodes. All sequences generated in this study were deposited in GenBank

Alignment of ITS-1 and ITS-2 sequences representing <i>Anisakis</i> spp.

<p>Dots indicate identity with the first sequence, dashes are inferred insertion-deletion events and * represents our sample.</p

Alignment of mtDNA <i>cox</i>-2 sequences representing <i>Hysterothylacium</i> and <i>Iheringascaris</i> taxa.

<p>Dots indicate identity with the first sequence, dashes are inferred insertion-deletion events and * represents our samples.</p

A–H: <i>Hysterothylacium</i> sp. larvae: SEM and CLSM microscopy.

<p>A- SEM of anterior end with alae and excretory pore; B- Detail of L3 lips with inconspicuous boring tooth and papillae; C- Detail of lips of L4 with dorsal lip showing double papilla; D- CLSM of esophagus; E- CLSM reconstruction with ventriculus, intestinal caecum and esophagus; F: CLSM reconstruction with nerve ring and excretory pore; G- SEM of tail; H- SEM micrograph with a detail of the digitiform tip with terminal mucron. Abbreviations: a - alae; ep - excretory pore; p – papilla; t - tooth; dl - dorsal lip; e - esophagus; ic - intestinal caecum; v - ventriculus; n - nervous ring and m - mucron.</p

Maximum likelihood reconstruction between sequences of <i>Anisakis typica</i> obtained in this study (*) and sequences of <i>Anisakis</i> species from the GenBank, with the tree inferred from the ITS data set.

<p>The numbers on the tree branches represent the percentage of bootstrap resampling. <i>Ascaris lumbricoides</i> was used as an out group.</p

Maximum likelihood reconstruction between sequences of <i>Anisakis typica</i> obtained in this study (*) and sequences of <i>Anisakis</i> species from the GenBank, with the tree inferred from mtDNA <i>cox</i>-2 and LSU data sets.

<p>The numbers on the tree branches represent the percentage of bootstrap resampling. <i>Ascaris lumbricoides</i> was used as an out group.</p

A–F: <i>Anisakis typica</i> larvae: light, CLSM and SEM microscopy.

<p>A- Aanterior end with boring tooth; B- SEM of lips with papilla, boring tooth and excretory pore; C- Esophagus and ventriculus; D- Position of excretory pore; E- CLSM reconstruction with detail boring tooth and excretory pore; F- SEM of tail with mucron terminal. Abbreviations: e - esophagus; ep - excretory pore; t - tooth; p - papilla; v - ventriculus; m - mucron.</p

Maximum likelihood reconstruction between sequences of <i>Hysterothylacium</i> sp. larvae obtained in this study (*) and sequences of other anisakid species from the GenBank inferred from the ITS dataset.

<p>The numbers on the tree branches represent the percentage of bootstrap resampling. <i>Heterocheilus tunicatus</i> was used as an out group.</p

Ecological data of <i>Anisakis typica</i> and <i>Hysterothylacium</i> sp.: mean intensity (no. of parasites/parasitized fish); the bars represent the standard deviation.

<p>Ecological data of <i>Anisakis typica</i> and <i>Hysterothylacium</i> sp.: mean intensity (no. of parasites/parasitized fish); the bars represent the standard deviation.</p

List of species from the <i>Genbank</i> used for comparison in phylogenetic analysis and alignments.

<p>List of species from the <i>Genbank</i> used for comparison in phylogenetic analysis and alignments.</p