Introduction
⌅Chagas disease is considered a neglected parasitic disease that affects the seven countries of Central America; it is estimated that around 12% of the population lives in risk areas, where Triatoma dimidiata (Hemiptera: Reduviidae) is the main vector (Peterson et al., 2019Peterson, J. K., Hashimoto, K., Yoshioka, K., Dorn, P. L., Gottdenker, N. L., Caranci, A., Stevens, L., Zuniga, C., Saldaña, A., Rodriguez, S., y Monroy, C. (2019). Chagas Disease in Central America: Recent Findings and Current Challenges in Vector Ecology and Control. In Current Tropical Medicine Reports. Springer Verlag. https://doi.org/10.1007/s40475-019-00175-0 ). Despite the elimination of Rhodnius prolixus (Hemiptera: Reduviidae), as a result of the Initiative for Chagas Disease Control in Central America, “IPCA” (Hashimoto y Schofield, 2012Hashimoto, K., & Schofield, C. J. (2012). Elimination of Rhodnius prolixus in Central America. Parasites & Vectors, 5(1), 45. https://doi.org/10.1186/1756-3305-5-45 ; Ponce, 2007Ponce, C. (2007). Current situation of Chagas disease in Central America. Memorias Do Instituto Oswaldo Cruz, 102, 41-44.), the domiciliary infestation by T. dimidiata and its presence in sylvatic ecotopes continues to be highly significant (Dorn et al., 2022Dorn, P. L., Monroy, M. C., & Stevens, L. (2022). Sustainable, integrated control of native vectors: The case of Chagas disease in Central America. Frontiers in Tropical Diseases, 3. https://doi.org/10.3389/fitd.2022.971000 ). For this reason, the search for effective control tools for T. dimidiata, as alternatives to traditional insecticide spraying, represents a priority in Central American countries (Dorn et al., 2022Dorn, P. L., Monroy, M. C., & Stevens, L. (2022). Sustainable, integrated control of native vectors: The case of Chagas disease in Central America. Frontiers in Tropical Diseases, 3. https://doi.org/10.3389/fitd.2022.971000 ).
Microhymenopterans in the family Scelionidae are oophagous parasitoids of insects and arachnids, and, in the case of the subfamily Telenominae, specifically of triatomine bug eggs (Reduviidae: Triatominae) (Austin et al., 2005Austin, A. D., Johnson, N. F., & Dowton, M. (2005). Systematics, evolution, and biology of Scelionid and Platygastrid wasps. Annual Review of Entomology, 50(1), 553-582. https://doi.org/10.1146/annurev.ento.50.071803.130500 ). Telenomus farai Costa-Lima 1927Ramírez-Ahuja, M. D. L., Davila-Barboza, J., Talamas, E., Moore, M., Bobadilla, C., Ponce, G., Rodriguez-Sanchez, I., y Flores, A. (2021). First record of Telenomus fariai Costa Lima, 1927 (Hymenoptera, Scelionidae, Telenominae) as a parasitoid of Triatoma dimidiata (Latreille, 1811) (Hemiptera, Reduviidae, Triatominae) eggs in Mexico. Journal of Hymenoptera Research, 87, 309. https://doi.org/10.3897/jhr.87.73546 has been reported as a parasite of eggs in various species of the genera Triatoma and Panstrongylus (Bosque y Rabinovich, 1979Bosque, C., & Rabinovich, J. E. (1979a). Population dynamics of Telenomus fariai (Hymenoptera: Scelionidae), a parasite of Chagas’ disease vectors: vii. Oviposition behavior and host discrimination. The Canadian Entomologist, 111(2), 171-180. https://doi.org/10.4039/Ent111171-2 ;Costa-Lima, 1928Costa Lima, A. D. (1928). Notas sobre a biologia do Telenomus fariai Lima, parasito dos ovos de Triatoma. Memórias Do Instituto Oswaldo Cruz, 21(1), 201-218. https://doi.org/10.1590/S0074-02761928000100005 ; Peláez, 1944Peláez, D. (1944). Algunas notas sobre el hallazgo en México de un microhimenóptero parásito de huevos de Triatoma pallidipennis (Stal). Ciencia, 5(1), 29-33.; Pellegrino, 1950aPellegrino, J. (1950a). Nota sôbre o parasitismo de ovos de Triatoma infestans e Panstrongylus megistus pelo microhimenóptero Telenomus fariai Lima, 1927. Memórias Do Instituto Oswaldo Cruz, 48(1), 669-673. https://doi.org/10.1590/S0074-02761950000100024 , 1950bPellegrino, J. (1950b). Parasitismo experimental de ovos de várias espécies de Triatoma pelo microhimenóptero Telenomus fariai Lima, 1927. Memorias Do Instituto Oswaldo Cruz, 48, 1-12. https://doi.org/10.1590/S0074-02761950000100025 ; Zeledón, 1957Zeledón, R. (1957). Sobre la biología del Telenomus fariai Lima, 1927 (Hymenoptera: Scelionidae), parásito endófago de huevos de algunos Triatominae. Revista de Biología Tropical, 5(1), 1-17.).
