1969 Journal of the Lepidopterists' Society 109 A SIMPLE TECHNIQUE FOR HAND-PAIRING LIMENITIS BUTTERFLIES (NYMPHALIDAE) AUSTIN P. PLATT Department of Biology, Wesleyan University, Middletown, Connecticut The development of various methods for hand-pairing butterflies has been reviewed by Clarke and Sheppard (1956). Such methods have been very useful in genetic and hybridization studies, particularly among the Papilionidae and the Pieridae. However, the Nymphalidae and other families having relatively small copulatory structures are known to be quite difficult to hand-pair. Clarke and Sheppard state that Lorkovic had successfully hand-paired three genera of Nymphalids, Limenitis, Neptis, and Melitaea. In addition, Remington (1958) has pro­ duced interspecific hybrids between Limenitis wiedemeyerii Edwards and L. astyanax (Fabricius) by hand-pairing. The present paper will report a simple hand-pairing technique developed during the past two summers which has proved useful for breeding Limenitis in the lab­ oratory. The method involves stunning the male and female to be paired in a large sodium cyanide insect killing bottle from one to five minutes at room temperature, and then everting the male claspers (often partially accomplished by the cyanide treatment) by applying slight pressure to the lateral and ventral portions of the male's abdomen with the thumb and forefinger. Meanwhile, the stunned female is held in the other hand, and her abdomen is curved downward, so that it is exposed beneath the wings. The butterflies are positioned so that the male can clasp the ventral portion of the female's abdomen one segment in front of the ovipositor. During this procedure the male is moved constantly in a small circle to stimulate eversion of the genitalia and clasping. \Vhile still unconscious the pair is laid on its side with the male's abdomen positioned at an angle of about 135 degrees in relation to the female's. The male involuntarily opens and closes his valvae, thereby inserting the aedeagus and initiating the rhythmic contractions of copulation. Once the male has clasped the abdomen of the female and the butter­ flies begin to recover from the cyanide (usually within four to seven minutes after stunning), the pair is placed on a rectangular piece of moist cellulose sponge set on a paper towel, and the male's hindwings are positioned inside the female's, using a pointed probe. The pair is then covered with a transparent plastic shoe box above which are placed incandescent and fluorescent lamps, and the butterflies are left undis­ turbed while recovelY and mating proceed. 110 PLATT: Hand-pairing Limenitis Vol. 23, no. 2 Occasionally during recovery from the cyanide one or both butterflies may begin to flap their wings violently, and the pair may break apart; however, if the pair is clasping properly, only slow rhythmic wing move­ ments occur as the butterflies bask in the warmth of the lights. Since developing this technique in August, 1965, I have been able to carry out 35 matings involving the species and forms of Limeniti51 occur­ ring in New England. Table 1 lists the numbers and types of intra­ specific and interspecific crosses which have been completed to date. The brood data obtained from those crosses made during 1966 have been reported elsewhere (Platt & Brower, 1968). As the table indicates, mated females could not always be induced to oviposit. Bred females were confined in silk organza bags outdoors on boughs of trees for egg laying. Females of L. arthemis (Drury), including its form "pmserpina" Edwards, and L. astyanax (Fabricius) were placed on wild cherry, Prunus serotina Ehrl. and females of L. archippus ( Cramer) were put on black willow, Salix nigra Marsh. Extended periods of cold, windy, and rainy weather, as well as predation by stink bugs, (Hemiptera, Pentatomidae) which occasionally pierce the abdomens of females through the bags, accounted for the deaths of nine females before they had laid eggs. In eight other instances eggs which were laid were found to be infertile. Therefore, after death all hand-paired females were dissected (using the method employed by Bums, 1966) to determine whether or not a sper­ matophore had been formed within the bursa copulatrix of each female. The presence of a spermatophore within the female was considered evidence that the pairing itself had been successful. Successful matings were found to last from 45 minutes to over two hours; in no case has a spermatophore been found in females which have been in copula for only 25 minutes or less. So far there have been only five cases in which no spermatophore was found in females which had been in copula for longer than one hour. In all successful pairings to date only a single spermatophore has been