Obtention of pupae
Pupae were obtained from fruits of Campomanesia adamantium collected at the Research Center of the Agency for Agricultural Development and Rural Extension in the town of Campo Grande (537 m a.s.l.; 20º 27' S; 54º 37' W) from plants grown in the area. Fruits were placed on a wooden platform covered with a 9-mm-diameter wire mesh and put inside plastic boxes containing a water layer of 15 mm to retain the last stage larvae, according to Uchôa-Fernandes and Zucchi (1999). The maggots were collected from the water layer by pouring it through 1-mm-diameter meshes every 24 hours. Afterwards, larvae were placed in Petri dishes with sterile grit to stimulate pupae formation. After emergence of the parasitoids, the hosts’ pupae were preserved in 90% alcohol.
Dna Extraction
DNA extraction was performed under the conditions described by Lacruz-Ramos et al. (2019), with modifications. Host pupa samples were removed from 90% alcohol and dried at room temperature for 24 hours, then samples were macerated in liquid nitrogen, 150 µl of molecular grade chelating resin (Chelex 100 Resin Bio-Rad®) and 6 µl of proteinase K, followed by incubation in a heating block at 56°C for 4 hours and centrifugation at 10,000 RPM for 10 minutes. Subsequently, 40 µl of supernatant were obtained, DNA concentration was assessed by Biodrop Duo UV-Vis Spectrophotometer, and samples were stored at -20°C.
Pcr Optimization, Amplification, Purification, And Sequencing (Dup: Abstract ?)
The universal barcoding primer pair LCO1490 (5'-GGTCAACAAATCATAAAGATATTG-3') and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994) was used to amplify mitochondrial cytochrome oxidase c subunit I (COI). Reactions were performed in 25µL volumes and final concentrations of 1x buffer, 25 mM MgCl2, 0.2 mM each dNTP, 0.5 µM each primer, BSA 10%, and 0.005 units/µL of Taq DNA polymerase.
PCR amplifications were performed with an initial denaturation step at 94°C (2 minutes) and 32 cycles at 94°C (30 seconds), 51°C (30 seconds) and extension at 68°C (45 seconds), with a final extension step at 68°C (5 minutes). PCR products were purified using Illustra™ ExoProStar 1-Step (GE Healthcare Life Sciences, Little Chalfont, UK) according to the manufacturer's instructions.
Sequencing of samples was performed by ACTGene Análises Moleculares Ltd. (Center for Biotechnology, UFRGS, Porto Alegre, RS, Brazil) using the automatic sequencer AB 3500 Genetic Analyzer equipped with 50-cm capillaries and POP7 polymer (Applied Biosystems). The sequences obtained were analyzed and processed. To identify host pupae, the percentages of sequence identity and coverage were compared with the sequences available on GenBank (http://www.ncbi.nlm.nih.gov), using BLASTn to search for the most closely matching sequences. Sequence data from this study were submitted to GenBank under accession numbers OP021150 and OP021151.
Determination Of Genetic Distance Among Hosts’ Pupae
The edited sequences were aligned using the MEGA program (Tamura et al. 2011) version 8.0, with grouping by neighbor-joining method (Saitou and Nei, 1987), using the p-distance matrix with the pairwise gap deletion option and with 10,000 bootstrap (BP) repetitions. The consensus sequences were aligned with the sequences available on GenBank using ClustalW (Thompson et al. 1994), and the dendrogram was made with version 8.0 of the MEGA program. The chosen outgroup was Xylocopa pubescens (GenBank accession number AY005236.1).