JHR 97: 207-228 (2024) ge, JOURNAL OF eet snnscnn ne doi: 10.3897/jhr.97.117514 RESEARCH ARTICLE () Hymenopter a https://jhr.pensoft.net Thelnternaonl Society of Hymenoptariss, RESEARCH Redescription of Apanteles mimoristae (Hymenoptera, Braconidae), a parasitoid of the native pyralid cactus moth Melitara cf. nephelepasa in central Mexico Renato Villegas-Lujan', Robert Plowes*, Lawrence E. Gilbert’, Julio Cesar Rodriguez', Ricardo Canales-del-Castillo*, Gabriel Gallegos-Morales', Martha P. Espafia-Luna’*, José Fernandez-Triana’, Sergio R. Sanchez-Pefia' | Universidad Auténoma Agraria Antonio Narro, Saltillo, Coahuila, Mexico 2. University of Texas at Austin, Austin, Texas, USA 3 Universidad Auténoma de Nuevo Leén, San Nicolds de los Garza, Nuevo Leon, Mexico 4 Universidad Auténoma de Zacatecas, Zacatecas, Mexico 5 Canadian National Collection of Insects, Ottawa, Ontario, Canada Corresponding author: Sergio R. Sanchez-Pefia (sanchezcheco@gmail.com) Academic editor: J.M.Jasso-Martinez | Received 18 December 2023 | Accepted 1 March 2024 | Published 26 March 2024 Attps://zoobank. org/1B729C13-0B3A-44 1 4-AFB0-BCB1A6 130342 Citation: Villegas-Lujan R, Plowes R, Gilbert LE, Rodriguez JC, Canales-del-Castillo R, Gallegos-Morales G, Espafia-Luna MP, Fernandez-Triana J, Sanchez-Pefia SR (2024) Redescription of Apanteles mimoristae (Hymenoptera, Braconidae), a parasitoid of the native pyralid cactus moth Melitara cf. nephelepasa in central Mexico. Journal of Hymenoptera Research 97: 207-228. https://doi.org/10.3897/jhr.97.117514 Abstract Novel trophic associations have sometimes resulted in fortuitous and significant biological control. After the invasion of North America by the South American cactus moth, Cactoblastis cactorum (Berg) (Pyrali- dae: Phycitinae), it is pertinent to characterize the assemblage of local natural enemies that could utilize this moth in new host-parasitoid associations. Herein we report on Apanteles mimoristae Muesebeck (Bra- conidae: Microgastrinae), a North American gregarious endoparasitoid wasp attacking the caterpillar of the phycitine cactus moth Melitara cf. nephelepasa (Dyar) (Pyralidae: Phycitinae, also known as zebra worm), also native to North America; both collected in Opuntia ficus-indica (L.) Mill. (Cactaceae) culti- vated fields at rural areas of Mexico City. We provide an updated morphological account for A. mimoristae visualized with light microscopy and scanning electron microscope (SEM); a fragment of its cytochrome oxidase subunit I (COI) gene sequence data is reported for the first time. Additionally, we analyze its taxo- nomical position relative to other Apanteles species from the Americas including those attacking cactus- feeding moths. Our analyses place A. mimoristae (from Mexico) in a clade with A. esthercentenoae Fernan- dez-Triana (from Costa Rica), a parasitoid of both Cromarcha stroudagnesia Solis (Pyralidae) and Palpita venatalis (Schaus) (Crambidae) (non cactus-feeding), and in a sister clade to A. opuntiarum Martinez & Copyright:This is an open access article distributed under the terms of the CCO Public Domain Dedication. 208 Renato Villegas-Lujan et al. / Journal of Hymenoptera Research 97: 207-228 (2024) Berta (from Argentina) and A. alexanderi Bréthes (from Argentina and Uruguay), parasitoids of the cactus- feeding phycitines Cactoblastis and Tucumania respectively. Finally, we provide an updated key for the identification of Apanteles species recorded parasitizing cactus moth caterpillars in the American continent. Keywords Agriculture, biological control, ecosystem, invasive insect, North America, Opuntia, South America Introduction Novel trophic associations can result after dispersal and expansion of the geographi- cal distribution of organisms. Some novel associations (e.g., infection, parasitism, parasitoidism, predation) sometimes bring about significant levels of mortality of phytophagous insects (Torres-Acosta et al. 1916; Felipe-Victoriano et al. 1917; Durocher-Granger et al. 2021) resulting (in the case of pests) in fortuitous or “new association” biological control (Sterling 1978; Hokkanen and Pimentel 1989). The South American cactus moth Cactoblastis cactorum (Berg) (Pyralidae: Phycitinae) is one of the most important herbivores of Opuntia Miller and related genera (prickly pears, Cactaceae) (Morrison et al. 2021). It poses a serious threat