Banca de DEFESA: LUCAS AGUIAR ROSA MACHADO

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : LUCAS AGUIAR ROSA MACHADO
DATE: 16/02/2022
TIME: 09:30
LOCAL: Plataforma Zoom
TITLE:

Expansion and Improvement of  an Online Tool Based on Use of PCR-RFLP Technique for the Species level  Identification of ticks of the Genus Amblyomma Koch, 1844 (Acari: Ixodidae)

KEY WORDS:

Acarology, Restriction enzymes, Molecular biology

PAGES: 90
BIG AREA: Ciências Agrárias
AREA: Medicina Veterinária
SUBÁREA: Medicina Veterinária Preventiva
SPECIALTY: Doenças Parasitárias de Animais
SUMMARY:

During the last 20 years, Brazilian researchers have played a leading role in efforts to improve our knowledge of the bioecology, the presence and importance of pathogens and the dynamics of tick populations associated with wild animals in South America. Many of these findings were provided in the form of molecular data, principally sequencing of molecular markers amplified using the polymerase chain reaction (PCR) technique. However, despite the advances achieved so far, there are still a number of barriers that prevent molecular techniques from becoming the standard approach for identifying ticks associated with wild animals in Brazil. Research carried out by our group within the PPGCV/UFRRJ, between 2018-2019, resulted in the development of a robust and low-cost system (which serves as an alternative to sequencing) for the identification, at the species level, of ticks of the Ixodidae family. The system is based on the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism technique (PCR-RFLP), using a fragment of the mitochondrial gene that encodes 16S ribosomal RNA as target. To facilitate access to this system and to promote its use by other Brazilian researchers, an online tool called “TickCutter” was developed. The present project aimed to expand and improve this tool on two fronts. Firstly, a new tool called “COIsearcher” was developed using an alternative molecular marker, specifically Cytochrome C Oxidase I (COI), in order to address some of the limitations associated with the use of a single molecular marker. Secondly, through the inclusion of PCR-RFLP data (band patterns determined by in silico digestion of virtual “amplicons” (~460 nucleotides) of the 16S rDNA gene with the enzymes DraI and VspI), derived from tick species of the Argasidae family and/or from novel representatives of the Ixodidae family. A total of 35 new banding patterns were identified among 363 previously unpublished 16S rDNA sequences derived from ticks of the Ixodidae family (deposited in the GenBank between 06/09/2019 to 08/03/2021. The inclusion of these data served to increase the number of species identified by the tool. Regarding the Argasidae family of ticks, 31 banding profiles were identified among the 75 sequences obtained from GenBank (which represented 22/25 of the Argasidae species recognized in Brazil). However, it was observed that patterns derived from six species (Antricola guglielmonei, Ornithodoros capensis, Ornithodoros guaporensis, Ornithodoros kohlsi, Ornithodoros marinkellei and Ornithodoros mimon) generated conflicting identifications with banding profiles derived from the following species of the Ixodidae family: Amblyomma coelebs, A. geayi, A. tigrinum, Dermacentor nitens, Haemaphysalis leporispalustris, Ixodes auritulus and Rhipicephalus microplus. The new “COIsearcher” tool was developed in a very similar manner to the “TickCutter 16S” tool, and took advantage of some of the solutions developed to solve problems encountered during the development of the 16S module. However, some modifications were necessary due to the differences between the markers, particularly in terms of the size of the amplicons. The database of the new module was established based on the in silico digestion of virtual “amplicons” (709 nucleotides) of the COI gene, derived from tick species of the Ixodidae family, with the enzymes AluI and MboI. A total of 65 banding profiles were recorded among a total of 281 sequences derived from 27 of the 33 species of the genus Amblyomma. In addition, 23 additional banding patterns were identified from within 570 sequences deposited for the species Dermacentor nitens, Haemaphysalis juxtakochi, H. leporispalustris, I. auritulus, R. microplus and Rhipicephalus sanguineus. The interspecific discriminatory power of the “COIsearcher” tool was high. However, some conflicting identifications were detected, in a manner similar to what was previously observed with the “TickCutter 16S” tool. The solution found to resolve these conflicts was the identification of a third enzyme capable of generating discriminatory patterns at the species level. The application of this strategy allowed the differential identification of 86/88 of the banding patterns present within the “COIsearcher” database, a result considered to be highly satisfactory. It was concluded that the modifications introduced to the “TickCutter” platform will provide greater flexibility and discriminatory power to the current identification system and will offer a solution to most of the limitations associated with the use of a single molecular marker.

 

 

 

BANKING MEMBERS:
Presidente - 1354903 - DOUGLAS MCINTOSH
Interno - 3701492 - HUARRISSON AZEVEDO SANTOS
Externa ao Programa - 3103478 - MARISTELA PECKLE PEIXOTO
Externo à Instituição - DANIEL PAIVA BARROS DE ABREU - UFRJ
Externo à Instituição - HERMES RIBEIRO LUZ - UFMA