The presence of T. fariai in Central America has been documented. Previous studies performed in Costa Rica with insects collected in El Salvador revealed phenotypic characteristics, levels of infestation, progeny structure, and the ability of these microhymenopterans to parasitize eggs of T. dimidiata of the region (Zeledón, 1957Zeledón, R. (1957). Sobre la biología del Telenomus fariai Lima, 1927 (Hymenoptera: Scelionidae), parásito endófago de huevos de algunos Triatominae. Revista de Biología Tropical, 5(1), 1-17.). Considering that T. dimidiata is an important vector of Chagas disease in Latin America, the density-dependent population regulation between these triatomines and T. fariai represents a potential tool for the biological control of these vectors (Costa Costa-Lima, 1927Ramírez-Ahuja, M. D. L., Davila-Barboza, J., Talamas, E., Moore, M., Bobadilla, C., Ponce, G., Rodriguez-Sanchez, I., y Flores, A. (2021). First record of Telenomus fariai Costa Lima, 1927 (Hymenoptera, Scelionidae, Telenominae) as a parasitoid of Triatoma dimidiata (Latreille, 1811) (Hemiptera, Reduviidae, Triatominae) eggs in Mexico. Journal of Hymenoptera Research, 87, 309. https://doi.org/10.3897/jhr.87.73546 ; Zeledón, 1957Zeledón, R. (1957). Sobre la biología del Telenomus fariai Lima, 1927 (Hymenoptera: Scelionidae), parásito endófago de huevos de algunos Triatominae. Revista de Biología Tropical, 5(1), 1-17.). In this context, studies by Zeledón (1957)Zeledón, R. (1957). Sobre la biología del Telenomus fariai Lima, 1927 (Hymenoptera: Scelionidae), parásito endófago de huevos de algunos Triatominae. Revista de Biología Tropical, 5(1), 1-17. described that the temperature-dependent development time of T. fariai inside T. dimidiata eggs was approximately 35 days, and that the average number of microhymenopterans per egg was 6.66 females and 1.15 males, with a relative frequency of adult emergence of 15% for males and 85% for females (Zeledón, 1957Zeledón, R. (1957). Sobre la biología del Telenomus fariai Lima, 1927 (Hymenoptera: Scelionidae), parásito endófago de huevos de algunos Triatominae. Revista de Biología Tropical, 5(1), 1-17.). Reports of T. fariai parasitizing field-collected eggs of T. dimidiata have been recorded in Mexico (Arisqueta-Chablé et al., 2022Arisqueta-Chablé, C., Ramírez-Ahuja, M. de L., Delfín-González, H., Gómez-Govea, M. A., Peña-Carrillo, K. I., Vazquez-Prokopec, G., Rodríguez-Sanchez, I. P., & Manrique-Saide, P. (2022). Identity and report of Telenomus fariai parasitizing