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
1
Department
Department
3
Department
4
Department
2
of
of
of
of
Biological Sciences, Kent State University at Stark, North Canton, Ohio 44720
Agricultural and Environmental Sciences, Clemson University, Clemson, South Carolina 29634
Mathematical Sciences, Clemson University, Clemson, South Carolina 29634
Materials Science and Engineering, Clemson University, Clemson, South Carolina 29634
ABSTRACT Most butterflies and moths (Lepidoptera)
use modified mouthparts, the proboscis, to acquire fluids. We quantified the proboscis architecture of five
butterfly species in three families to test the hypothesis
that proboscis structure relates to feeding guild. We
used scanning electron microscopy to elucidate the fine
structure of the proboscis of both sexes and to quantify
dimensions, cuticular patterns, and the shapes and
sizes of sensilla and dorsal legulae. Sexual dimorphism
was not detected in the proboscis structure of any species. A hierarchical clustering analysis of overall proboscis architecture reflected lepidopteran phylogeny,
but did not produce a distinct group of flower visitors
or of puddle visitors within the flower visitors. Specific
characters of the proboscis, nonetheless, can indicate
flower and nonflower visitors, such as the configuration
of sensilla styloconica, width of the lower branches of
dorsal legulae, presence or absence of dorsal legulae at
the extreme apex, and degree of proboscis tapering. We
suggest that the overall proboscis architecture of Lepidoptera reflects a universal structural organization
that promotes fluid uptake from droplets and films. On
top of this fundamental structural organization, we
suggest that the diversity of floral structure has
selected for structural adaptations that facilitate entry
of the proboscis into floral tubes. J. Morphol. 000:000–
C
2015 Wiley Periodicals, Inc.
000, 2015. V
KEY WORDS: butterfly; flower visiting; fluid uptake;
mouthparts; nectar feeding; sap feeding
INTRODUCTION
Approximately 28% of all fluid-feeding insects
are butterflies and moths in the Lepidoptera
(Adler and Foottit, 2009). More than 95% of the
Lepidoptera acquire fluids by means of a tubular
proboscis (Krenn, 1990, 2010). The feeding device
consists of two medially concave, elongated maxillary galeae joined dorsally and ventrally by cuticular projections, termed “legulae,” forming a food
canal (Eastham and Eassa, 1955; Hepburn, 1971;
Krenn, 1997). Spaces between the dorsal legulae
facilitate capillary action, supporting the withdrawal of liquids from pools and porous substrates, such as rotting fruit, into the food canal
C 2015 WILEY PERIODICALS, INC.
V
(Monaenkova et al., 2012). A pump in the head
then forces the liquid up the food canal to the gut
(Eberhard and Krenn, 2005; Borrell and Krenn,
2006; Lee et al., 2014).
Feeding guilds (i.e., groups of species with similar feeding habits) have long been recognized in
the Lepidoptera and have been associated with
higher taxa, such as nymphalid subfamilies or
tribes (Gilbert and Singer, 1975; Krenn et al.,
2001). Adult Lepidoptera are conventionally categorized into at least two broad feeding guilds:
flower visitors (nectar feeders) and nonflower visitors (nonnectar feeders), the latter drinking from
wetted surfaces, such as sap flows and rotting
fruit. Within the flower-visiting guild, the males of
some species routinely drink from damp soil
(“puddling” sensu Arms et al., 1974).
Selected features of proboscis architecture have
been associated with adult-feeding habits (Krenn
et al. 2001; Monaenkova et al., 2012; Lehnert et al.,
2013; Kwauk et al., 2014; Tsai et al., 2014). Tearing
and rasping spines and other cuticular modifications, for example, are on the proboscises of moths
that routinely feed on lachrymal secretions and
pierce animal tissue to feed on blood (B€
anziger,
1971; B€
uttiker et al., 1996; Hilgartner et al., 2007;
Zaspel et al., 2011). Flower-visiting butterflies have
darker proboscises (Krenn et al., 2001) and significantly longer proboscises relative to their bodies
than do nonflower visitors (Kunte, 2007). Enlarged,
densely arrayed chemo-mechanoreceptors, that is,
Contract grant sponsor: National Science Foundation (award
number 1354956); Contract grant sponsor: NIH; Grant number:
5P02GM103444; Contract grant sponsor: NIFA/USDA.
*Correspondence to: Matthew S. Lehnert; 6000 Frank Ave. NW,
North Canton, OH 44720. E-mail: [email protected]
Received 27 August 2015; Revised 10 October 2015;
Accepted 18 October 2015.
Published online 00 Month 2015 in
Wiley Online Library (wileyonlinelibrary.com).
