Pterourus multicaudatus (Kirby) (Lepidoptera: Papilionidae) (Fig. 1a), is a butterfly distributed from Canada and the United States through Mexico and Guatemala (NaturaLista, CONABIO, 2020). In Mexico this papilionid was reported from Chiapas, Durango, State of Mexico, Guanajuato, Hidalgo, Morelos, Oaxaca and Puebla (Cibrián-Tovar et al., 1995). Larvae of P. multicaudatus feed on leaves of Citrus aurantium L. (Rutaceae), Fraxinus uhdei (Wenz.) Lingelsh., Ligustrum japonicum Thunb. (Oleaceae), Salix spp. (Salicaceae), Prunus persicae (L.) Batsch. and Prunus serotina Ehrh (Rosaceae) (Cibrián-Tovar et al., 1995; Ramos et al., 2011). In rural communities of Mexico, people use adults of P. multicaudatus as foodstuff (Ramos et al., 2011). There is little information about natural mortality of P. multicaudatus (Jiménez-Galván et al., 2017). According to Gómez & Concha (2017), knowledge of insects natural enemies is a basic requirement from a science perspective, as well as a transcendental element in economic entomology. Santhosh & Basavarajappa (2017) point out that around 95% individuals in every Lepidoptera species die before adulthood due to the direct effects of entomopathogens, predators and parasitoids.

The most common parasitoids of Papilionidae are Hymenoptera (Chalcidoidea and Ichneumonoidea) and Diptera (Tachinidae) (Krishna et al., 2014; De Souza et al., 2015; Kan & Kan, 2015). The genus Pteromalus Swederus (Chalcidoidea: Pteromalidae) includes 504 species, 47 of them reported in North America; the majority are parasitoids of larvae and pupae of Coleoptera, Lepidoptera and Diptera (Baur, 2015; Noyes, 2018). The biology of this group of parasitoids is not well known, except that of P. puparum L., which is the most well-studied species. Native to Europe, P. puparum has been introduced into numerous countries for biological control of cabbage butterflies (Pieris spp.) (Lepidoptera: Pieridae) (Barron, 2004). Currently, it is registered in Canada, the United States, New Zealand, Japan, China, Iran, Russia, Spain, India, and Egypt (Abbas & El- Dakroury, 1985; Barron, 2004; Razmi et al., 2011; Liu et al., 2014), parasitizing pupae of 17 families of lepidopterans, including Nymphalidae, Papilionidae and Pieridae (Takagi, 1985; McDonald & Kok, 1990; Barron, 2004; Noyes, 2018).

Pteromalus puparum L. has been reported as parasitoid of 11 species of Papilionidae (Noyes, 2018). To our knowledge, the genus has got comparatively little attention in Mexico. Until now the only known record for P. puparum in Mexico was for the state of Guanajuato parasitizing Pieris rapae L. pupae (Marín-Jarillo & Bujanos-Muñiz, 2001). A preliminary biological study about P. multicaudatus published by Jiménez-Galván et al. (2017) reported a wasp of genus Pteromalus associated with pupae of this papilionid in the state of Mexico, but the authors were not able to determine the species. In an attempt to its identification, we received help from Dr. Hannes Baur (expert taxonomist in the genus) and, in this note, we report the first record of P. puparum parasitizing pupae of P. multicaudatus in the state of Mexico contributing to the knowledge of the biotic agents involved in P.multicaudatus mortality.

From September to November 2015, 13 P. multicaudatus pupae were collected from Santa Cruz Atzcapotzaltongo (19.1908° N, 99.3916° W; 2,621 m.a.s.l.), Toluca, State of Mexico. Pupae were transported to the laboratory, where they were placed individually in Petri dishes (9 cm diameter) under laboratory conditions. The pupae were checked daily until the emergence of the parasitoids. Adult parasitoids were collected and preserved in 70% ethanol and sent to Dr. H. Baur (Institute of Ecology and Evolution, University of Bern, Switzerland), who provided species determination. Voucher specimens were deposited in the Insectarium of the Facultad de Ciencias Agrícolas at the Universidad Autónoma del Estado de Mexico.

A search for natural enemies of immature stages of P. multicaudatus was carried from March to September 2016; 48 eggs and 397 larvae in different stages of development were collected from leaves of P. serotina trees growing in private yards from two localities of the State of México: 1) El Cerrillo, Piedras Blancas (19.2432° N, 99.4120° W; 2,614 m.a.s.l.), municipality of Toluca, and 2) Agua Bendita (19.5230° N, 99.4406° W; 2,658 m.a.s.l.), municipality of Timilpan. In the laboratory, eggs were individually placed in Petri dishes (5.5 cm diameter) with absorbent paper moistened with distilled water as an incubation substrate. Larvae were placed in groups of three in plastic jars (14.0 × 10.5 cm diameter) with P. serotina leaves as a food source and they were replaced every three days. The jars were covered with cheesecloth to avoid larvae escape. Eggs and larvae were incubated at 26 ± 1 °C, 70% relative humidity, photoperiod of 10:14 h, and were checked daily to determine the presence of parasitoids.

