FIOCRUZ | Accessing the sand fly diversity in caves of Tocantins, Northern Brazil: species delimitation using COI DNA barcoding

Study Extent

This study was performed in the Municipality of Palmas, capital of the state of Tocantins. Entomological collections were carried out on private properties, with prior authorization from the owners, using HP light traps installed from 6:00 pm to 6:00 am, at 1.5 meters above ground level, for three consecutive nights. Captures began in January 2022 and ended in May 2022, totaling five months of captures. Two caves were also surveyed in a district of the Municipality of Palmas, Taquaruçu, named ‘Boa Esperança’ cave (10º3'06”S; 48º05'35”W) and Evilson’s cave (10º2'31”S; 48º 12' 22”W). At each of the aforementioned collection sites, two traps were installed, one in the aphotic zone and the other in the photic zone of each cave. In addition, two other traps were installed on farms in the North of Palmas, in the ‘Serra do Carmo’ farm (10º4'39”S; 48º20'8”W) and Iago’s farm (10º7'17”S; 48º19'48”W ), with one trap on each farm.

Sampling

Sand flies were placed in 1.5 mL tubes containing 70% alcohol, and then the legs of each specimen were collected and frozen at -20°C for subsequent DNA amplification. For morphologicalidentification, the females were dissected, separating the head and the final portion of the abdomen (last three segments), which were fixed in Berlese mounting medium between a slide and coverslip. The males were mounted without dissection.

Quality Control

The morphological identification was carried out under a biological microscope following the identification keys and classification proposed by Galati (2018). COI=PCR assyas were performed with negative and positive controls.item-0

Method steps

1. We manually checked the electropherograms to remove primer sequences and assemble contigs using SeqTrace v0.9. The COI barcodes were submitted to the Barcode of Life Data System – BOLD (Ratnasingham and Hebert 2007), and then to the NCBI GenBank (Sayers et al. 2022). Sequences were assigned to the Process IDs TOBAR001-23 – TOBAR132-23, and Accession Numbers OR773186 – OR773317 for BOLD and GenBank, respectively.

Sand flies (Diptera, Psychodidae, Phlebotominae)

1. Phlebotominae
   common name: sand fly rank: subfamily

Sand flies collected in Tocantins state, Brazil.