to Opuntia cacti in North America, both in their native natural communities and in commercial plantations (Starmer et al. 1988; Hight and Carpenter 2009). Opuntia (called no- pal in Mexico) is widely consumed by the Mexican people since prehispanic times. The areas involved are of a continental scale and this implies that intensive meas- ures like chemical control and physical destruction of C. cactorum are not feasible, making biological control by permanently established natural enemies (classical biological control) the most viable option (Habeck and Bennet 1990; Vigueras and Portillo 2001). North American Opuntia species are attacked by caterpillars of native cactus moths in the Pyralidae. Among these, the genus Melitara Walker is widespread in the deserts of northern Mexico and the southern and western United States; these insects usually bore into pads, are solitary and ocassionally cause economic damage or plant dieback (Mann 1969; personal observations). The study of local natural enemies is a key aspect in biocontrol of native and exotic insects (Morales-Galvez et al. 2022). Several species of Microgastrinae (Bra- conidae) are among the potential natural enemies considered for biological control of C. cactorum. Some species of Apanteles Foerster have been reported as parasitoids of pyralid cactus feeding moths, such as Cactoblastis Ragonot in South America, and the closely related genus Melitara in North America as well as Loxomorpha Amsel (=Mimorista) in the related family Crambidae (Muesebeck 1921). This family and the Pyralidae form the superfamily Pyraloidea. About half of described species of Apanteles are gregarious endoparasitoids of the host larval stage (Fernandez- Triana et al. 2014; Varone et al. 2015; Figueroa et al. 2021). Apanteles alexanderi Bréthes, Updated description of Apanteles mimoristae 209 A, mimoristae Muesebeck, A. opuntiarum Martinez & Bertha, and Iconella etiellae Viereck (a genus closely related to Apanteles) are among native Microgastrinae species parasitizing pyralid Opuntia-feeding moths in the American continent (Muesebeck 1921; Martinez et al. 2012; Fernandez-Triana et al. 2013). Of those, only A. opun- tiarum from Argentina has been studied as biological control agent of C. cactorum (Martinez et al. 2012). The most frequent reported hosts of another related wasp, Apanteles megathymi Riley, are butterfly larvae in the Hesperiidae (giant skippers like Megathymus spp.), but it has also been reported attacking the cactus zebra worm, Melitara nephelepasa (Dyar) and Laniifera cyclades (Druce) (Crambidae) in Opuntia spp (Mann 1969). Apanteles mimoristae was described in the early 1900s based on four females and one male (Muesebeck 1921). However, the taxon is not well defined taxonomically: the original description is very short and incomplete, and it lacks illustrations and molecular information. Another important aspect in the Microgastrinae, as in other parasitoid wasp families, is the existence of cryptic species (Whitfield 1997; Hoy et al. 2000) making accurate species identification difficult even for specialists. Indigenous parasitoids and other natural enemies may associate to invasive spe- cies (cactus moth in this context) to create new trophic relationships, exemplifying the so-called “new association biological control” (Hokkanen and Pimentel 1989). It is possible that indigenous Apanteles species parasitizing Melitara, like A. mimor- istae, might exploit the invasive moth C. cactorum as a host. Our objective is to clarify and refine the description of A. mimoristae to better characterize the species and support its identification. DNA barcode sequences complemented by morpho- logical comparison were used to investigate species boundaries. A key is provided to highlight the differences between Apanteles species reported to attack phycitine cactus months in the American continent. The information presented here will serve as a foundation for investigations on the possible interactions of A. mimoristae in the zones invaded by C. cactorum in North America as an element in a biological control approach. Materials and methods Rearing material. Seventy caterpillars (larval stage) of zebra worm, Melitara cf. neph- elepasa ranging from half-grown to fully grown were collected from prickly pear pads (Opuntia ficus-indica L. var. Milpa Alta) at commercial plots in Mexico City, central Mexico (19.191444, -99.003810) (Fig. 1) in the summer and fall of 2019 and 2020. These caterpillars were transported to the laboratory of the Universidad Auténoma Agraria Antonio Narro, and were placed individually in plastic containers, on a layer of moist filter paper at room temperature (25°+2C), 100% RH, and 12:12 h L:D, and fed daily fresh fragments of O. ficus-indica pads until they reached the adult stage or parasitoid emergence. 