eggs of Triatoma dimidiata at Yucatan, Mexico. Southwestern Entomologist, 47(2), 345-352. https://doi.org/10.3958/059.047.0210 ; Ramírez-Ahuja et al., 2021Ramírez-Ahuja, M. D. L., Davila-Barboza, J., Talamas, E., Moore, M., Bobadilla, C., Ponce, G., Rodriguez-Sanchez, I., y Flores, A. (2021). First record of Telenomus fariai Costa Lima, 1927 (Hymenoptera, Scelionidae, Telenominae) as a parasitoid of Triatoma dimidiata (Latreille, 1811) (Hemiptera, Reduviidae, Triatominae) eggs in Mexico. Journal of Hymenoptera Research, 87, 309. https://doi.org/10.3897/jhr.87.73546 ) and Costa Rica (Zeledón et al., 1970Zeledón, R., Valerio, C. E., y Valerio, J. E. (1970). Enemies of Triatoma dimidiata Latreille, 1811 in an Endemic Area of Chagas’ Disease in Costa Rica (Hemiptera, Reduviidae). Journal of Medical Entomology, 7(6), 722-724. https://doi.org/10.1093/JMEDENT/7.6.722 ). In addition, recent studies based on molecular analyses have confirmed that populations of T. fariai associated with T. dimidiata in Central America and Mexico correspond to at least three different haplotypes (Ramírez-Ahuja et al., 2021Ramírez-Ahuja, M. D. L., Davila-Barboza, J., Talamas, E., Moore, M., Bobadilla, C., Ponce, G., Rodriguez-Sanchez, I., y Flores, A. (2021). First record of Telenomus fariai Costa Lima, 1927 (Hymenoptera, Scelionidae, Telenominae) as a parasitoid of Triatoma dimidiata (Latreille, 1811) (Hemiptera, Reduviidae, Triatominae) eggs in Mexico. Journal of Hymenoptera Research, 87, 309. https://doi.org/10.3897/jhr.87.73546 ), and multiple genetic sequences of T. fariai from the Pailas Frías - Rincón de la Vieja Volcano (Guanacaste province, Costa Rica) appear deposited in systems such as BOLD BINS, but not associated with any publication. This paper reports the infection of field-collected T. dimidata eggs with T. fariai in Alajuela, Costa Rica, with combined morphological and molecular analyses to confirm the species. The information presented could support future biological control strategies and contributes to the limited knowledge of this species in the region.
Materials and methods
⌅Collection Site and Entomological Data
⌅As part of a project that receives arthropod samples of medical importance, on June 28, 2022, the Vector Research Laboratory (LIVE) received a group of T. dimidiata eggs collected manually from the peridomicile of a house, with a previous positive history of infestation by T. dimidiata, located in the town of Tuetal Sur, province of Alajuela, 10,0265 N and -84,2318 W (Figure 1).