DOI 10.1002/jmor.20487
2
M.S. LEHNERT ET AL.
sensilla styloconica (Altner and Altner 1986) form a
brushy tip in nonflower-visiting butterflies (Krenn
et al., 2001; Knopp and Krenn, 2003; Petr and
Stewart, 2004; Krenn, 2010), which takes up fluid
more effectively from liquid films (Molleman et al.,
2005). We recently discovered that these sensilla
are hydrophilic, and function like a sponge when
arranged as a brush (Lehnert et al., 2013), a mechanism further studied by Lee and Lee (2014). In
addition, some nonflower-visiting butterflies have a
more elliptical proboscis in cross-section, compared
with the condition of flower visitors (Lehnert et al.,
2013). When an elliptical proboscis is dipped into a
liquid, the meniscus rises to a higher level compared with that of a circular proboscis (Lehnert
et al., 2013; Alimov and Kornev, 2014), which might
help Lepidoptera to engage more interlegular
spaces for liquid uptake (Kwauk et al., 2014).
In accord with the hypothesis that structural
organization of an organismal device is matched to
its functional demand (Weibel et al., 1991), we
looked for a testable framework to facilitate the
prediction of general feeding habits (guilds) of butterflies. Therefore, we asked the following question: Can visible structural features of the
proboscis predict feeding guild? To test the hypothesis that species with similar feeding habits have
structurally similar proboscises, we examined 21
proboscis characters for three guilds of butterflies:
flower visitors, flower-visiting puddlers, and nonflower visitors. To provide a robust analysis of the
overall proboscis landscape, we built on the study
of nymphalid butterflies by Krenn et al. (2001),
and included characters recently associated with
fluid uptake, such as attributes of the dorsal legulae (Monaenkova et al., 2012; Lehnert et al.,
2013), as well as characters with unknown functions. We also tested the hypothesis that males
and females differ in the structure of their proboscises, and predicted differences in the puddling
group because predominantly males exhibit this
behavior (Arms et al. 1974). In analyzing the
structural characters, we considered only their
geometrical features and not their materials
properties.
MATERIAL AND METHODS
Species
Five species representing three families served as test subjects. We selected the monarch butterfly, Danaus plexippus
(Linnaeus, 1758) (Nymphalidae), to represent Lepidoptera with
predominantly flower-visiting (i.e., nectar-feeding) habits
(Brower, 1961); the cabbage butterfly, Pieris rapae (Linnaeus,
1758) (Pieridae), and the eastern tiger swallowtail, Papilio
glaucus Linnaeus, 1758 (Papilionidae), to represent flower visitors with males that also routinely puddle (Arms et al., 1974;
Adler and Pearson, 1982); and the red-spotted purple, Limenitis
arthemis astyanax (Fabricius, 1775) (Nymphalidae), and the
question mark, Polygonia interrogationis (Fabricius, 1798)
(Nymphalidae), to represent nonflower visitors that feed chiefly
Journal of Morphology
from porous substrates, such as rotting fruit and tree sap
(Scott, 1986).
Danaus plexippus was received as pupae or adults from
Shady Oak Butterfly Farm (Brooker, Florida) or were laboratory reared on milkweed (Asclepias spp.). Larvae of P. rapae,
from Carolina Biological Supply Co. (Burlington, NC) were
reared on artificial diet. Adults of P. glaucus, L. a. astyanax,
and P. interrogationis were captured as adults or reared from
larvae collected in Clemson, South Carolina (N348390 , W828500 ),
from April to September 2011. Voucher specimens were deposited in the Clemson University Arthropod Collection.
Scanning Electron Microscopy
Lepidopteran heads were secured and proboscises straightened with insect pins on polystyrene foam where they remained
through a series of ethanol washes (80%, 95%, 100%; ca. 24 h
each) followed by chemical drying in hexamethyldisilazane.
Specimens were attached to a scanning electron microscope
(SEM) mount with carbon-graphite adhesive tape and sputtercoated with gold or platinum for 1–3 min. A Hitachi TM-3000
SEM was used to photograph the dorsum of each proboscis at
503 magnification, 15 kV, and full vacuum. Selected areas were
observed at 1503 or higher magnifications. Composite images
R Photoshop CS2
of each proboscis were assembled in AdobeV
(Adobe Systems) and used for measurements and illustrations.
ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.
gov/ij/, 1997-2015) was used to acquire measurements.
Measurements
We used SEM to ensure accuracy of proboscis measurements
and character assessments. Overall, we quantified or categorized a total of 21 characters for 11–20 (typically 20) specimens
of each of the five species (Tables (1–3)). We compared characters between sexes for each species [P. glaucus 11 females and
9 males for all measured characters, except number of handswitches (10f, 8m); D. plexippus 10f, 10m; L. a. astyanax 11f,
9m, except number of handswitches (6f, 6m), widths of Zone 1
(8f, 8m) and Zone 2 (10f, 7m); P. interrogationis 5f, 14m, except
for handswitches (3f, 11m), widths of Zone 1 (5f, 13m) and Zone
2 (5f, 13m); P. rapae 8f, 12m, except for handswitches (2f, 5m),
and sensilla styloconica stylus length and peg length (6f, 9m)].