210 Renato Villegas-Lujan et al. / Journal of Hymenoptera Research 97: 207-228 (2024) WN ne ne, ha — 4 - mtr 8 Av. Espana } ¥ ie ad 5 4 Figure |. Collection site of Apanteles mimoristae from Melitara cf. nephelepasa in cultivated fields of Opun- tia ficus-indica (Cactaceae) in San Jerénimo Miacatlan (SJM), in the municipality of Milpa Alta, Mexico City, MX (left). The cactus fields are partially surrounded by houses and other agricultural areas (right). Morphological analysis Parasitoid wasp. After emergence, adult wasps were preserved in 70% ethanol (for mounting and photographing) and 96% ethanol (for DNA analysis). Morphological terms and diagnostic structures followed Wharton (1997) and Wharton et al. (1997). Features were compared with the original description of A. mimoristae (Muesebeck 1921), and also Martinez et al. (2012) and Fernandez-Triana et al. (2013, 2014). Due to costs and travel limitations, it was not possible for the first author to examine the holotype of A. mimoristae. The Mexican material listed was examined by author JFT and compared to paratypes of A. mimoristae in the Canadian National Collection of Insects (CNC). They were also compared to the brief but valid description of the species by Muesebeck (1921). The geographical distribution of Apanteles species was obtained from Fernandez-Triana et al. (2020). Morphological analysis and imaging used a micro- scope eye-piece Dino-Lite AM7025x camera (Dino-Lite, Los Angeles, USA) adapted on an Olympus SZ51stereoscope plus an Olympus 110 AL2X-2WD38 magnifying lens (Olympus, Fukuoka, Japan). Images were also obtained with a Hitachi TM-3000 scanning electron microscope (SEM) (Hitachi High-Tech Corp., Fukuoka, Japan). For measurements, the DinoXcope software version 2.3 (Dino-Lite) was used. Images were edited on ImageJ (National Institutes of Health, USA). All measurements are expressed in mm. Morphological terms and their abbreviations used are: SV = surface of the ver- tex, [1 = mediotergite 1, and T2 = mediotergite 2. Abbreviations of depositories are: UNAM — Coleccién Nacional de Insectos, Instituto de Biologia, UNAM, Ciudad de México, México CNC Canadian National Collection of Insects, Arachnids and Nematodes, Ot- tawa, Canada NMNH National Museum of Natural History, Washington D.C., United States UT University of Texas at Austin, United States Updated description of Apanteles mimoristae 211 Melitara cf. nephelepasa caterpillars were identified following the description and geographical range reported in Mann (1969), collected in the host plant O. ficus-indica. DNA extraction and PCR Extraction, PCR conditions, and sequencing for the cytochrome oxidase subunit 1 (COD) were performed following Lopez-Monzon et al. (2019). Barcode sequences were obtained both at Brackenridge Field Laboratory, University of Texas (UT) at Austin, US (5 specimens) and at Facultad de Agronomia, Universidad Auténoma de Zacatecas (UAZ) at Zacatecas, MX (1 specimen). Phylogenetic analysis There were no known A. mimoristae sequences available before this work. A frag- ment of 628 base pairs (bp) of the cytochrome oxidase subunit I (COI) gene was obtained from all specimens at UT. The raw sequences were edited in Codon Code ver. 5.0.1 (Codon Code Corporation, Dedham, MA, US). The obtained sequences were aligned using ClustalW within the Molecular Evolutionary Genetics Analy- sis (MEGA) Version X software (Kumar et al. 2018), alongside a total of 152 se- quences from Apanteles (Smith et al. 2008), supplemented with sequences from other Apanteles and related genera and species in the Braconidae retrieved through the Basic Local Alignment Search Tool (BLAST). Utilizing various exploratory phyloge- netic trees derived from the Neighbor-Joining (NJ) and Maximum Likelihood (ML) algorithms, the alignment was refined to a subset of 33 sequences to ascertain the taxonomic and phylogenetic placement of A. mimoristae (Table 