We describe the wasp Iconella melitaraevora sp. nov. (Braconidae, Microgastrinae), a parasitoid of Melitara subumbrella caterpillars (Lepidoptera, Pyralidae, Phycitinae). Multiple wasp cohorts were reared from hosts collected in a montane dryland habitat of New Mexico, USA. This is the first case of gregariousness in Iconella. An updated key to American Iconella species is provided. We present a maximum likelihood tree using the cytochrome c oxidase subunit I (COI) locus of this species relative to other described Iconella species from the Americas. We also describe the biology and environmental conditions where Iconella melitaraevora sp. nov. was discovered in the context of its prickly pear cactus (Opuntia Mill.) herbivore host. The potential biological control capacity of microgastrine wasps which attack prickly pear specialist herbivores is discussed.

CO1, gregarious, maximum likelihood tree, Nearctic, Opuntia, Phycitinae, species description, wasp

Microgastinae is a lineage of parasitoids wasps (Hymenoptera: Braconidae) that attack Lepidoptera with approximately 3,000 named species worldwide and is estimated to comprise 30,000 to 50,000 species when undescribed species are included (Rodriguez et al. 2013; Fernandez-Triana et al. 2020). Iconella (Mason, 1981), is a small genus within Microgastrinae with four previously described species from North America, the Caribbean, and South America (Fernandez-Triana et al. 2013). Iconella are known to be solitary, koinobiont, endoparasitoids of shelter-forming microlepidopteran larvae (Whitfield et al. 1997; Fernandez-Triana et al. 2013). Besides this fundamental natural history, the ecology of Iconella species is not well understood aside from the Neotropical species I. isolata (Muesebeck, 1955) which has been investigated as a biological control agent of crop pests (Bennett 1960; Bartlett 1978).

Until now, two species were known from North America, I. canadensis Fernández-Triana 2013 and I. etiellae (Viereck 1911). Here, we describe a new species from high-elevation, dryland habitat in the southern Rocky Mountains, Iconella melitaraevora sp. nov. We present information on its biology and compare it with other species in the genus. We conclude this contribution with a discussion of biological control implications of microgastrine wasps that attack Opuntia specialist Lepidoptera.

Opuntia phaeacantha Engelm. (caterpillar host plant) pads and parasitized Melitara subumbrella (Dyar 1925) caterpillars which fed upon O. phaeacantha were collected on 23 August 2023 in Santa Fe County, New Mexico on the property of Hillary and David Armstrong (35.2216, -106.2080). The collections were made at 2225 m a.s.l. in piñon-juniper woodland near the transition from piñon-juniper to Rocky Mountain conifer forest ecoregion. The vegetation of this semi-arid ecosystem is characterized by piñon pine (Pinus edulis Engelm., Pinaceae), one-seed juniper (Juniperus monosperma (Engelm.) Sarg., Cupressaceae), desert prickly pear (Opuntia phaeacantha, Cactaceae), tree cholla (Cylindropuntia imbricata (Haw.) F.M.Knuth, Cactaceae), and Gambel oak (Quercus gambelii Nutt., Fagaceae). Temperature range varies dramatically in this region, both daily and seasonally. The average annual maximum temperature is 17 °C, with high temperatures exceeding 38 °C occasionally, and extended periods below -18 °C from November to March. The average yearly minimum temperature is 4 °C. Precipitation is driven by monsoons from June to August, with the area receiving an average of 50 cm rainfall annually. The area receives 152 cm of snow on average from November to March.

The M. subumbrella larvae were brought to The University of Texas at Austin, Brackenridge Field Laboratory on 03 September 2023 within pads of their host plant. The pads were maintained in containment at room temperature, with indirect sunlight. Pads with actively feeding M. subumbrella larvae were placed inside large plastic boxes on top of 5 cm of ProMix BX mycorrhizae growth media (Québec, Canada) within white mesh, emergence cages. Emergent wasps were collected with an aspirator and preserved in 100% ethanol between 27 November and 25 December 2023.