Composite SEM images (503 magnification) were used to
define areas (i.e., zones) of the proboscis to assist in structural
comparisons among species. These structural zones correspond
with well-defined functional zones, that is, the drinking and
nondrinking regions (Monaenkova et al., 2012; Lehnert et al.,
2013). Upper and lower branches of dorsal legulae (Lehnert
et al., 2013) were present along more than 95% of the length of
each galea of all species, and the width of the upper branch
was used to designate structurally defined zones. In ImageJ
software, a straight line was drawn from the base to the tip of
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 consistently enlarged, signifying the end of one zone and the beginning of a second zone (cf. Fig. 1b of Lehnert et al., 2013). A
third zone was designated on proboscises that lacked dorsal
legulae at the distal tip (Figs. 1 and 2).
Structural comparisons within and among species included
total proboscis lengths, zone lengths, percent zone lengths (503
magnification), galeal widths (measured at the middle of Zones
1 and 2 and including sensilla styloconica when present, 1503
magnification), and average widths of upper and lower legular
branches in Zones 1 and 2 (based on five randomly chosen dorsal legulae near the middle of each zone per specimen, 1503
magnification) (Fig. 1). The medial tips of dorsal legulae were
examined in Zone 2 for the presence of a toothlike projection.
The forewing length of each specimen was measured as a possible covariate of proboscis length. Zones 1 and 2 each were
No
P. interrogationis
19
20
20
20
20
n
31.21 6 0.39d
42.45 6 0.82c
49.65 6 0.74b
24.55 6 0.35e
56.2 6 0.95a
Forewing
length (mm)
12.77 6 0.26c
13.17 6 0.18c
14.39 6 0.26b
9.46 6 0.16c
17.95 6 0.40a
Proboscis
length (mm)
15.29 6 0.35a
(85.2%)D
8.75 6 0.16d
(92.5%)A
13.06 6 0.24b
(90.8%)B
10.74 6 0.17c
(81.5%)E
11.33 6 0.23c
(88.7%)C
Zone 1
2.65 6 0.08a
(14.7%)B
0.70 6 0.02d
(7.5%)E
1.31 6 0.04c
(9.1%)D
2.43 6 0.04b
(18.5%)A
1.44 6 0.04c
(11.3%)C
Zone 2
NA
0.01 6 0.0b
(0.1%)A
0.02 6 0.0a
(<0.1%)A
0.01 6 0.0b
(0.1%)A
NA
Zone 3
20
20
20
20
19
P. glaucus
P. rapae
D. plexippus
L. a. astyanax
P. interrogationis
37.91 6 0.98a
13.7 6 0.47b
7.56 6 0.58d
11.05 6 0.56c
10.2 6 0.32c
46.68 6 1.02c
45.46 6 1.46c
43.02 6 1.26c
77.22 6 2.88a
60.17 6 2.13b
Lower
branch
(Zone 1)
71.7 6 2.23b
26.03 6 1.03e
45.81 6 1.23d
81.18 6 1.88a
60.04 6 1.89c
Upper
branch
(Zone 2)
7.17 6 1.5b (18)
105.86 6 27.96a (7)
0.15 6 0.08b
2.92 6 1.06b (12)
2.86 6 0.98b (14)
No. of
handswitches
in Zone 1 (n)
*Arising from base of lower branch (1) or galeal wall (2), or well-developed along proboscis length, except in Zone 3 (3).
Lowercase letters indicate significant differences (P < 0.05) within columns, as determined using Fisher’s LSD test.
Sample size (n) is given in parentheses only when it differs from the sample size presented in the n column.
n
Species
Upper
branch
(Zone 1)
Dorsal legulae width (mm)
Not overlapping
Overlapping
Not overlapping
Overlapping
Overlapping
Dorsal legulae
arrangement
(Zone 2)
TABLE 2. Measurements (means 6 SE) and characteristics of dorsal legulae for five butterfly species
Lowercase and uppercase letters indicate significant differences (P < 0.05) within columns, as determined using Fisher’s LSD test.
p, Puddling by males; NA, Zone 3 does not exist.
Sample size (n) is given in parentheses only when it differs from the sample size presented in the n column.
