Hindawi Publishing Corporation Psyche Volume 2011, Article ID 940280, 6 pages doi:10.1155/2011/940280 Research Article ARearingMethodforArgynnis (Speyeria) diana (Lepidoptera: Nymphalidae) That Avoids Larval Diapause Carrie N. Wells, Lindsey Edwards, Russell Hawkins, Lindsey Smith, and David Tonkyn Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634, USA Correspondence should be addressed to Carrie N. Wells, [email protected] Received 25 May 2011; Accepted 4 August 2011 Academic Editor: Russell Jurenka Copyright © 2011 Carrie N. Wells et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We describe a rearing protocol that allowed us to raise the threatened butterfly, Argynnis diana (Nymphalidae), while bypassing the first instar overwintering diapause. We compared the survival of offspring reared under this protocol from field-collected A. diana females from North Carolina, Georgia, and Tennessee. Larvae were reared in the lab on three phylogenetically distinct species of Southern Appalachian violets (Viola sororia, V. pubescens,andV. pedata). We assessed larval survival in A. diana to the last instar, pupation, and adulthood. Males reared in captivity emerged significantly earlier than females. An ANOVA revealed no evidence of host plant preference by A. diana toward three native violet species. We suggest that restoration of A. diana habitat which promotes a wide array of larval and adult host plants, is urgently needed to conserve this imperiled species into the future. 1. Introduction larvae in cold storage blocks and storing them under con- trolled refrigerated conditions for the duration of their The Diana fritillary, Argynnis (Speyeria) diana (Cramer overwintering period [10]. Scott [11]rearedA. atlantis from 1775), is a threatened butterfly species that has been extir- Colorado under a 24 h photoperiod and was able to stimulate pated from large portions of its former distribution [1–5]. first instar larvae to feed on Viola nephrophylla leaves after a The subgenus Speyeria, now incorporated into the genus Arg- shortened diapause period of several days to several weeks. ynnis [6], has been described as challenging to rear, primarily James [12] compared acceptance of V. adunca, V. glabella, due to the six- to nine- month overwintering period required V. labradorica, and V. tricolor by first instars of five Greater by first instar larvae. During winter diapause, tiny first instar Fritillaries from the Pacific Northwest, Argynnis. cybele leto, larvae are most vulnerable to environmental extremes such A. coronis simaetha, A. zerene picta, A. egleis mcdunnoughi, as freezing temperatures, flooding, and fire [7]. Almost 140 and A. hydaspe rhodope. Ours is the first study of host plant years ago, Edwards noted the challenges of rearing the larvae acceptance and survival of an eastern Argynnis species. of Argynnis cybele (Fabricius 1775),A.aphrodite(Fabricius Argynnis (Speyeria) diana is an eastern North American 1789), and A. diana [8]. Edwards obtained hundreds of A. fritillary species that is rare to uncommon across much of diana ova from 60 females captured outside of Coalburgh, its distribution in the southeastern United States (Figure 1). West Virginia, but none of the larvae survived to pupation Violets are the sole food plant used by Argynnis caterpillars; [8]. The first description of bypassing larval diapause in however, specific associations or preferences for Viola spp. Argynnis came nearly a century later from W.H. Evans’ notes have not been reported. In late fall, adult female A. diana will on rearing and breeding A. atlantis (Drury 1773) [9]. Evans each lay thousands of eggs singly on the forest floor near or suggested the use of mechanical stimulation to encourage on Viola spp. First instar larvae eat their way out of the egg feeding by “listless” A. atlantis larvae [9]. casing, and immediately burrow into the litter layer of the First instar Argynnis larvae are notorious for not surviv- forest floor. There, they remain inactive through the winter ing overwintering when reared in a lab, regardless of their months. The following spring, first instar larvae emerge from care and treatment [9]. To address this, Matoon et al. [10] the litter layer to feed on violets, Viola spp., both nocturnally developed a rearing protocol that requires packing Argynnis and diurnally [13, 14]. 2 Psyche (a) (b) Figure 1: Argynnis diana male (top, orange and black) and female (bottom, blue and black) adults are sexually dimorphic and striking with their bright coloration. Photos by Connie Wells. 