蝶と蛾 Lepidoptera Science 67（2）: 58-66, October 2016 Biology and chemical ecology of Eupackardia calleta（ Saturniidae） in southern Texas Joseph H. LOUWAGIE III and Richard S. PEIGLER Department of Biology, University of the Incarnate Word, 4301 Broadway, San Antonio, Texas 78209-6397 USAi Abstract Field and lab observations were made on Eupackardia calleta, a large saturniid that flies in southern Texas. Leucophyllum langmaniae is newly reported as a hostplant in nature. An oviposition was found in nature comprising 17 eggs on 8 leaves. Samples of cocoons preyed upon by a mammal are illustrated. Three parasitoids are reported: the euplemid Anastatus from eggs, the tachinid Lespesia sp. near texana, and the chalcidid Conura maria from cocoons, the latter two for the first time. Predation on a third-instar larva by the vespid Polistes exclamans was observed. Phototactic behavior of males（ which are normally diurnal） and a multiple mating by a female are newly reported. Three new county records for Texas are cited. Biochemical analyses were done on unfed first-instar larvae, fed second-instar larvae, mature larvae, and adult moths, and the following compounds were found: 3,4-dihydroxyphenylalanine, dopamine, norepinephrine, hydroquinone, 2-（dimethylamino）ethylacetate, trehalose, and 3,5-dimethylphenol, some of which probably provide protection from predators. Trials on larvae were conducted in the lab and field with five species of ants（ Hymenoptera: Formicidae） to test for chemical protection against predation. It was observed that larvae have some chemical protection from ants, and because some of the above mentioned biogenic chemicals were found to be carried into the adult stage, it is proposed that moths of E. calleta may be Müllerian instead of Batesian mimics of Battus（ Papilionidae）. Key words ants, biochemical defenses, Formicidae, Leucophyllum, mass spectrometer, mimicry, parasitoids, predators. Introduction likely also serves as a model for E. calleta（ Waldbauer and Sternburg 1976）. Battus philenor and B. polydamas are common The large saturniid moth Eupackardia calleta（ Westwood） in Texas and southern Arizona where E. calleta occurs（ Collins （Figs 1, 2） ranges across southern Texas from the Coastal Bend 2007; d’Abrera 2016）. as far north as Cuero and Victoria in the east to the Big Bend and Trans-Pecos sections of the state in the west. It was long The brightly colored larvae of E. calleta（ Figs 6 , 7） are assumed that the distribution in eastern and western Texas was ostensibly aposematic, because they contain biogenic chemicals disjunct（ Tuskes et al. 1996）, but new records to the south and that repel predators（ Collins and Weast 1961; Deml and Dettner southwest of San Antonio reveal that the range is continuous. 1993, 1995, 1997）. Some of these biogenic compounds are The moth also ranges down to the Lower Rio Grande Valley in identified and discussed below in our results and discussion. The the southern tip of Texas and into Mexico. The primary hostplant purpose of this paper is to document new observations on the in southern Texas is cenizo（ Leucophyllum frutescens, range, foodplants, parasitoids, predators, adult behavior, and the Scrophulariaceae）. Most of what is known about E. calleta in biochemical ecology of the larvae and adults. Texas was summarized by Collins and Weast（ 1961）, Ferguson （1972）, Miller（ 1976）, Tuskes et al.（ 1996）, and Peigler and Materials and methods Maldonado（ 2005）. We made five field trips to areas south of San Antonio to collect Because of their black ground color with red and white cocoons and larvae. Most of our research material of E. calleta markings, the adult moths of E. calleta are believed to be was collected on plantings of the hostplant in Devine（ Medina Batesian mimics of the papilionid Battus philenor（ Linnaeus）, County） and Pleasanton（ Atascosa County）. The main study like diurnal males of the saturniid Callosamia promethea site in Devine consisted of an unpruned hedge of cenizo shrubs （Drury） and the butterflies Speyeria diana（ Cramer）, Limenitis more than 3 m tall and 40 m long, in a vacant lot adjacent to an arthemis astyanax（ Fabricius）, Papilio troilus Linnaeus, Papilio old house. The males were attracted to captive females in San polyxenes asterius（ Stoll）, and the dark form of Papilio glaucus Antonio, Bexar County. The species has not previously been Linnaeus. In southern Texas, Battus polydamas（ Linnaeus） reported in literature from Atascosa, Bexar, and Medina counties. We reared larvae in cages using sprigs of cenizo inserted in i [email protected] bottles of water. Biology and chemical ecology of Eupackardia calleta（ Saturniidae） in southern Texas 59 To examine possible presence of several compounds that have once, and Crotch（ 1956） reported that a female of Samia ricini been previously reported（ Deml and Dettner 1993） in E. calleta （Wm. Jones） emitted