No
L. a. astyanax
Yes (p)
P. rapae
Yes
Yes (p)
P. glaucus
D. plexippus
Flower
visitor
Species
Zone length (mm)
(Percent total proboscis length)
TABLE 1. Forewing and proboscis measurements (means 6 SE) for five butterfly species
3
3
2
2
1
Absent
Absent
Absent
Present
Present
Toothlike
projections
201.39 6 6.38a
(17)
114.06 6 2.82b
(18)
297.81 6 9.54a
(16)
201.53 6 4.69c
(18)
Origin of
dorsal
legulae*
106.83 6 2.75b
53.61 6 2.34c
113.87 6 3.86b
241.91 6 4.39b
136.62 6 2.86d
229.78 6 6.05b
Zone 2
Galea width (lm) (n)
Zone 1
STRUCTURE OF BUTTERFLY PROBOSCIS
3
Journal of Morphology
Journal of Morphology
*Chemosensilla are reduced in size and do not meet our classification criteria for sensilla styloconica; a stylus longer than its peg indicated a sensillum styloconicum, whereas
a stylus shorter than its peg indicated a sensillum basiconicum.
NA, Not applicable.
Sample size (n) is given in parentheses only when it differs from the sample size presented in the n column.
NA
5.01 6 0.18c (15)
NA
20.95 6 0.66a
8.76 6 0.24b
NA
5.32 6 0.17c (15)
NA
117.57 6 2.46a
67.28 6 1.93b
NA
Sparse
NA
Dense
Dense
20
20
20
20
19
P. glaucus
P. rapae
D. plexippus
L. a. astyanax
P. interrogationis
P
P
P
P
P (14)
A*
P
A*
P
P
P
P
P
P
P (11)
NA
Scattered
NA
Uniform
Uniform
Peg length (n)
Stylus length (n)
Arrangement
Basiconica (n)
Styloconica
Trichodea (n)
n
Species
Presence (P) or absence (A*) of sensilla
Distribution
Styloconica
M.S. LEHNERT ET AL.
TABLE 3. Arrangement of sensilla trichodea, basiconica, and styloconica (means 6 SE) in Zone 2 on proboscises of five butterfly species
4
divided into three sections of equal length and the presence of
sensilla was determined in the middle section of each zone if
they were within 20 mm (basiconica, styloconica) or 50 mm (trichodea) of the base of the dorsal legulae. The average widths of
five randomly chosen sensilla styloconica were determined for
each specimen (stylus and peg separately). The galeal surface
sculpture was observed at 503 and higher magnifications in
Zone 1. The number of switches in handedness of the dorsal
legulae was determined throughout Zone 1 at 1503
magnification.
Statistics
Analysis of covariance was used to compare species with
regard to proboscis length, adjusting for forewing length as a
proxy for body size (Miller, 1977). Analysis of variance was
used to test for significant differences (P < 0.05) in proboscis
lengths, zone lengths and widths, percent zone lengths, legular
widths, handswitches, and stylus and peg lengths of sensilla
styloconica between sexes within and among species. Significant differences in means among species were compared using
Fisher’s least significant difference (LSD) test.
An agglomerative hierarchical clustering algorithm using
average linkage distance (Johnson and Wichern, 2007) was
employed to simultaneously evaluate the means of 13 proboscis
characters for the five species. A dendrogram depicting the
results of the hierarchical cluster analysis was produced, calibrated from 1.0 to 1.5, representing a matrix of distances.
Characters used for analysis included proboscis length, percent
length of Zone 2, number of handswitches in Zone 1, width of
galea in Zones 1 and 2, percentage change in width between
Zones 1 and 2, width of the lower and upper branches of dorsal
legulae in Zone 1, percentage of lower branch of dorsal legulae
covered by upper branch in Zone 1, width of upper branch of
dorsal legulae in Zone 2, dorsal legular arrangement, presence/
absence of a toothlike projection at medial tips of dorsal legulae, and presence/absence of styloconica sensilla. Nonordinal
categorical variables and variables based on measured structures that were absent in certain species were omitted from the
clustering analysis. All statistical analyses were conducted in
SAS version 9.3.
Terminology
Studies of the lepidopteran proboscis have produced a mix of
functional, structural, and positional terminology (Eastham and
Eassa, 1955; Hepburn, 1971; Kingsolver and Daniel, 1979;
Krenn, 1990, 1998; Walters et al., 1998; Krenn and M€
uhlberger,
2002; Krenn and Kristensen, 2004; Petr and Stewart, 2004;
Zaspel et al., 2011; Monaenkova et al., 2012; Lehnert et al.,
2013). We, therefore, explicitly define the terms that we use.