1). The phylogenetic relationship for the Apanteles species attacking cactus-feeding moths was then con- structed by Maximum Likelihood (ML) algorithm using MEGA version X (Kumar et al. 2018) and parameter as GTR+I+G (General Time Reversible with invariant sites and a gamma distribution) an evolutionary model with 1,000 replicates. Model selection was performed using statistical and evolutionary analysis of multiple se- quence alignments TOPALi v2 (Milne et al. 2009). In MrBayes ver.3.2.5 5 (Ron- quist et al. 2012) we set partitions, first, second and third positions of COI, and models as selected by Partition Finder v1.1.1 (Lanfear et al. 2012) under the Bayesian information criterion (BIC) and the “all” search algorithm. For the first partition and second position the best model was GTR+I+G, and for the third partition the best model was HKY+1+G (Hasegawa-Kishino-Yano with invariant sites and a gamma distribution). We conducted the Bayesian phylogenetic analysis with nucmodel = 4by4, nruns = 2, nchains = 4, and sampled freq = 1000 (Sanchez-Pefa et al. 2017), for one billion generations. We assessed convergence and stationarity in the Bayes- ian analysis using the “sump” command to examine log marginal likelihood plots, average standard deviation of split frequencies among runs, and the potential scale reduction factor for all parameters. Nodes that had posterior probabilities greater than 0.95, were considered well supported. 212 Renato Villegas-Lujan et al. / Journal of Hymenoptera Research 97: 207-228 (2024) Table |. Dataset of selected COI sequences of 33 species of Microgastrinae (Apanteles Forster, Dolicho- genidea Viereck, Glyptapanteles Ashmead, Iconella Mason, and Parapanteles Ashmead) and their GenBank accession numbers utilized in the phylogenetic analysis. DNA voucher numbers [DHJPAR = Daniel H. Janzen and Winnie Hallwachs database at University of Pennsylvania; CNIN = Coleccién Nacional de Insectos, Universidad Nacional Aut6noma de México (UNAM); USNM, National Museum of Natural History] * = Apanteles species parasitizing cactus moths in the Phycitinae on the American continent. A recent revision of the genus Parapanteles (Parks et al. 2020) indicated that it is not monophyletic, with species interspersed among Apanteles and closely related genera of Braconidae. Microgastrinae species with COI sequence Collection and voucher code = GenBank Accession number Apanteles sp. Rodriguez48 DHJPAR0002317 KF462163 Apanteles sp. Rodriguez48 DHJPAR0047068 KF462208 Apanteles sp. Rodriguez106 DHJPAR0049396 KF462061 Apanteles sp. Rodriguez12 DHJPAR0047067 KF462166 Apanteles sp. Rodriguez169 DHJPAR0041984 JQ575692 Apanteles sp. Rodriguez69(3) DHJPAR0039707 HQ926377 Apanteles sp. Rodriguez47 DHJPAR0002260 MT469770 Apanteles sp. Rodriguez47 DHJPAR0045169 KF462233 Apanteles sp. Rodriguez107 DHJPAR0034228 JQ853633 Apanteles sp. Rodriguez32 DHJPARO048151 KF462206 Apanteles sp. Rodriguez158 DHJPAR0049161 KF461918 Apanteles sp. Rodriguez168 DHJPARO0045255 KF462252 Apanteles sp. Rodriguez68(2) DHJPARO0004091 EU397563 Apanteles sp. Rodriguez33 DHJPARO0048132 KF462068 Apanteles sp. Rodriguez67 DHJPARO0049140 KF462152 ‘Apanteles alexanderi CNIN1122 JX566790 “Apanteles opuntiarum CNIN1113 JX566778 Apanteles sp. Rodriguez105 (A. esthercentenoae) DHJPARO0005185 EU396687 “Apanteles Milpa Alta DF (A. mimoristae) OQ676887 Parapanteles sp. Whitfield 133 DHJPAR0020673 JQ850314 Parapanteles sp. Whitfield44 DHJPAR0030780 JQ854565 Parapanteles sp. Whitfield45(2) DHJPARO0020128 EU397378 Apanteles sp. Rodriguez110 DHJPARO005168 EU396741 Parapanteles sp. Whitfield102 DHJPARO0041787 MN645414 Parapanteles sp. Whitfield303 DHJPARO0012759 EU396804 Parapanteles sp. Whitfield302 DHJPARO0012793 EU396799 Parapanteles sp. Whitfield70 DHJPAR0020653 JQ853731 Apanteles sp. Rodriguez167 DHJPAR0045315 KF462011 Apanteles sp. Rodriguez169 DHJPAR0041984 JQ575692 Iconella sp. Whitfield05 DHJPAR0045362 KC685306 Dolichogenidea sp. Whitfield11 USNM00496786 JQ852381 Glyptapanteles sp. Whitfield175 DHJPARO0040014 JQ574612 Results Material examined Mexico, 159, 154 of A. mimoristae; Mexico City, Milpa Alta, San Jerénimo Mia- catlan; 19.191444, -99.003810, 2384 masl; 21.xi.2020; Renato Villegas leg.; zebra worm, Melitara cf. nephelepasa in commercial plots of Opuntia ficus-indica (Cactaceae) (prickly pear or nopal); GenBank: OQ676887.1 and OQ561741.1. Updated