pheromone on three occasions 24 h apart, and cenizo leaves, we used an Advion® mass spectrometer of and mated each time. Morton（ 2009） recorded multiple matings the Single Stage Quadrupole model. For MS detection, the in females of Callosamia promethea, a saturniid that has diurnal ionizing method is ESI+ mode, and the sample injection method males and nocturnal females like E. calleta and S. pavonia. He was direct injection. It has a mass range of 10 m/z to 1,200 m/z, proposed various hypotheses for this polyandry, including and a scanning speed of 10,000 m/z units sec－1 and a mass increased fecundity and reduced predation. The latter was based resolution of 0.5-0.7 m/z units（ FWHM） at 1,000 m/z units sec－ on the hypothesis that because males of C. promethea are 1 over the entire acquisition range. The mass accuracy is ±0.1 mimics of toxic papilionids（ see Introduction above）, but m/z units over the entire acquisition range. For mass sensitivity, females are not, pairs mating during the day may afford the scan sensitivity is 100 pg Reserpine, and the SIM sensitivity additional protection to females from avian predators. However, is 10 pg Reserpine. although they are nocturnal, females of E. calleta also have the mimetic phenotype, so the reduced predation hypothesis Hemolymph samples from caterpillars and moths were extracted probably would not apply to this species. using micropipettes after puncturing the integument. We tested six samples: unfed larva（ first instar）, 1 week old fed larva A captive pair of E. calleta reared from cocoons collected in （second instar）, mature larva hemolymph, mature larva scoli Devine mated in a cage on 30 March 2014. The female deposited （glandular secretions）, female moth hemolymph, and cenizo many eggs the next two nights in a paper bag, and those eggs leaf. We used a 10 μl sample（ sample + 40 μl de-ionized water） hatched 10 days later. She was placed in a cenizo shrub in the direct injection with a 70% methanol pump infusion. The cenizo front yard（ in northern San Antonio） of the second author after leaf preparation was slightly different than the larvae and moth nightfall on 1 April, and late on 2 April was observed to be samples. We placed one cenizo leaf into a mortar and pestle mating with another male that she had attracted. The next bowl, then added 2-3 drops of de-ionized water. The cenizo leaf morning she was gone, but the male was perched under a porch was crushed and the liquid from the leaf secretions mixed with light that was ca. 3 m from the shrub. It has long been accepted the water, and 10 μl of the sample were extracted. Once the data that males are diurnal and females are nocturnal, with only the were collected, we looked to see if there were certain chemicals latter coming to lights, but Howe（ 1963） illustrated a male he identified in E. calleta by Deml and Dettner（ 1993）. The collected at light in Cuero, DeWitt County, Texas, and our biogenic chemicals were tentatively assigned on the basis of observation verifies his report that males are occasionally molecular ions（ m/z） by using an on-line database called attracted to lights. We do not believe that males normally fly at MassBank, maintained by the National Bioscience Database night, and the above two instances of phototaxis possibly Center, Japan（ Horai et al. 2010）. occurred only because those males were very near lights when mating ceased. It is also possible that females occasionally fly to We ran some brief trials in the field and lab using five species of lights with males still attached to them, leaving the males at the ants to test the hypothesis that larvae possess some chemical lights when copulation ceases and then flying away to oviposit. protection. Larvae were placed beside entrances of ant nests in the field, and along with captive ants in petri dishes in the lab. We did not have the opportunity to take females of E. calleta to We were able to keep ants alive in plastic and glass containers field sites where high populations exist. However, in northern for a week or longer by providing small pieces of tissue paper San Antonio, males were attracted on the following dates and soaked with honey and water. The trials included offering first- times（ Central Daylight Time） in 2014 : 10 : 22 on 14 March; instar larvae（ 4 mm long） that had never fed, second-instar 9:34, 10:39, and 10:47 on 15 March; 9:52 on 16 March（ but larvae（ 8 - 9 mm long） that had fed for several days, mature windy after 10:00）; 13:00 on 21 March; 10:26 on 22 March; and larvae（ 65 mm long）, and mealworms（ Tenebrio molitor 9:30 on 29 March. The temperature was 7° Celsius on 29 March. Linnaeus）（ 12-15 mm long）. The latter were considered to be a Emergences from the cocoons occur around midnight, so that good control because they are palatable to a wide range of adults are ready for courtship behavior when the sun rises.