From the larvae maintained under the aforementioned conditions, a sample of 28 “sentinel” pupae were exposed to parasitism. In three private yards (PY), pupae were adhered at a height of 1.75 m to the exterior walls of houses that had a P. serotina tree nearby. Eight pupae were evaluated in El Cerrillo, Piedras Blancas (June 25^th, 2016; PY 1), ten pupae in Santa Cruz Atzcapotzaltongo (August 9^th, 2016; PY 2) and ten in Timilpan (September 8^th, 2016; PY 3). The behavior of parasitoids toward exposed pupae was observed daily during ten days from 08:00 to 20:00 h in Santa Cruz Atzcapotzaltongo (PY 2). Fifteen days later, pupae were removed and placed in individual Petri dishes to await the emergence of parasitoids or butterflies. The measured variables were the number of pupae parasitized and the number of parasitoids emerging per pupa, and a X² test was used to determine sexual proportion of the parasitoids. Statistical test was conducted using the software SAS (SAS, 2009).

No parasitoids were recorded on eggs or larvae in the sampled localities during 2016. Instead, five of the 13 pupae collected in 2015 in Santa Cruz Atzcapotzaltongo, all (ten) of the exposed pupae in Santa Cruz Atzcapotzaltongo, and one of the ten exposed pupae in Timilpan in 2016, were parasitized by P. puparum (Figs. 1b and 1c). No pupae parasitoids were recorded in El Cerrillo, Piedras Blancas. The distinctive morphological characters of P. puparum are mentioned by Askew & Shaw (1997) and Baur (2015).

Pteromalus puparum wasps arrived at the pupae beginning on the second day of exposure, usually one female, though at times up to three, interacted with a pupa for a period of one to five hours. In Santa Cruz Atzcapotzaltongo a prepupa of P. multicaudatus of wild origin not belonging to our “sentinel” sample was observed near to our experimental sentinel pupae. Three females of P.puparum were observed moving along its body for eight hours; probably these females may have been waiting for the individual to transition to the pupal stage in order to parasitize it. Takagi (1985) reported similar behavior in P. puparum on prepupae of Papilio xuthus L. (Lepidoptera: Papilionidae), though it was described there as a case of phoresis. In this study, P. puparum oviposited in the non-sclerotic intersegmental areas between the thorax and abdomen of the pupae (Fig. 1d). Finally, on the seventh day of exposure, no more P. puparum wasps were observed on pupae of P. multicaudatus.

Pteromalid larvae consumed the entire internal tissues and organs of the host. The wasps chewed from the interior of the pupal wall outward and they exited the pupae over a period of one to seven days through one to three holes (Fig. 1e). In this study, we recorded sixteen P. multicaudatus pupae parasitized, out of 41, representing a parasitism rate of 39%. In average, adult parasitoids emerged 46.15 ± 7.99 days after oviposition, with a range of 39-53 days (N = 10 pupae) during October and November. The number of parasitoids that emerged varied from seven to 498 wasps per pupa. Females outnumbered males at a ratio of 4:1 (χ² = 172.16, DF = 15, p ˃ 0.01) (Table I). Lasota & Kok (1986) have reported a rate of parasitism of 48.3% in pupae of P. rapae attributed to P. puparum, with an average of 52.3 wasps per pupa and a 1:1 female: male ratio. Razmi et al. (2011), calculated a 47.89% rate of parasitism by P. puparum on Pieris brassicae (L.) with an average of 42.36 wasps per pupa, but in some cases, they recorded up to 200 wasps per pupa. According to the importance of P. puparum as a cause of mortality in pupae of P. multicaudatus, additional studies are needed to determine its influence on population dynamics of this lepidopteran associated to P. serotina, that according to Cibrián-Tovar et al. (1995), is classified as a pest of forestry interest in Mexico.

Acknowledgments

The first author received a scholarship from the Consejo Nacional de Ciencia y Tecnología (CONACyT) to complete a Master’s degree in the Posgrado de Ciencias Agropecuarias y Recursos Naturales of the Universidad Autónoma del Estado de México (UAEMéx). The authors thank Hannes Baur for identification of Hymenoptera parasitoid and Lynna Marie Kiere for assistance in the revision of our manuscript. This research was financed in part by the Secretaría de Investigación y Estudios Avanzados de la UAEMéx, project #3796/2014/CID.

REFERENCES

Abbas, M.S.T., & El-Dakroury, M.S.I. (1985) Studies on Pteromalus puparum L. (Hymenoptera: Pteromalidae), a pupal parasite of the cabbage worm Pieris rapae L. (Lepidoptera: Pieridae) in Egypt. Zoosystematics and Evolution, 61, 339-344.

Askew, R.R., & Shaw, M.R. (1997) Pteromalus apum (Retzius) and other pteromalid (Hym.) primary parasitoids of butterfly pupae in Western Europe, with a key. Entomologistʼs Monthly Magazine, 133, 67-72.