Dorsal legula (pl., legulae) (Figs. 1–12)—cuticular extension(s) along the dorsal midline of the proboscis. Each dorsal
legula consists of an upper and a lower branch. The lower
branches of opposing galeae overlap in the nondrinking region
of the proboscis, but are more widely spaced in the drinking
region (Lehnert et al., 2013) where they overlap little or not at
all. The upper branches are enlarged and more widely spaced
in the drinking region and can have a toothlike projection at
the medial tip. The upper branches are either well developed
along the length of the proboscis or appear to originate from
other structures in the nondrinking region, such as bumps at
the bases of the lower branches of dorsal legulae or as small,
triangular spines on the galeal wall adjacent to the lower
branch.
Longitudinal groove (Figs. (5 and 12))—trough of variable
width that runs lengthwise along the dorsum of each galea for
part of the proboscis length, usually next to a longitudinal ridge
that separates the trough and dorsal legulae. The longitudinal
groove is not present in all species.
Microbumps (Fig. 12)—minute tubercles of various shapes
on the galeal surface. The distribution pattern of microbumps
STRUCTURE OF BUTTERFLY PROBOSCIS
5
Fig. 1. Schematic of the proboscis of P. glaucus, with scanning electron micrographs (insets A–E), showing measurements of linear
features. (A) Upper (ub) and lower (lb) branches of dorsal legulae were measured from their bases to the distal tip at the midpoint of
Zone 1 (indicated by red box on illustration). (B) The transition from Zone 1 to Zone 2 (indicated by black line) is based on dorsal
legular (dl) widths (Monaenkova et al., 2012). Sensilla trichodea (st) on the galea (ga) were counted if they were within 50 mm of the
dorsal legulae. (C) The presence of Zone 3 was based on absence of dorsal legulae; the boundary of Zones 2 and 3 is indicated by a
black line. Sensilla basiconica (sb) were recorded if they were within 20 mm of the dorsal legulae. (D) Midpoint of Zone 1 indicating
where galeal width was measured (red line). (E) Galeal width also was measured at the midpoint of Zone 2. Scale as in Figure 2.
can change along the length and width of the proboscis, but
tends to be expressed as a series of microbumps perpendicular
to the proboscis midline.
Microvalley (Fig. 12)—gap between neighboring microbumps
within a series of microbumps.
Macrovalley (Fig. 12)—trough between each series of microbumps. Macrovalleys are typically wider and deeper than
microvalleys.
Proboscis handedness (Fig. 5)—arrangement determined by
which galea has dorsal legulae overlapping the dorsal legulae
of the adjacent galea. A proboscis is considered left-handed, for
instance, if the dorsal legulae on the organism’s left galea over-
lap those on the right galea. Switches in handedness can occur
multiple times along the proboscis.
Sensillum styloconicum (pl., sensilla styloconica) (Figs. (3 and
6), and 8211)—sensory organ with a stylus longer than its peg.
Sensilla styloconica can be sparse (more than one sensillum
apart) or dense (less than one sensillum apart), and their distribution can be uniform (in a single row) or scattered (not in a single row). Various criteria are used to distinguish sensilla
styloconica from sensilla basiconica (Petr and Stewart, 2004;
Bauder et al., 2013; Faucheux, 2013), which with the presence of
potentially intermediate forms of sensilla styloconica (Paulus
and Krenn, 1996), confound classification of sensory structures.
Journal of Morphology
6
M.S. LEHNERT ET AL.
Fig. 2. Butterflies and their proboscises (distal region to right). The top two proboscises indicate the relative zone lengths of nonflower visitors (P. interrogationis and L. a. astyanax), followed by the proboscis of the flower visitor, D. plexippus. The two bottom
proboscises represent flower visitors with male puddling habits (P. glaucus and P. rapae). The black bars indicate 1 mm.
We adopted the terminology and criteria used by Petr and Stewart (2004), and use an operational classification scheme based
solely on the size of the stylus in relation to the peg.
Sensillum basiconicum (pl., sensilla basiconica) (Figs. (3 and
6), and 9)—sensory organ with a stylus shorter than its peg.
Sensillum trichodeum (pl., sensilla trichodea) (Figs. (6 and
9), and 12)—hair-like or spine-like sensory organ without a stylus or peg. Sensilla trichodea are treated here as synonymous
with sensilla cheatica (Xue and Hua, 2014), although Faucheux
(2013) suggested functional differences.
Zone 1 (Figs. 1–426, and 12)—nondrinking region of the proboscis that extends from the junction of the proboscis with the
head to where the upper branches of dorsal legulae begin to
enlarge. This zone is hydrophobic (Lehnert et al., 2013).
Zone 2 (Figs. 124, 628, and (10 and 11))—drinking region of
the proboscis that extends from where the upper branches of
dorsal legulae begin to widen to where dorsal legulae are no
longer present. Zone 2 is characterized by larger, more widely
Journal of Morphology
spaced upper branches of dorsal legulae than those in Zone 1.
This zone is hydrophilic (Lehnert et al., 2013).