Wasp and caterpillar DNA extractions were performed at the Brackenridge Field Laboratory using the DNeasy Blood & Tissue Kit (Qiagen, Germany). A region of approximately 450–750 bp of the mitochondrial gene CO1 was amplified with degenerate primer sets LCO1490-F/HCO2198-R (Vrijenhoek 1994), followed by Sanger sequence analysis at Eton Bioscience Inc (California USA). Melitara subumbrella was identified as the caterpillar host following description and ecological context provided by Mann (1969) as well as via BLAST search query of the CO1 region on GenBank (Process ID NACMA004-25). CO1 sequences of other Nearctic and Neotropical Iconella species were downloaded from GenBank and the Barcode of Life Data System (BOLD; (Ratnasingham and Hebert 2007) (Table 1). Iconella CO1 sequences were aligned using MUSCLE version 5 (Edgar 2004) with default parameters in Geneious version 8 (Kearse et al. 2012). Maximum likelihood trees were constructed in MEGA 11 with a Tamura-Nei substitution rate model (Tamura et al. 2021) and 10,000 bootstrap replicates to infer generic placement. Iconella melitaraevora CO1 sequences were accessioned on BOLD (Process ID NACMA001-24).

Morphological terms follow Huber and Sharkey (1993), Whitfield et al. (1997), and Fernandez-Triana et al. (2014), which are included in the Hymenoptera Anatomy and Ontology (HAO) website (http://portal.hymao.org/projects/32/public/ontology/). The abbreviations F2 and F14 are used for antennal flagellomeres 2 and 14; T1 and T2 are used for metasomal mediotergites 1 and 2; and L and W refer to length and width. A diagnostic description is provided, followed by some measurements of morphological traits commonly used in Microgastrinae (Fernandez-Triana et al. 2014). The holotype and most paratype specimens are deposited in The University of Texas at Austin Insect Collection in Austin, TX (UTIC), with male and female paratypes also deposited in the Canadian National Collection of Insects, Arachnids and Nematodes in Ottawa, Canada (CNC) and the Smithsonian Institute in Washington D.C., USA (USNM).

Videos and images of the interaction are provided in Suppl. material 1 available on GitHub via Zenodo at https://doi.org/10.5281/zenodo.14589691 (Morrison et al. 2024).

In November, temperatures fall below freezing daily, with permanent snow on the ground, in Santa Fe County, New Mexico. This extreme environmental condition provides some limitation on what the possibilities are for I. melitaraevora overwintering activity. One possibility is that I. melitaraevora overwinter as pupae within M. subumbrella puparia buried in the soil, until temperatures rise enough for the snow to thaw in the spring. Some portion of the wasps must be able to tolerate this cold period as pupae (Langer and Hance 2000), unless they can remain active as adults during the winter by attacking alternative hosts (Eoche‐Bosy et al. 2016). In the spring, we predict that moths which survived winter diapause emerge, mate, and produce a new generation of larval hosts. In general, cactus moth flight activity does not commence until their Opuntia host plants begin growing new tissue that caterpillar larvae require for development (Morrison et al. 2025). The wasps documented here emerged in room temperature conditions in November and December 2023. Thus, the average immature development time that we measured may be considerably faster than what happens in the field. We predict that I. melitaraevora developmental phenology responds to favorable conditions, presumably those which facilitate host plant growth.

Until now, all Iconella species with known host information were solitary parasitoids (Whitfield et al. 1997). This is the first time that a gregarious Iconella species has been documented. The wasps in each cohort were probably oviposited by a single female, although it is possible they experienced super parasitism (Bakker et al. 1985; Varone et al. 2024), which is a rare phenomenon in the Micrograstrinae (Fernandez-Triana, personal observations). Regardless, all adults in each cohort emerged within 24 hours of each other which is within the fecundity range of gravid microgastrine females (Harvey et al. 2014).

All known American Iconella species attack shelter-forming lepidopteran larvae in the Pyralidae, Crambidae (Pyraloidea), and Tortricidae (superfamily Tortricoidea) (Whitfield et al. 1997; Fernandez-Triana et al. 2013). Most species in these superfamilies have shelter-building larvae (Lill et al. 2007), but these host superfamilies are not closely related within the lepidopteran clade Ditrysia (Regier et al. 2013). This suggests that Iconella are adapted to circumvent the behavioral manipulations that their hosts do to their food plants (Murdoch 1969; Ghimire and Phillips 2014). Host-finding mechanisms that Iconella species may use to locate suitable hosts include following volatile chemical compounds emitted by damaged host plants (Mbata et al. 2017; Turlings and Erb 2018), and homing in on caterpillar frass volatiles (Van Leerdam et al. 1985; Ngi-Song and Overholt 1997). A tight association with shelter-building larvae is also known from the braconid subfamily, Cheloninae. Chelonine females inspect leaf surfaces to find concealed host eggs into which they oviposit directly into developing larvae (Stireman III and Shaw 2022).