Barron, M.E. (2004) Population ecology of the red admiral butterfly (Bassaris gonerilla) and the effects of non-target parasitism by Pteromalus puparum. Ph.D. dissertation, Lincoln University, New Zealand.

Baur, H. (2015) Pushing the limits – two new species of Pteromalus (Hymenoptera, Chalcidoidea, Pteromalidae) from Central Europe with remarkable morphology. Zookeys, 514, 43-72.

Cibrián-Tovar, J., Méndez-Montiel, M.T., Campos-Bolaños, R., Yates, H.O. III, & Flores-Lara, J.E. (1995) Insectos Forestales de México. Universidad Autónoma Chapingo, Chapingo, México.

De Souza, S.A.G., Loiácono, M.S., Margaría, C.B., & Ferreira, M.R. (2015) A new species of Telenomus Haliday (Hymenoptera: Platygastridae) egg parasitoid of Parides ascanius (Cramer) (Lepidoptera: Papilionidae) a threatened species from Brazil. Zootaxa, 3986, 387-392.

Gómez, C.A., & Concha, M.I. (2017) First record of the natural enemy Acalanthis quadrisignata (Coleoptera: Trogossitidae) associated with pupal chambers of Pissodes castaneus (Coleoptera: Curculionidae), in Chubut province, Patagonia, Argentina. Revista de la Sociedad Entomológica Argentina, 76, 54-56.

Jiménez-Galván, E., Castañeda-Vildózola, A., Sánchez-Pale, J.R., & Aguilar-Medel, S. (2017) Estudio biológico de Pterourus multicaudatus W.F. Kirby (Lepidoptera: Papilionidae) en dos localidades del Estado de México. Entomología Mexicana, 4, 498-502.

Kan, P., & Kan, B. (2015) Biological observations on Buquetia musca (Robineau-Desvoidy) (Diptera: Tachinidae), a parasitoid of Papilio machaon Linné (Lepidoptera: Papilionidae). Filming VarWild, 1-12.

Krishna, R.R., Rajanikumari, T.V.S., Devaki, K., & Sarada, G. (2014) Extent of parasitisation of citrus butterfly, Papilio demoleus (Linnaeus) (Lepidoptera: Papilionidae) in sweet orange (Citrus sinensis Swingle) and acid lime (Citrus aurantifolia) Swingle. Pest Management in Horticultural Ecosystems, 20, 123-126.

Lasota, J.A., & Kok, L.T. (1986) Parasitism and utilization of imported cabbageworm pupas by Pteromalus puparum (Hymenoptera: Pteromalidae). Environmental Entomology,15, 994–998.

Liu, Y.Q., Shi, Z.H., Zalucki, M.P., & Liu, S.S. (2014) Conservation biological control and IPM practices in Brassica vegetable crops in China. Biological Control, 64, 37-46.

Marín-Jarillo, A., & Bujanos-Muñiz, R. (2001) Insectos plaga de brócoli y coliflor y sus enemigos naturales en la región del Bajío, México. Publicación especial núm. 2. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, México.

McDonald, R.C., & Kok, L.T. (1990) Postrefrigeration viability of Pteromalus puparum (Hymenoptera: Pteromalidae) prepupas within host chrysalids. Journal of Entomological Science, 25, 409-413.

NaturaLista, CONABIO (2020) Mariposa Cometa Xochiquetzal (Papilio multicaudata). Available in https://www.naturalista.mx/taxa/68263-Papilio-multicaudata (accessed 06 March 2020).

Noyes, J.S. (2018) Universal Chalcidoidea database. Available in http://www.nhm.ac.uk/chalcidoids (accessed 06 March 2020).

Ramos, E.J., Moreno, M.P.G., Vázquez, I.A., Landero, I., Oliva, R.H., & Camacho, H.M.V. (2011) Edible Lepidoptera in Mexico: geographic distribution, ethnicity, economic and nutritional importance for rural people. Journal of Ethnobiology and Ethnomedicine, 7, 1-22.

Razmi, M., Karimpour, Y., Safaralizadeh, M.H., & Safavi, S.A. (2011) Parasitoid complex of cabbage large white butterfly Pieris brassicae (L.) (Lepidoptera, Pieridae) in Urmia with new records from Iran. Journal of Plant Protection Research, 51, 248-251.

Santhosh, S., & Basavarajappa, S. (2017) Record of natural enemies of few butterfly species amidst ecosystems of Chagaramajanar district, Karnataka, India. Research Journal of Life Sciences, Bioinformatics, Pharmaceutical and Chemical Sciences, 2, 1-18.

SAS (2009) SAS Institute, Inc, user guide: Statistical Analysis System, version 9.0. Cary, NC, USA.

Takagi, M. (1985) The reproductive strategy of the gregarious parasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae). Oecologia, 68, 1-6.

Author notes

acastanedav@uaemex.mx