Zone 3 (Figs. 1–3)—distalmost region of the proboscis that
lacks dorsal legulae; it is not present in all species. This zone is
hydrophilic and, hence, a functional subset of Zone 2, the drinking region (Lehnert et al., 2013).
RESULTS
Sexual Dimorphism
No significant sexual dimorphism was detected
for any variable of any species (df 5 4, P > 0.05).
Proboscis Length
Proboscis lengths differed significantly among
species (F 5 136.41; df 5 4, 94; P < 0.0001) and
STRUCTURE OF BUTTERFLY PROBOSCIS
7
Fig. 3. Scanning electron micrographs indicating the presence or absence of Zone 3 on butterfly proboscises. A and B show the dorsal and lateral view of Zone 3 on a galea of P. rapae, respectively. Zone 3 is characterized by a proboscis tip (tp) that lacks dorsal
legulae (dl). A also indicates the sensilla styloconica (ss) and basiconica (bs), and ventral legulae (vl) on proboscises of P. rapae. C
and D represent the tips of proboscises of nonflower visitors, L. a. astyanax (C) and P. interrogationis (D). C shows the toothlike projection that extends from the medial tip of the dorsal legulae. The nonflower visitors have dorsal legulae extended to the tip and,
therefore, lack Zone 3. B shows that Zone 3 extends beyond the food canal (fc).
were longest in P. glaucus (Table 1). Forewing
length differed among species (F 5 345.51; df 5 4,
94; P < 0.0001), and was not a significant covariate
of proboscis length in P. rapae or P. interrogationis, but was in the other species.
Zone Dimensions
Zone 1 occupied more than 80% of the total proboscis length for all species (Table 1; Fig. 2). Zone
lengths differed significantly among species (Zone
1, F 5 108.17; df 5 4, 94; P < 0.0001), as did the
percent of proboscis length represented by each
zone (Zone 1, F 5 280.20; df 5 4, 94; P < 0.0001;
Table 1). The percent of the proboscis length designated as Zone 2 was greatest in L. a. astyanax
and shortest in P. rapae. Pieris rapae and P. glaucus had the shortest and longest average proboscis
lengths, respectively, and did not have similar proportions of the proboscis represented by each zone
(Table 1; Fig. 2). Zone 3 was absent in nonflower
visitors (Fig. 3). The proboscis of P. rapae had a
significantly greater percentage length for Zone 3
than did other flower visitors (F 5 164.61; df 5 2,
57; P < 0.0001; Table 1).
The galeal widths in Zone 1 (F 5 106.57; df 5 4,
89; P < 0.0001) and Zone 2 (F 5 189.82; df 5 4, 90;
P < 0.0001) differed significantly among species,
with L. a. astyanax having the widest Zones 1 and
2 (Table 1). Measurements in the middle of Zones
1 and 2 indicated that the proboscis tapered distally in all species, with the greatest reduction of
width (>50%) in D. plexippus, P. glaucus, and P.
rapae. Polygonia interrogationis had a 43.4%
reduction in width and L. a. astyanax a 32.4%
reduction, when including sensilla styloconica.
Dorsal Legulae—Upper Branches (Zone 1)
The upper branches of the dorsal legulae varied
in width, shape, and point of origin among species
(Table 2, Fig. 4). Widths of the upper branches in
Zone 1 differed significantly among species
(F 5 392.80; df 5 4, 94; P < 0.0001) and were widest
in P. glaucus and P. rapae. In P. glaucus, they
were nearly as wide as the lower branches
(Fig. 4A). The upper branches of dorsal legulae in
Journal of Morphology
8
M.S. LEHNERT ET AL.
or pentagonal and nearly flush with the ridge of
the galeal surface.
Dorsal Legulae—Lower Branches (Zone 1)
The lower branches of dorsal legulae in Zone 1
were pointed in the Nymphalidae and spatula
shaped in P. rapae, but were partially covered by
the upper branches in P. glaucus (Figs. 4 and 5).
The number of handswitches differed significantly
among species (F 5 32.62; df 5 4, 66; P < 0.0001).
Handswitches occurred along Zone 1 an average of
<7 times in all species except P. rapae, which had
significantly more handswitches, producing a
zipper-like appearance (Table 2; Fig. 5B). Handswitches in P. glaucus uniquely occured among the
upper branches of dorsal legulae (Fig. 5A).
Dorsal Legulae—Widths
All species exhibited a structural change of the
upper branches of dorsal legulae, demarcating
Zones 1 and 2 (Table 2; Fig. 6); however, the
changes in overall dorsal legular widths between
these zones differed among species, regardless of
whether the upper or the lower branches were
wider. Dorsal legulae of P. glaucus were approximately 50% wider in Zone 2 than in Zone 1, were
of similar width between zones in the Nymphalidae, and decreased in width in P. rapae (Table 2).
