Giving eyespots a shiner: Pharmacologic manipulation of the Io moth wing pattern

Introduction

While our understanding of the mechanisms involved in butterfly wing pattern development has been increasing exponentially in the recent two decades, the work has been largely limited to butterflies such as Junonia, Heliconius, Papilio and Bicyclus. Thanks to these ‘model’ genera, we now understand homologies among wing pattern elements and the adaptive radiation that led to the kaleidoscope of intriguing ‘designs’ found among ca. 160,000 Lepidoptera species (Martin & Reed, 2010).

Natural and artificially generated aberrations serve as windows into the developmental mechanisms and evolutionary history of animals. In addition to many naturally occurring melanic aberrations and some melanic recessive phenotypes that can be obtained and/or maintained through inbreeding, the dark markings of Lepidoptera wings can sometimes be amplified by the timely application of a colder regime to the immature stages (e.g., Sourakov, 2015 and references within). Serfas & Caroll (2005) first demonstrated that injections of heparin into the early pupal stage can simulate cold shock and alter wing patterns in similar ways. Martin & Reed (2014) utilized heparin injections to understand genetic controls and homologies among separate wing pattern elements.

Eyespots are characteristic of many Lepidoptera, and considerable advances have been made towards understanding their evolution (Monteiro et al., 2006). In Automeris io, a species whose name, if anglicized (‘Eye’‘Oh’!), invokes associations with its pair of magnificent dorsal hindwing eyespots that are exposed when the moth (otherwise cryptic) is threatened. Several recessive mutations causing deformations of the black ring surrounding the dark blue eyespot with a white center have been obtained through inbreeding, conducted first by Thomas Manley (1978, 1990) and, more recently, myself (Table 1 below). However, the most dramatic aberration, which involves the melanization of almost the entire hindwing, was found in an A. io male collected in 1966. It was noticed only recently while the MGCL Saturniidae collection was being re-curated (Covell, 2012).

In the present study, an attempt has been made to replicate these aberrations with heparin injections to the prepupal and pupal stages, as well as by cold shock. The results of the former manipulations, while not replicas of previously known aberrations, are quite dramatic and are illustrated here along with some other aberrations, both known and those previously unrecorded.

Methods

Representatives of five broods of Automeris io from local stock (over 300 caterpillars) were reared on sugarberry under ambient condition in Gainesville, Florida, in the fall of 2016 resulting in 130 pupae. While the caterpillars were pupating in November, ten randomly selected pupae were injected, using a 10µl syringe, under a wing with ≈5mg (10µl (1 drop) of heparin dissolved in distilled water). Additionally, one prepupa was injected a day before pupation with half of that amount, and a dozen were subjected to cold shock in the refrigerator (4–5 C°) overnight. Ten other pupae were injected with 10µl of mannitol (saturated solution), and the rest were left untreated. All pupae were kept under ambient conditions during diapause, until they began emerging in May 2017.

Results and discussion

While most of the pupae that were injected did not emerge, one female with a strongly modified wing pattern emerged from a pupa injected with 5mg of heparin (Figure 1A.i). Injection must have damaged the right hindwing, so it did not spread properly (Figure 1A.ii), but the left side was structurally intact. The control sibling female is illustrated in Figures 1B.i and B.ii for comparison.

A less aberrant male, whose prepupa was injected with 5mg of heparin a day before pupation, also emerged (Figures 2A.i, 2A.ii), different in its dorsal hindwing eyespots from all control siblings (e. g., Figure 2B). Most of the cold-shocked and the mannitol-injected individuals showed no obvious deviation from expected wing patterns. The slight wing pattern changes (Figures 2C and 2D) exhibited by two females, cold-shocked as prepupae, suggest that cold shock may have some melanization-inducing effect and perhaps, if administered differently, may potentially have a greater effect on the phenotype.

Figure 1.

A. “Black eye” aberration. A female of the Io Moth, Automeris io, with wing pattern altered by injection 5mg of heparin (sulfated polysaccharide) into the early pupal stage. Voucher FLMNH-MGCL#289216. B. A normal A. io female from the same brood. (i) dorsal, (ii) ventral surface. Photos by Andrei Sourakov.

Figure 2.

A. “Comet eye” aberration. A male of the Io Moth, Automeris io, from prepupa injected with 2.5mg of heparin a day prior to pupation. FLMNH-MGCL#289217. B. A normal A. io male from the same brood. Voucher FLMNH-MGCL#289218. C, D. Slight aberrations (“Comet eye” and “Barley eye”) of the black ring around eyespots in two A. io females cold-shocked as prepupae. Photos by Andrei Sourakov.

On the dorsal hindwing of aberrant female (Figure 1A.i), the blue eyespot center is smaller than that of the control siblings (e.g., Figure 1B.i), due to the infraction of melanin from the surrounding black ring. Heparin injection must have enhanced or prolonged the process of expansion of black pigment once it formed in the black ring around the blue scales. Judging by the wild aberrant male, such melanization process can go as far as eliminating the eyespot entirely (Figure 3A). In the heparin-injected aberrant female, the process of expansion mostly occurred outwards, so that the black ring around the eyespot merged with the normally thin black EIII line of the hindwing margin, which too had widened (Figure 1A.i).

Figure 3.

A. “Caecus” aberration. A wild-collected Automeris io male exhibiting a unique melanic aberration in which most wing pattern elements are obscured by melanin. (i) Dorsal side, (ii) Ventral side, (iii) Close-up of the eyespot area. Collected in Wascott Township, Douglas Co., Wisconsin, on 24 June 1966 by J.L. Boughner; Voucher FLMNH-MGCL#1000907. B, C. “Broken eye” aberrations resulting from recessive mutation reared by Thomas R. Manley (1978, 1990). (B) Male 67#3 resulting from cross of wild “broken eye” female crossed with sibling non-aberrant male; Collection of Peabody Museum; YPM No. 843761; (C) Female 67#1 sibling with the above male; YPM No. 843765. D. “Teardrop” aberration resulting from a recessive mutation reared by Thomas R. Manley. Cross 13-86 (1986) of normal “teardrop” brood male 10-85 with sib “teardrop” female 27-85. Collection of Peabody Museum, YPM No. 843755. E. “Winking eye” aberration resulting from a recessive mutation (expressed through 2^nd consecutive full-sib crossing) reared by A. Sourakov in 2014. F. “Barley eye” aberration resulting from a recessive mutation (expressed through full-sib crossing) reared by A. Sourakov in 2014. Voucher FLMNH-MGCL #166627. Photos by Andrei Sourakov.

A name “Black Eye” is proposed for the aberration shown in Figure 1A, following the tradition started by Manley (1978, 1990), who gave genetic aberrations of Automeris io names, such as “Broken eye,” (Figures 3B and 3C) and “Teardrop” (Figure 3D). Names of all aberrations, old and newly proposed, are summarized in Table 1.

As can be observed by comparing the “Black Eye” to its sibling (Figure 1B.i), the DI element of the forewing also underwent modification, as if it were smudged from its center along the veins toward the distal part of the wing. The ventral surface of the wing in “Black Eye” shows considerable expansion and diffusion of the small and compact black ring of control specimens around the small white ventral eyespot (Figures 1A.ii and 1B.ii).

Scholars engaged in wing pattern research have recently identified 27 genes associated with wing melanin production (Zhang et al., 2017). It is hoped that the present publication, while documenting unique aberrations in a single species, will be useful in the future work directed at understanding wing pattern evolution and development in general.