description of Apanteles mimoristae 213 Redescription Apanteles mimoristae Muesebeck, 1921 Note. Measurements from reared specimens in this work. Female. Body length of x=2.94 (2.805—3.097) (Figs 2-4). Head. Transverse; antennae shorter than body, X=2.45 (2.282—2.605), face smooth and eyes moderately setose in frontal view (Fig. 3A). SV finely wrinkled around the ocelli, but smooth and opaque between the ocelli (Figs 3B, 3C). Ocular—ocellar line/ posterior ocellus diameter: 2.13—2.32. Intercellular distance/posterior ocellar diameter: 2.15—2.33. Anten- nal flagellomere 2 length/width: 2.06—2.46. Antennal flagellomere 14 length/width: 1.06-— 1.40. Length of antennal flagellomere 2/length of antennal flagellomere 14: 2.08-2.18. Mesosoma. Dull black. Mesoscutum: indistinctly/irregularly punctate, profusely setose and markedly rugose, the roughness does not cover its surface in dorsal view (Fig. 3D). Scutellum: flat, dull, smooth, and setose. Pits in the scutoscutellar sulcus: 10. Mesopleura: anterior half punctate and pilose; posterior half smooth and polished. Propodeum: with rugae; with a well-defined broad median areola, inside of areola with marked wrinkles, and a transversal carina which does not reach the spiracle (Fig. 3D, E). = — Figure 2. Apanteles mimoristae Muesebeck (Braconidae: Microgastrinae) female. Habitus. 214 Renato Villegas-Lujan et al. / Journal of Hymenoptera Research 97: 207-228 (2024) 250 pm Figure 3. Apanteles mimoristae Muesebeck A-C head A frontal view B dorsal view C ocelli D, E meso- soma D dorsal view E lateral view F—H legs F anterior G medium H posterior. Updated description of Apanteles mimoristae 215 Figure 4. Apanteles mimoristae Muesebeck, female wings: forewing (left) and hindwing (right) Legs: all coxae blackish; profemur: with less of the basal half dark brown and the rest dark yellow or light brown; mesofemur: more than half blackish and the rest dark brown; metafemur: entirely black; protibia: evenly light yellowish; mesotibia: basally yellowish and mostly light brown towards the apex; metatibia: color pattern varies from dark brown fading to light yellow from the base to the apex (Fig. 3F, H). Metatibia inner spur length slightly longer than half of metatarsus (Fig. 3H). Metafemur length/ width: 2.92—3.16. Metatibia inner spur length/metabasitarsus length: 0.48—0.50. Wings: hyaline; translucent pterostigma with dark brown margins; veins transparent to dark brown (Fig. 4). Fore wing length: 3.61—-3.65 mm. Fore wing veins length: 1/2RS: 1.56-1.62, 2RS: 1.11-1.19, 2M/(RS+M)b: 0.78-0.85. Pterostigma length/ width: 3.19-3.66. Anterior half of humeral complex whitish yellow, posterior half is light to dark brown; tegula pale to dark. Metasoma.1 1: elongated from above, wider at the base than at the apex, with marked wrinkles across all surface, mainly in the median area; barely setose, with two depressions on the posterior margin, more or less of the same size (Fig. 5A). T1 length/width at posterior margin: 2.42—2.50. T2: wider than long, smooth, with- out rugosities, with little setae in dorsal view and two groups of setae in lateral view (Fig. 5B, C). Width at posterior margin/length: 2.90—3.05. Ovipositor: mean length of sheaths = 0.895 mm (0.863-0. 935); black, slightly wider at the base than at the apex, almost as long as the abdomen, smooth, opaque, moderately setose, and covering all the ovipositor (Fig. 5D). Ovipositor sheaths length/metatibial length: 0.833—1.02. Pleats in the hypopygium: at least 4. Male. Very similar to female (Fig. 6), excepting smaller body size, x = 2.696 (2.599—2.785); antennae longer than body, 3.008 (2.752—3.187). Distribution. Mexico (Mexico City), United States (Texas and Florida). Biology. Gregarious larva-prepupa koinobiont endoparasitoid. Hosts. Melitara cf. nephelepasa (Pyralidae) feeding on Opuntia ficus-indica (Cac- taceae); Melitara junctolineella Hulst (Pyralidae); Loxomorpha flavidissimalis (Grote) (Crambidae). A list of selected morphological differences between A. mimoristae and four other Apanteles species parasitic on Pyralidae: Phycitine stem- and cladode borer larvae are summarized in Table 2. The dichotomous key provided below includes selected Apan- teles species parasitizing phycitine cactus moths in North and South America. Renato Villegas-Lujan et al. / Journal of Hymenoptera Research 97: 207-228 (2024) 216 — gowrdop—~—“i=