In P. rapae, the upper branches of dorsal legulae
approximately doubled in width from Zone 1 to
Zone 2; however, the lower branches decreased in
width in Zone 2, resulting in a reduction in overall
dorsal legular width. The upper and lower
branches of P. rapae formed a groove between
them in Zone 2 (Fig. 7B).
Fig. 4. Scanning electron micrographs of the origin and transition of the upper branches of dorsal legulae. A shows enlarged
upper branches (ub) of dorsal legulae in Zone 1 of proboscises of
P. glaucus, which enlarge and demarcate Zones 1 and 2 (inset).
B and C show smaller upper branches of dorsal legulae in Zone
1 of L. a. astyanax and P. interrogationis, respectively. The lower
branches (lb) of dorsal legulae are apparent in Zone 1, whereas
the upper branches of dorsal legulae in L. a. astyanax (shown in
B) appear as triangular projections from the galeal wall. The
upper branches on proboscises of P. interrogationis appear as
small bumps at the base of the lower branches of dorsal legulae
in Zone 1. All species have upper branches of dorsal legulae
enlarged, demarcating Zones 1 and 2 (insets).
Zone 1 of the three Nymphalidae were not well
developed. The upper branch of a dorsal legula of
P. interrogationis in Zone 1 was a small bump at
the base of the lower branch, whereas the upper
branch of L. a. astyanax was a small, triangular
protrusion on the adjacent galeal wall (Fig. 4B,C).
The upper branch of D. plexippus was rectangular
Journal of Morphology
Dorsal Legulae—Shapes of Upper Branches
in Zone 2
The dorsal legulae in Zone 2 of L. a. astyanax, P.
interrogationis, and P. rapae overlapped in
shingle-like fashion (Table 2, Fig. 7), resulting in
interlegular spacing that was not completely visible from the dorsum (Fig. 8). The dorsal legulae of
D. plexippus and P. glaucus were positioned sideby-side and were nearly flat, with the enlarged
interlegular spaces visible from the dorsum (Fig.
8). Polygonia interrogationis and L. a. astyanax
had a toothlike projection on the tip of each dorsal
legula (Fig. 3; Table 2). The upper branches of L.
a. astyanax were serrated distally (Figs. 7 and 8).
Sensilla
Sensilla basiconica and trichodea were present
on all proboscises (Fig. 9). Sensilla styloconica,
however, were found only on proboscises of L. a.
astyanax, P. interrogationis, and P. rapae (Figs. 9
and 10). The short chemosensilla in Zones 2 and 3
on proboscises of P. glaucus and D. plexippus were
STRUCTURE OF BUTTERFLY PROBOSCIS
9
Fig. 5. Scanning electron micrographs showing handswitches of dorsal legulae in Zone 1. (A) Handswitches (hs) on proboscises of
P. glaucus occur with the upper branches of dorsal legulae (dl). Handswitches in other species occur solely with the lower branches,
shown here with proboscises of P. rapae (B) and L. a. astyanax (C). Proboscises of P. rapae have a large number of handswitches
that appear zipper-like. A galea (ga) is shown as a reference in each image. The longitudinal groove (lg) on the proboscis of P. glaucus
appears near the dorsal legulae.
classified as sensilla basiconica (according to criteria outlined in our Terminology section above).
The sensilla styloconica were dense and uniformly
distributed in rows in L. a. astyanax and P. interrogationis, giving Zone 2 an overall brush-like
appearance (Fig. 11, Table 3), but were sparse and
scattered in P. rapae. The stylus and peg lengths
of sensilla styloconica differed significantly among
species (F 5 768.92; df 5 2; P < 0.0001 and
F 5 334.35; df 5 2; P < 0.0001, respectively) and
were of different shapes (Fig. 10, Table 3).
Microbumps, Microvalleys, and Macrovalleys
Each species had unique shapes and patterns
of microbumps and micro- and macrovalleys
(Fig. 12). These patterns also changed along the
proboscis length and width within species. Papilio
glaucus, D. plexippus, and P. interrogationis had
similar microbump patterns on the lateral surface
of the galeae, which consisted of rows of circular
microbumps, with microvalleys interspersed by
macrovalleys. The rows of circular microbumps
continued medially to the longitudinal groove near
the dorsal legulae of D. plexippus, but transitioned
to a zigzag pattern in P. glaucus and were spinelike in P. interrogationis. The microbump pattern
of L. a. astyanax produced a wrinkled appearance,
which became spine-like near the dorsal legulae
(Fig. 12). In P. rapae, it consisted of crosswise
ridges with a series of spike-like microbumps and
broad macrovalleys (Fig. 5B) that transitioned into
Fig. 6. Scanning electron micrographs showing the boundary of Zones 1 and 2. All proboscises have upper branches (ub) of dorsal
legulae that abruptly enlarge, indicating the transition (tr) from Zone 1 to 2. The lower branches (lb) of dorsal legulae are prevalent
throughout Zone 1 in all species except P. glaucus, where they are hidden by the upper branches. Zone demarcation was observed in
proboscises with relatively smooth galeae (ga) along the proboscis length (e.g., D. plexippus, A) and proboscises with sensilla styloconica (ss) (e.g., L. a. astyanax, B). Sensilla trichodea (ts) are on the galeal wall, but are more abundant in Zone 2.
