JOURNAL OF MORPHOLOGY 00:00–00 (2015) Structure of the Lepidopteran Proboscis in Relation to Feeding Guild Matthew S. Lehnert,1,2* Charles E. Beard,2 Patrick D. Gerard,3 Konstantin G. Kornev,4 and Peter H. Adler2 1Department of Biological Sciences, Kent State University at Stark, North Canton, Ohio 44720 2Department of Agricultural and Environmental Sciences, Clemson University, Clemson, South Carolina 29634 3Department of Mathematical Sciences, Clemson University, Clemson, South Carolina 29634 4Department of Materials Science and Engineering, Clemson University, Clemson, South Carolina 29634 ABSTRACT Most butterflies and moths (Lepidoptera) (Monaenkova et al., 2012). A pump in the head use modified mouthparts, the proboscis, to acquire flu- then forces the liquid up the food canal to the gut ids. We quantified the proboscis architecture of five (Eberhard and Krenn, 2005; Borrell and Krenn, butterfly species in three families to test the hypothesis 2006; Lee et al., 2014). that proboscis structure relates to feeding guild. We Feeding guilds (i.e., groups of species with simi- used scanning electron microscopy to elucidate the fine structure of the proboscis of both sexes and to quantify lar feeding habits) have long been recognized in dimensions, cuticular patterns, and the shapes and the Lepidoptera and have been associated with sizes of sensilla and dorsal legulae. Sexual dimorphism higher taxa, such as nymphalid subfamilies or was not detected in the proboscis structure of any spe- tribes (Gilbert and Singer, 1975; Krenn et al., cies. A hierarchical clustering analysis of overall pro- 2001). Adult Lepidoptera are conventionally cate- boscis architecture reflected lepidopteran phylogeny, gorized into at least two broad feeding guilds: but did not produce a distinct group of flower visitors flower visitors (nectar feeders) and nonflower visi- or of puddle visitors within the flower visitors. Specific tors (nonnectar feeders), the latter drinking from characters of the proboscis, nonetheless, can indicate wetted surfaces, such as sap flows and rotting flower and nonflower visitors, such as the configuration fruit. Within the flower-visiting guild, the males of of sensilla styloconica, width of the lower branches of dorsal legulae, presence or absence of dorsal legulae at some species routinely drink from damp soil the extreme apex, and degree of proboscis tapering. We (“puddling” sensu Arms et al., 1974). suggest that the overall proboscis architecture of Lepi- Selected features of proboscis architecture have doptera reflects a universal structural organization been associated with adult-feeding habits (Krenn that promotes fluid uptake from droplets and films. On et al. 2001; Monaenkova et al., 2012; Lehnert et al., top of this fundamental structural organization, we 2013; Kwauk et al., 2014; Tsai et al., 2014). Tearing suggest that the diversity of floral structure has and rasping spines and other cuticular modifica- selected for structural adaptations that facilitate entry tions, for example, are on the proboscises of moths of the proboscis into floral tubes. J. Morphol. 000:000– that routinely feed on lachrymal secretions and 000, 2015. VC 2015 Wiley Periodicals, Inc. pierce animal tissue to feed on blood (Banziger,€ KEY WORDS: butterfly; flower visiting; fluid uptake; 1971; Buttiker€ et al., 1996; Hilgartner et al., 2007; mouthparts; nectar feeding; sap feeding Zaspel et al., 2011). Flower-visiting butterflies have darker proboscises (Krenn et al., 2001) and signifi- cantly longer proboscises relative to their bodies than do nonflower visitors (Kunte, 2007). Enlarged, INTRODUCTION densely arrayed chemo-mechanoreceptors, that is, Approximately 28% of all fluid-feeding insects are butterflies and moths in the Lepidoptera (Adler and Foottit, 2009). More than 95% of the Contract grant sponsor: National Science Foundation (award number 1354956); Contract grant sponsor: NIH; Grant number: Lepidoptera acquire fluids by means of a tubular 5P02GM103444; Contract grant sponsor: NIFA/USDA. proboscis (Krenn, 1990, 2010). The feeding device consists of two medially concave, elongated maxil- *Correspondence to: Matthew S. Lehnert; 6000 Frank Ave. NW, lary galeae joined dorsally and ventrally by cuticu- North Canton, OH 44720. E-mail: [email protected] lar projections, termed “legulae,” forming a food canal (Eastham and Eassa, 1955; Hepburn, 1971; Received 27 August 2015; Revised 10 October 2015; Krenn, 1997). Spaces between the dorsal legulae Accepted 18 October 2015. facilitate capillary action, supporting the with- Published online 00 Month 2015 in drawal of liquids from pools and porous sub- Wiley Online Library (wileyonlinelibrary.com). strates, such as rotting fruit, into the food canal DOI 10.1002/jmor.20487 VC 2015 WILEY PERIODICALS, INC. 2 M.S. LEHNERT ET AL. sensilla styloconica (Altner and Altner 1986) form a from porous substrates, such as rotting fruit and tree sap brushy tip in nonflower-visiting butterflies (Krenn (Scott, 1986). et al., 2001; Knopp and Krenn, 2003; Petr and Danaus plexippus was received as pupae or adults from Shady Oak Butterfly Farm (Brooker, Florida) or were labora- Stewart, 2004; Krenn, 2010), which takes up fluid tory reared on milkweed (Asclepias spp.). Larvae of P. rapae, more effectively from liquid films (Molleman et al., from Carolina Biological Supply Co. (Burlington, NC) were 2005). We recently discovered that these sensilla reared on artificial diet. Adults of P. glaucus, L. a. astyanax, are hydrophilic, and function like a sponge when and P. interrogationis were captured as adults or reared from 0 0 arranged as a brush (Lehnert et al., 2013), a mech- larvae collected in Clemson, South Carolina (N34839 , W82850 ), from April to September 2011. Voucher specimens were depos- anism further studied by Lee and Lee (2014). In ited in the Clemson University Arthropod Collection. addition, some nonflower-visiting butterflies have a more elliptical proboscis in cross-section, compared Scanning Electron Microscopy with the condition of flower visitors (Lehnert et al., Lepidopteran heads were secured and proboscises straight- 2013). When an elliptical proboscis is dipped into a ened with insect pins on polystyrene foam where they remained liquid, the meniscus rises to a higher level com- through a series of ethanol washes (80%, 95%, 100%; ca. 24 h pared with that of a circular proboscis (Lehnert each) followed by chemical drying in hexamethyldisilazane. et al., 2013; Alimov and Kornev, 2014), which might Specimens were attached to a scanning electron microscope help Lepidoptera to engage more interlegular (SEM) mount with carbon-graphite adhesive tape and sputter- coated with gold or platinum for 1–3 min. A Hitachi TM-3000 spaces for liquid uptake (Kwauk et al., 2014). SEM was used to photograph the dorsum of each proboscis at In accord with the hypothesis that structural 503 magnification, 15 kV, and full vacuum. Selected areas were organization of an organismal device is matched to observed at 1503 or higher magnifications. Composite images its functional demand (Weibel et al., 1991), we of each proboscis were assembled in AdobeVR Photoshop CS2 (Adobe Systems) and used for measurements and illustrations. looked for a testable framework to facilitate the ImageJ software (Rasband, W.S., ImageJ, U. S. National Insti- prediction of general feeding habits (guilds) of but- tutes of Health, Bethesda, Maryland, USA, http://imagej.nih. terflies. Therefore, we asked the following ques- gov/ij/, 1997-2015) was used to acquire measurements. tion: Can visible structural features of the proboscis predict feeding guild? To test the hypoth- Measurements esis that species with similar feeding habits have We used SEM to ensure accuracy of proboscis measurements structurally similar proboscises, we examined 21 and character assessments. Overall, we quantified or catego- proboscis characters for three guilds of butterflies: rized a total of 21 characters for 11–20 (typically 20) specimens of each of the five species (Tables (1–3)). We compared charac- flower visitors, flower-visiting puddlers, and non- ters between sexes for each species [P. glaucus 11 females and flower visitors. To provide a robust analysis of the 9 males for all measured characters, except number of hand- overall proboscis landscape, we built on the study switches (10f, 8m); D. plexippus 10f, 10m; L. a. astyanax 11f, of nymphalid butterflies by Krenn et al. (2001), 9m, except number of handswitches (6f, 6m), widths of Zone 1 and included characters recently associated with (8f, 8m) and Zone 2 (10f, 7m); P. interrogationis 5f, 14m, except for handswitches (3f, 11m), widths of Zone 1 (5f, 13m) and Zone fluid uptake, such as attributes of the dorsal legu- 2 (5f, 13m); P. rapae 8f, 12m, except for handswitches (2f, 5m), lae (Monaenkova et al., 2012; Lehnert et al., and sensilla styloconica stylus length and peg length (6f, 9m)]. 2013), as well as characters with unknown func- Composite SEM images (503 magnification) were used to tions. We also tested the hypothesis that males define areas (i.e., zones) of the proboscis to assist in structural comparisons among species. These structural zones correspond and females differ in the structure of their probos- with well-defined functional zones, that is, the drinking and cises, and predicted differences in the puddling nondrinking regions (Monaenkova et al., 2012; Lehnert et al., group because predominantly males exhibit this 2013). Upper and lower branches of dorsal legulae (Lehnert behavior (Arms et al. 1974). In analyzing the et al., 2013) were present along more than 95% of the length of structural characters, we considered only their each galea of all species, and the width of the upper branch was used to designate structurally defined zones. In ImageJ geometrical features and not their materials software, a straight line was drawn from the base to the tip of properties. a single dorsal legula at the base of the proboscis (Fig. 1). The line was held at constant length and moved distally along the bases of the dorsal legulae until the width of the legulae consis- MATERIAL AND METHODS tently enlarged, signifying the end of one zone and the begin- Species ning of a second zone (cf. Fig.