Identification of fruit fly (Tephritidae) pupae parasitized by hymenoptera parasitoids

One of the major problems in orchards is the presence of Tephritidae fly maggots, especially in Mirtaceae species. Various biological control tools include the use of Hymenoptera parasitoids, which naturally control fly populations. However, identification of the host species of parasitoids is challenging, because only the puparium remains after the parasitoid hatches. Therefore, the aim of this work was to determine the host species of Hymenoptera parasitoids, through the puparium. Host species were identified by amplification and sequencing of cytochrome C oxidase subunit I (COI) of mitochondrial DNA. The results of the present study show that COI DNA sequencing was able to reliably identify host pupae to the genus level, allowing the identification of the Anastrepha genus in this study. This suggests that this method can be used to identify agriculturally relevant hosts through its puparium remains, since this genus has already been described as a pest of Campomanesia adamantium. Knowledge of the species that cause damage to crop is extremely important to study the biological and ecological aspects of these pests and their control. In this way, our results suggest that this method can be used to identify economically relevant host pupal species since the genus identified in this study has already been described as a pest of C. adamantium.

fruit fly species; therefore, identification of their hosts is critical to understand the biodiversity of parasitized fruit flies and the life cycle of their parasitoids, supporting the development of new tools for their management and control (Araújo et al., 2014;Almeida et al., 2019;Bomfim et al., 2007;Chan-Canché et al., 2020;Souza et al., 2022).
The identification of the fruit flies is based mostly on the adults' morphological features using aspects such as wings patterns, size and length of the ovipositor in identification keys.Therefore, it is really challenging to precisely attribute a correlation between the species of parasitoids and its host species, because the parasitoid wasp kills the host fly during the pupae phase, remaining only the puparium after the parasitoid emerges.In this context, the use of DNA tags might serve as an alternative for the identification of these specimens with the material from the hosts' pupae.
Thus, the purpose of this work is to identify the species of hosts by DNA amplification and sequencing, using the cytochrome C oxidase subunit I (COI) regions of mitochondrial DNA obtained from puparium remains.

| Obtention of puparium remains
Puparium remains were obtained from fruits of Campomanesia adamantium (Cambess.)O. Berg (Myrtaceae) 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.; 20°27′S; 54°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 Uchoa and Zucchi (1999).The maggots were collected from the water layer by pouring it through 1-mm-diameter meshes every 12 h.Afterwards, larvae were placed in Petri dishes with sterile grit to stimulate pupae formation.After the emergence of the parasitoids, identified as Utetes sp., Doryctobracon sp. and Opius sp.(Unpublished data) 220 remaining host pupariums were preserved in 90% ethanol.

| DNA extraction
DNA extraction was performed under the conditions described by Lacruz-Ramos et al. (2019), with modifications.Two host puparium remains samples were removed from 90% ethanol and dried at room temperature for 24 h, 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 h and centrifugation at 10,000 RPM for 10 min.Subsequently, 40 μL of supernatant was obtained, DNA concentration was assessed by Biodrop Duo UV-Vis Spectrophotometer and samples were stored at −20°C.
PCR amplifications were performed with an initial denaturation step at 94°C (2 min) and 32 cycles at 94°C (30 s), 51°C (30 s) and extension at 68°C (45 s), with a final extension step at 68°C (5 min).PCR products were purified using Illustra™ ExoProStar 1-Step (GE Healthcare Life Sciences) according to the manufacturer's instructions.
Sequencing of samples was performed by ACTGene Análises Moleculares Ltd. (Center for Biotechnology, UFRGS) using the automatic sequencer AB 3500 Genetic Analyser equipped with 50-cm capillaries and POP7 polymer (Applied Biosystems).The sequences obtained were analysed 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.

| 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 neighbour-joining method (Saitou & 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.

| DNA extraction
DNA extraction using a chelating resin (Chelex 100 Resin Bio-Rad®) proved to be very efficient in identifying fruit flies (Tephritidae) parasitized by Hymenoptera parasitoids using its puparium remains (Figure 1).The DNA concentrations were determined as 31.74μg/mL for sample 1 and 59.10 μg/mL for sample 2.

| PCR optimization, amplification, purification and sequencing
The two samples tested were successfully amplified, confirming that amplification of hosts' pupae COI genes is possible (Figure 2).

| Molecular identification and determination of genetic distance among hosts' pupae
Using sequences obtained from mitochondrial cytochrome oxidase c subunit I (COI), BLASTn analysis against the GenBank database identified the hosts' pupae (Table 1), with Sample 1 having 100% identity with Anastrepha fraterculus and Sample 2 having 97.79% identity with Anastrepha fraterculus.
The phylogenetic analysis separated the hosts' pupae into one clade composed only of representatives from the genus Anastrepha grouped with 100% BP (Figure 3).

| DISCUSS ION
The use of molecular tools for insect identification and classification has become an alternative to morphological studies (Adeniran et al., 2021).The marker that has been used to identify species is mitochondrial cytochrome oxidase c subunit I (COI).This mitochondrial DNA-specific conserved region has been reported to be amplified and sequenced with ease (Hebert & Gregory, 2005;Zhang & Hewitt, 1997).
The results of the present study show that the sequencing of mitochondrial COI can be used to reliably identify hosts' pupae to the genus level, with Anastrepha sp.being identified in this study.
Larvae from the genus Anastrepha show great economic importance because they cause fruit damage that can lead to 100% losses in untreated orchards (Costa & Silva, 2016).This genus is divided into 21 groups (Norrbom et al., 2012), with the species A. fraterculus being the most economically important (Uchoa & Zucchi, 1999), as this group can infest innumerable fruit varieties, including the fruits of C. adamantium (Coelho & Uchoa, 2023).Thus, our results suggest that this method can be used to identify economically relevant host pupal species since the genus identified in this study has already been described as a pest of C. adamantium.
Nevertheless, further studies need to be conducted to improve this protocol and increase the accuracy of host pupal identification.

F
I G U R E 2 PCR products of hosts' pupae.L, ladder 1 KB; 1, sample (1); 2, sample (2).[Colour figure can be viewed at wileyonlinelibrary.com]TA B L E 1 DNA sequence matching of the identified hosts' pupae and the identity percentage found on the NCBI (National Center for Biotechnology Information) website.