formed. Refinement of this hand-pairing method during the past summer has permitted me to pair practically any two individuals, provided that the male is at least five days old, and that its abdomen has hardened; also, the female, which is usually larger than the male, must be stunned in the killing bottle before the male is put into it, and must remain in the bottle while the male is being stunned. Further, I have found that if the first pairing attempt is unsuccessful, the butterflies can be stunned several more times without danger of killing them. However, the individuals apparently develop a higher to 1- 1969 Journal of the Lepidopterists' Society 111 TABLE 1. HAND-PAIRED Limenitis CROSSES CARRIED OUT AT WAKEFIELD, R. 1. AND MIDDLETOWN, CONN. (1966-67). Dissection of the 35 females involved in these crosses revealed that each contained a single spermato­ phore. P, Cross No. of Eggs Eggs Eggs not Male Female broods fertile infertile obtained Intraspecific1 : arthemis X arthemis 2 1 1 0 astyanax X astyanax 8 3 32 2 archippus X archippus 5 4 0 1 proserpina" X "proserpina" 3 3 0 0 " proserpina" X arthemis 1 0 P 0 proserpina" X astyanax 2 1 0 1 arthemis X astyanax 4 1 1 2 astyanax X arthemis 3 2 1 0 Totals 28 15 7 6 Inter -species: archippus X arthemis 1 0 0 1 arc hippus X astyanax 3 2 0 1 arthemis X arc hippus 2 1 0 1 astyanax X archippus 1 0 1 0 Totals 7 3 1 3 1 All breeding and collecting evidence accumulated to date supports the contention that L. arthe­ tnis, proserpina, and astyanax represent intergrading forms within a single species complex. 2 These three crosses involved a single male which was bred to three different females; the male and two of the females were F'r siblings of a wild-caught female from Stanton, N. J., which laid 47 eggs, only 8 of which hatched; the third female was an F 1 from a wild female captured near Spring­ dale, N. J.; the author believes this infertility has resulted from the use of an inviable llstyanax strain. :3 A very old male, which died in copula, was used in making this cross. erance to the cyanide each time that they are stunned, so that with each successive attempt they must remain in the bottle for a longer period of time. This method has enabled me to breed several males three or four separate times, using different females each time. In such cases the males breed best on alternate, rather than consecutive days. Between pairings the males are kept in labeled, transparent envelopes in total darkness at room temperature. Virgin butterflies to be paired are kept the same way, and are fed a solution of dilute honey and water once daily, except on the day that they are to be bred. Apparently females will breed most readily a day or two after emer­ gence from the chrysalids; however, successful first matings have been accomplished with some females more than two weeks after they have hatched. The viability of both parents and progeny seems to be unaffected by 112 PLATT: Hand-pairing Limenitis Vol. 23, no. 2 the cyanide treahnent, for sex ratios and total numbers in the hand-paired broods compare favorably with those in broods obtained from wild fe­ males, and from females bred in outdoor cages. Only in the interspecific crosses in which L. archippus has been paired to either L. arthemis or L. astyanax is there evidence of inviability in the heterogametic (female) sex: all Fl progeny in these crosses so far have been males. This simple method of hand-pairing has proved extremely useful in crossing Limenitis butterflies, and has provided a means of using male individuals in a series of crosses with females having different phenotypes. With modifications this technique may prove useful for breeding other species of Lepidoptera, and for effecting hybrid crosses between closely related forms. ACKNOWLEDGMENTS This study has been supported in part by summer grants-in-aid of research from the Society of Sigma Xi (1966) and from Wesleyan U niver­ sity (1967). I am grateful to Dr. L. P. Brower of Amherst College for basic instruction in the technique of hand-pairing, and to Dr. J. M. Bums of Wesleyan University for instruction and assistance in the dissection of females for spermatophore detection. I wish also to thank Mr. J. Muller of Lebanon, N. J. and Mr. F. Rutkowski of New York City for providing live wild-caught females, whose progeny were used in this study. LITERATURE CITED BURNS, J. M., 1900. Preferential mating versus mimicry: disruptive selection and sex-limited dimorphism in Papilio glauctls. Science, 153: 551-553. CLARKE, C. A., & P. M. SHEPPARD, 1956. Hand-pairing of butterflies. Lepid. News, 10: 47-53. PLATT, A. P., & L. P. BROWER, 1968. Mimetic versus disruptive coloration in inter­ grading populations of Limenitis arthemis and astyanax butterflies.