Specimens. Parasitism by T. dilophonotae was detected from egg collections of E. ello carried out in the 2017/18 and 2021 harvests in areas of commercial production of cassava in the municipality of Marechal Cândido Rondon (24°50’96,92”S, 54°30’80,31”W) and Entre Rios do Oeste, in the western region of Paraná (Fig. 1).
Sampling was performed every two weeks in Marechal Cândido Rondon from 15 Oct 2017 to 28 Mar 2018, by zigzag walking sampling 30 equidistant points, and inspecting 6 plants/point. In the 2021 harvest, the collections were sporadic and carried out in Entre Rios do Oeste. The eggs collected were transferred to the Laboratory of Biological Control of the Universidade Estadual do Oeste do Paraná (UNIOESTE), where they were identified, placed in gelatin capsules and kept in BOD type climate chambers at 25 ± 2ºC and a photophase of 14 hours. The parasitoids that emerged were placed in 1.5mL microtubes containing 70% ethanol. In addition, 16 specimens were obtained that were laboratory-reared in E. ello eggs (10♀, 6♂).
Generic identification was performed following Masner (1976), and at the species level according to Johnson (1990) and Noronha et al. (2020). Color images were taken with a Leica MC 190HD digital camera attached to the Leica stereomicroscope M205A, and the specimens illuminated with a Leica LED5000 HDI high-light diffused dome (Kerr et al. 2008). The final extended focusing image was combined with the software Leica LAS X. The distribution map was generated using SimpleMappr (Shorthouse 2010). Geographic coordinates of the species records, if not present in labels, were derived from geoLoc (speciesLink, CRIA).
The specimens of T. dilophonotae are deposited in the Coleção de Insetos Entomófagos “Oscar Monte” (IB-CBE), in Unidade Laboratorial de Referência em Controle Biológico do Instituto Biológico, in Campinas, São Paulo, Brazil, under the unique identifier numbers IB-CBE-003116 to IB-CBE-003132.
Molecular data. To obtain the DNA barcodes we used 3 females of T. dilophonotae. The extraction of total genomic DNA was performed by non-destructive methodology, following work by Wengrat et al. (2021). For amplification of the mitochondrial gene the Cytochrome C Oxidase Subunit I (COI) primers SCEL-F1 (5’- GCAATAATTCGAATAGAATTAAGAGT-3’) (Gariepy et al. 2014) e HCO-2198 (5’- TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994) were used.
The concentration and reagents of the Polymerase Chain Reaction (PCR), as well as the thermocycling conditions for the SCEL-F1/HCO-2198 primers, followed the protocol of Gariepy et al. (2014). The amplicons were observed after electrophoresis under ultraviolet light on a 1.5% agarose gel stained with SYBR Safe (LIFE TECHNOLOGIES). The subsequent purification process for 10 µl of the final PCR product was performed using 1 µL (20 U µL–1) of Exonuclease I (Thermo Fisher Scientific™) and 2 µL (1 U µL–1) of thermosensitive alkaline phosphatase FastAP™ (Thermo Fisher Scientific) per 10 µL of the final PCR product. The purification conditions were 37°C for 30 min, followed by 80°C for 15 min. Bidirectional Sanger sequencing was performed at the Animal Biotechnology of ESALQ.
After sequencing, the material was manually aligned and edited, when necessary, for 562 base pairs (bp) for the SCEL-F1/HCO-2198 primer set. The chromatograms of each of the individual sequences were checked, edited, and aligned to produce the consensus sequence in Geneious Prime 2022.1 (https://www.geneious.com). The presence of NUNTs (nuclear parallels of mitochondrial origin) was observed in MEGA X, following the steps described in Corrêa et al. (2017). Sequences were submitted to NCBI.