The eggs collected were placed in a borosilicate glass vial and observed until T. dimidiata nymphs and/or parasitoids emerged. Most of these eggs showed a dark coloration when they were received in the laboratory, and the presence of some microhymenopterans was documented in the container after a couple of hours. After 24 h, the microhymenopterans that emerged were removed, sexed according to the morphology of the antennae, and freeze-inactivated. A small fraction of the females was transferred to Eppendorf tubes for further analysis by polymerase chain reaction (PCR) and sequencing of the cytochrome c oxidase subunit 1 mitochondrial gene (COI). The remaining eggs of T. dimidiata were cleared in lactophenol and used for the estimation of the intensity of parasitism. The measurement of the length of 34 emerged females was recorded; each specimen was placed on a microscope slide with a drop of Hoyer’s medium and measured with a calibrated slide micrometer. The morphological corroboration of the species was done by dissection and observation of the male genitalia, according to criteria previously established (Polaszek y Kimani, 1990Polaszek, A., y Kimani, S. (1990). Telenomus species (Hymenopetra: Scelionidae) attacking eggs of pyralid pests (Lepidoptera) in Africa: a review and guide to identification. Bulletin of Entomological Research, 80, 57-71. https://doi.org/10.1017/S0007485300045922 ), as it is accepted that the male genitalia unquestionably provides the best means of species-level identification (Polaszek y Kimani, 1990Polaszek, A., y Kimani, S. (1990). Telenomus species (Hymenopetra: Scelionidae) attacking eggs of pyralid pests (Lepidoptera) in Africa: a review and guide to identification. Bulletin of Entomological Research, 80, 57-71. https://doi.org/10.1017/S0007485300045922 ). The dissection and mounting of two male genitalia were performed as follows: males were cleared in lactophenol for two weeks and placed in a drop of Hoyer’s medium for entomological manipulation. The pleural borders of the metasoma of each male were cut sagittally with minuten pins and then, the tergal and sternal sclerites were separated to expose the genitalia. Each genitalia was transferred to a new drop of Hoyer’s medium and covered with a cover glass to complete the mounting process. Voucher specimens of T. fariai were deposited in the Medical Entomological Collection of the University of Costa Rica (Facultad de Microbiología, Sección de Entomología Médica).
Molecular identification
⌅To support morphological identification, genomic DNA from one microhymenopteran specimen was extracted using a NucleoSpin® tissue kit (Macherey-Nagel), according to the manufacturer's instructions. A one-step endpoint PCR protocol was applied to amplify a fragment of the COI gene with primers LCO1490 and HCO2198 (Folmer et al., 1994Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3(5), 294-299.). PCR conditions were 95 °C for 1 minute for primary denaturation, followed by 35 cycles at 94 °C for 1 minute, 55 °C for 1 minute, 72 °C for 1.5 minutes, and a final extension at 72 °C for 7 minutes. The product of amplification was visualized on 1.0% agarose gel electrophoresis. The amplicon was purified with Exonuclease I (EXO I) and FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific Inc.) and sequenced in both directions at Macrogen, Inc. (Seoul, South Korea). The software BioEdit Sequence Alignment Editor version 7.0.5.3 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) was used to assemble the sequences, and BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi) was used to search the closest sequences with the consensus sequence. First, the protein sequences of the COI gene were aligned using MAFFT (Katoh y Standley, 2013Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution, 30(4), 772-780. https://doi.org/10.1093/molbev/mst010 ). The corresponding nucleotide sequences downloaded from GenBank (MZ810544 and MZ810543; https://www.ncbi.nlm.nih.gov/genbank/) and BOLD BINS (BOLD:ADW5671, BOLD:ADB0583; https://v3.boldsystems.org/) of the aligned protein sequences, subsequently were aligned codon by codon, using the PAL2NAL (Suyama et al., 2006Suyama, M., Torrents, D., y Bork, P. (2006). PAL2NAL: Robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Research, 34(WEB. SERV. ISS.). https://doi.org/10.1093/nar/gkl315 ). Trissolcus brochymenae was included as an outgroup based on its position as sister to Telenomus sp. Phylogenetic analyses were performed on maximum likelihood (ML) to determine the relationship between Telenomus specimens. The TIM2+F was determined to be the most suitable model in maximum likelihood (ML) analyses using IQ-TREE web server, version 1.6.12; (Nguyen et al., 2015Nguyen, L. T., Schmidt, H. A., Von Haeseler, A., y Minh, B. Q. (2015). IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution, 32(1), 268-274. https://doi.org/10.1093/molbev/msu300 ). Branch support was estimated with 1000 ultrafast bootstrap replicates.
Results
⌅Morphological identification
⌅The sample of triatomine eggs received in the laboratory consisted of 29 eggs. After 24 hours, 36 wasps emerged from 6 eggs (Fig. 2), 16 were still parasitized, and 7 were uninfected. The total egg parasitism rate was 76%, with an average of 5.5 wasps per egg.