Iconella melitaraevora females may oviposit into hosts directly through Opuntia pads. The ovipositor is long enough (average I. melitaraevora ovipositor length = 1.12 mm) to penetrate an Opuntia epicuticle and reach the internal vascular tissue where Melitara larvae feed (average Opuntia cuticle width = 0.05–0.10 mm thick, (Morrison et al. 2021). However, we think that this is unlikely because Opuntia pads are difficult for insects to penetrate with ovipositors (Jezorek 2011). The alternatives are that females enter pads through holes where Melitara larvae excrete frass (Fig. 3C) and attack larvae inside larval feeding cavities, or they attack when larvae expose themselves to excrete frass from the holes (Fig. 3D). In Argentina, Apanteles opuntiarum Martínez & Berta, 2012 (Microgastrinae) females attack C. cactorum larvae (Pyralidae, Phycitinae) after entering pads through the frass excretion holes as well as when larvae expose themselves outside pads to excrete frass (L. Varone, personal observations). Direct observation is required to confirm how I. melitaraevora oviposition takes place.

Iconella melitaraevora is now the second Iconella species documented to use cactus moth larvae as hosts in North America. The host, M. subumbrella, is distributed from Baja California to west Texas, north along the foothills of the Rocky Mountains to Colorado, and east into the plains of Oklahoma (Mann 1969). Melitara systematics is discordant with multiple phylogenetic hypotheses making species limits uncertain (Simonsen 2008; Regier et al. 2012; Roe 2014). So, the host distribution may be incorrect, or it could be that the I. melitaraevora host availability is considerable given this documented host distribution. Iconella etiellae has been reared from Opuntia specialist Melitara junctolineella caterpillars in Eastern New Mexico, as well as a variety of other shelter-building Pyralidae (Rodriguez et al. 2013; Fernandez-Triana et al. 2020). This indicates that I. melitaraevora may also attack multiple hosts. More shelter-building lepidopteran larvae from the area should be reared to determine if I. melitaraevora is host-specific to Melitara subumbrella, specific to Melitara species, or attacks a range of shelter-building lepidopteran larvae like its sympatric congener I. etiellae. There remains much to be learned of cactus moth trophic interactions, and of shelter-building Lepidoptera enemies in general given that likely only 10% of microgastrine species are described (Rodriguez et al. 2013; Fernandez-Triana et al. 2020).

Parasitism by coevolved Microgastrinae and other hymenopteran parasitoids accounted for 20% of invasive Cactoblastis cactorum (Lepidoptera, Pyralidae, Phycitinae) mortality in Argentina, its native range (Varone et al. 2019). Native range host testing with several South American Microgastrinae demonstrated that A. opuntiarum is specific to C. cactorum (Martínez et al. 2012; Varone et al. 2015; Folgarait et al. 2018). Follow-up sequential no-choice tests with North American cactus moth larvae, and phylogenetic relatives, confirmed that A. opuntiarum will be host-specific to C. cactorum in North America as well (Florida Dept. Agriculture & Consumer Services, unpublished data). Large-scale rearing capacity for A. opuntiarum has advanced significantly over the past decade (Awad et al. 2019; Srivastava et al. 2019; Varone et al. 2024) and the wasp was in the final stages of regulatory approval for release in the U.S.A. at the time of publication.

North American Melitara are parasitized by wasps in at least four families, including: Braconidae (Cheloninae and Microgastrinae), Scelionidae, Eulophidae,and Chalcididae (Mann 1969; Rodriguez et al. 2013; Morales-Gálvez et al. 2022; Villegas-Luján et al. 2024) and have patchy distributions (authors, personal observations), which suggests that parasitoid pressure is high. Indeed, Mann (1969) also remarked how rare native cactus moth larvae were during a multi-decadal survey of Opuntia herbivores in the U.S.A. and Mexico. Given the shared biology and evolutionary proximity of American cactus moths, we hypothesize that some native North American cactus moth parasitoids in the Microgastrinae can utilize C. cactorum as a host and thus provide a level of natural biological control of this invasive species (Morrison et al. 2025). Ongoing studies and collections by groups at the Brackenridge Field Lab and Universidad Autónoma Agraria Antonio Narro (Saltillo, Mexico) are investigating this possibility.

Laura Springer and Zach Mann assisted with rearing of the wasps. Caroline Boudreault prepared the type specimens and took images. Jenna Haines performed DNA extraction and edited the CO1 sequences. Alex Wild accepted the holotype plus several paratypes at the UTIC. We also thank the USNM and CNC for accepting the paratypes for other workers to examine. We are grateful to Hillary and David Armstrong for preserving high quality land where we were able to discover the wasps.