2. Methods 24–48 hrs to prevent accumulation of mold, which can be detrimental to eggs and newly emerged larvae. We tallied ff We reared o spring of A. diana females collected from three the total number of ova produced daily by each of the eight Southeastern field sites on three native Viola species: Viola North Carolina A. diana females. In addition to these ova, sororia (Willd), Viola pubescens (Aiton), and Viola pedata colleagues kindly donated one hundred fifty A. diana ova (Linnaeus). There are approximately 80 species of Viola from three females captured in Carter County, Tennessee and native to North America, all of which are small herbaceous one hundred fifty ova from three females captured in Rabun plants producing five-petaled flowers with distinct petal County, Georgia in late September 2007. spurs [15]. The three Viola species chosen for our experiment Upon hatching, we evenly divided 1st instar larvae from are found throughout the geographic range of A. diana and each of our three field sites into petri dishes containing are easily distinguishable in appearance. These three species one of the three native violet species (Figure 2(a)). In order represent independent phylogenetic branches within the to bypass the six- to nine- month overwintering period, we genus, Viola [16]. Violets were purchased and donated from exposed all newly hatched larvae to continuous lighting, and a native nursery near our study area and transplanted into mechanically stimulated individual larvae 3 to 4 times daily one-gallon buckets containing organic potting soil mixed with a camel-hair brush to encourage feeding. Our lighting with worm castings and perlite. Plants were exposed to 12 h ◦ system consisted of a 1.2 m fluorescent grow light fixture photoperiod under 60 Watt grow bulbs at 72 Candwatered holding two 40 Watt, 122 cm fluorescent tubes, set 0.4 m daily. above the larvae and producing a constant temperature of 27◦C. We misted growing larvae twice daily with distilled 2.1. Butterfly Rearing. We captured eight A. diana females water and provided fresh filter paper and fresh Viola from a field site in Henderson County, North Carolina in clippings each day to reduce infection from pathogens. Once late September 2007. Live females were placed in glassine larvae reached the fifth instar, they were transferred to envelopes and held in a cooler for transport back to Clemson individual 0.25 L clear pupation chambers (Figure 2(a)). University, Clemson, SC. Within six hours of capture, we Once chrysalids formed, they were misted daily with distilled placed each female butterfly inside an individual paper bag water to prevent desiccation. Chrysalids were often observed containing clippings of fresh Viola foliage from V. soror ia, twitching and pulsing in response to the stimulation of V. pubescens,orV. pedata to trigger oviposition. We fed misting. Adult butterflies emerged in these containers. female butterflies twice daily by saturating cotton balls with Butterflies emerged in winter, and therefore could not be a commercially available sports drink, Gatorade, containing released to their maternal field sites. Instead, butterflies were a combination of water, sugar, salt, carbohydrates, and killed by freezing after their wings had fully expanded and electrolytes. Ova were deposited daily on the inside surface dried to preserve them for future study. of the paper bags, so we transferred butterflies to fresh paper bags every 24 h in order to collect and count ova each day. Small pieces of paper bag containing ∼10 to 50 3. Results ova were transferred to sterile petri dishes lined with 50 mm Whatman filter paper. We misted ova daily with distilled We were able to bypass first instar diapause with our rearing water to prevent desiccation and changed filter paper every protocol, reducing the 10–12-month life cycle of A. diana Psyche 3 Table 1: Egg production and survival of Argynnis diana females held in captivity for rearing and their progeny. Total Number of Number of Number of Number of Number of adults Butterfly ID number of hatched eggs larvae surviving larvae surviving pupating larvae (% ecolosion) eggs (hatch rate) to second instar to last instar NC-1 1605 746 (47%) 203 (27%) 54 24 6 (25%) NC-2 0 0 (0%) — — — — NC-3 1499 428 (29%) 115 (27%) — — — NC-4 1574 881 (56%) 155 (18%) 36 13 7 (54%) NC-5 1041 771 (74%) 211 (27%) 72 19 3 (16%) NC-6 1163 834 (72%) 427 (51%) 40 10 — NC-7 1265 816 (65%) 280 (34%) 55 8 2 (25%) NC-8 1386 963 (70%) 307 (32%) — — — NC Totals 9533 5440 (57%) 1698 (27%) 257 74 18 (24%) GA-1 50 25 (50%) 15 (60%) 9 7 5 (71%) GA-2 50 20 (40%) 6 (30%) 5 — — GA-3 50 29 (58%) 20 (69%) 16 9 3 (33%) ∗GA Totals 150 74 (49%) 41 (53%) 30 16 8 (50%) TN-1 50 28 (56%) 12 (43%) 4 — — TN-2 50 35 (70%) 29 (83%) 17 8 4 (50%) TN-3 50 30 (60%) 25 (83%) 13 5 3 (60%) ∗TN Totals 150 93 (62%) 66 (69%) 34 13 7 (54%) ∗ Ova collected from three females in Rabun County, GA (N = 150), as well as three females in Logan County, TN (N = 150), were donated from colleagues, E.