Emperors, admirals and giants, zebras, tigers and woolly bears: casting a broader net in exploring heparin effects on Lepidoptera wing patterns

Abstract

Background: Studies of heparin effects on Lepidoptera wing patterns have been restricted to a small number of species. I report observations from experiments on a broader range of taxa, including first results from swallowtails, tiger moths and microlepidoptera. Methods: Heparin injections were made in prepupae and pupae of Junonia coenia (common buckeyes), Agraulis vanillae (gulf fritillaries), Heliconius charithonia (zebra longwings), Asterocampa clyton (tawny emperors), Danaus plexippus (monarchs), Vanessa atalanta (red admirals); Heraclides cresphontes (giant swallowtails), Pterourus troilus (spicebush swallowtails), Protographium marcellus (zebra swallowtails), Battus polydamas (polydamas swallowtails); Hypercompe scribonia (giant leopard moths), Estigmene acrea (acrea moths), Hyphantria cunea (fall webworm moths), Utetheisa ornatrix (ornate bella moths); Glyphodes sibillalis (mulberry leaftier). Results: Heparin sometimes altered the entire pattern in a dramatic way, sometimes caused changes locally. In buckeyes, the previous heparin study conducted on pupae was compared to injections made at a prepupal stage. In gulf fritillaries, zebra longwings and tawny emperors, the dramatic changes occurred throughout their wings, while in monarchs, changes were restricted to wing margins. Changes achieved in red admirals, show that heparin action is unrelated to the original color. In swallowtails, transformations were restricted to border system, indicating higher levels of stability and compartmentalization of wing patterns. In mulberry leaftier, changes were restricted to the marginal bands. In tiger moths, elongation of black markings led to merging of spots; in the ornate bella moth, it was accompanied by an expansion of the surrounding white bands, and results were compared to the effects of colder temperatures. Conclusions: Using pharmaceutical intervention demonstrates that there are many similarities and some very significant differences in the ways wing patterns are formed in different Lepidoptera lineages. By creating a range of variation one can demonstrate how one pattern can easily evolve into another, aiding in understanding of speciation and adaptation processes.

Frizzled2 receives WntA signaling during butterfly wing pattern formation

“…However, current evidence suggests that extracellular WntA signaling mostly occurs in pupal rather than in larval wings. Indeed, injections of heparin, an extracellular Wnt interactor that result in Wnt-dependent pattern expansions (Martin and Reed, 2014;Mazo-Vargas et al, 2017;Sourakov, 2018;Sourakov and Shirai, 2020), are effective in the first 15% of pupal developmenttypically within 24 hr after pupa formation (APF) at optimal rearing temperatures. Further, scale organ precursor cell differentiation Development • Accepted manuscript occurs around 13-15% (Dinwiddie et al, 2014), and the competency of tissue grafts to induce ectopic patterns (Nijhout, 1991), or developmental studies of eyespot formation (Brunetti et al, 2001;Monteiro, 2015), all imply the existence of paracrine signals active during early pupal development.…”

Frizzled2 receives the WntA morphogen during butterfly wing pattern formation

“…However, current evidence suggests that extracellular WntA signaling mostly occurs after the onset of metamorphosis. Indeed, injections of heparin, an extracellular Wnt interactor that result in Wnt-dependent pattern expansions (Martin and Reed, 2014;Mazo-Vargas et al, 2017;Sourakov, 2018;Sourakov and Shirai, 2020), are effective within 15% of pupal development -typically within 24 hr after pupa formation (APF) at optimal rearing temperatures. Further, scale organ precursor cell differentiation also occurs around 13-15% (Dinwiddie et al, 2014), and the competency of tissue grafts to induce ectopic patterns (Nijhout, 1991), or developmental studies of eyespot formation (Brunetti et al, 2001;Monteiro, 2015), all imply the existence of paracrine signals active during early pupal development.…”

“…We thus hypothesize that Fz4 antagonizes Wnt in the extracellular space. It is worth noting that fz4 mKOs resemble the effect of extracellular heparin injections in early pupae, a treatment that emulates Wnt gain-of-function, including Discalis element expansions (and injection timing influencing inner vs. outer pattern expansions in these), as well as MBS pattern fusion and expansions in Junonia, Vanessa and Agraulis (Martin and Reed, 2014;Serfas and Carroll, 2005;Sourakov, 2018;Sourakov and Shirai, 2020). Importantly, heparin likely enhances the effects of WntA in the CSS, but fz4 mKOs only affected other heparin-sensitive patterns, implying that Fz4 is in fact unable to bind WntA.…”

Combinatorial Wnt signaling determines wing margin color patterns of the swallowtail butterfly ground plan

“…This division has also been observed in cold-shock experiments, drug perturbations, and naturally occurring phenotypes across Papilionidae (Tyler et al, 1994; Dabrowski and Dobranski, 1996; Umebachi and Osanai, 2003; Scriber et al, 2009; Perlman and Perlman, 2019). In contrast, heparin injections and cold shocks invariably expand the proximal patterns of other lepidopteran lineages, including Nymphalidae, Lycaenidae, and Arctiidae (Iwata et al, 2013; Gawne and Nijhout, 2019; Sourakov, 2020). Thus, the patterning of the proximal region appears to be divergent in Papilionidae, and we speculate that mechanisms other than Wnt signals or heparin-sensitive morphogen drive proximal stripe formation in swallowtail butterflies (Fig.…”

“…Pupal injections of heparin and dextran sulfate were done as previously described (Martin and Reed, 2014; Sourakov, 2020). Each individual was injected between 4-20 hours after pupation (Table S3), in the basal wing region, taking care to avoid tissue damage.…”