Journal of Morphology
10
M.S. LEHNERT ET AL.
Fig. 7. Scanning electron micrographs of the enlarged dorsal legulae in Zone 2. Upper branches (ub) of dorsal legulae of all species
are wider in Zone 2 than in Zone 1; however, the arrangement and shape of the dorsal legulae differs among species. Limenitis a.
astyanax (A) and P. rapae (B) have dorsal legulae that overlap shingle-like in Zone 2. The lower branches (lb) of dorsal legulae in L.
a. astyanax appear as large triangular projections, whereas the upper branches are serrated. Upper and lower branches of dorsal
legulae of P. rapae fuse together, forming a channel-like groove.
bumps in Zone 2. The microbump patterns of L. a.
astyanax and P. interrogationis in Zone 2 were
obscured by sensilla styloconica (Fig. 11).
Longitudinal Grooves
The proboscises of L. a. astyanax and P. interrogationis had a wide longitudinal groove near the
ridge that ran nearly the length of Zone 1 into
Zone 2. The longitudinal groove also was present
in D. plexippus and P. glaucus, but narrower, and
was absent in P. rapae. A single row of hair-like
setae (Fig. 2) occupied the longitudinal groove of
each galea near the base of the proboscis of
P. interrogationis for approximately 10% of the proboscis length, but was absent in the other species.
Grouping of Species Based on Proboscis
Structure
A dendrogram (Fig. 13) produced from the hierarchical clustering analysis of 13 proboscis characters showed that the nonflower visitors L. a.
astyanax and P. interrogationis clustered together.
The flower visitor D. plexippus clustered with
these two species, and the flower visitor and puddler P. rapae, in turn, clustered with this group of
three species. Papilio glaucus was the most distant from the other groups.
Fig. 8. Scanning electron micrographs showing the dorsal legular arrangement and interlegular spaces. Nonflower visitors have
dorsal legulae (dl) in Zone 2 that overlap (L. a. astyanax with sensilla styloconica [ss] shown in image A), whereas flower visitors
have dorsal legulae that do not overlap (P. glaucus shown in B). Differences in dorsal legular arrangement are coupled with differences in the arrangement of the corresponding interlegular spaces (il).
Journal of Morphology
STRUCTURE OF BUTTERFLY PROBOSCIS
11
Fig. 9. Scanning electron micrographs of sensilla on proboscises. Sensilla trichodea (ts) are on the galeae (ga) of all species (L. a.
astyanax shown in A). Sensilla styloconica (ss) are on nonflower visitors and P. rapae; however, they are denser and more uniform in
distribution in Zone 2 on proboscises of nonflower visitors (sensilla styloconica of L. a. astyanax shown in B). Sensilla basiconica (bs)
are present on all species (D. plexippus shown in C).
DISCUSSION
Does Overall Proboscis Landscape Reflect
Feeding Habits?
Although adult Lepidoptera typically feed opportunistically, they often are grouped as flower visitors and nonflower visitors (Gilbert and Singer,
1975; Krenn et al., 2001; Knopp and Krenn, 2003;
Lehnert et al., 2013). Our data, however, do not
support the hypothesis that external proboscis
architecture, based on an overall set of proboscis
characters, indicates a distinct guild of flower visitors. The smooth proboscis of D. plexippus, for
instance, groups with the brushy proboscises of
P. interrogationis and L. a. astyanax. Overall pro-
boscis architecture, however, does permit grouping
of the nonflower visitors.
The absence of a distinct grouping of flower visitors, based on an overall set of proboscis characters, does not mean that a flower-visiting species
cannot be categorized on the basis of selected
structural features. Our study taxa and many
examples in the literature (Krenn et al., 2001;
Knopp and Krenn, 2003; Molleman et al., 2005;
Lehnert et al., 2013) can be categorized as flower
visitors or nonflower visitors. How, then, can our
clustering analysis be reconciled with the ability
to recognize a flower visitor on the basis of visible
structural characters of its proboscis? The answer
Fig. 10. Structures measured and shapes of sensilla styloconica. Sensilla styloconica consist of a peg and stylus (A). The length of
the peg (pe) and stylus (st) and distance between sensilla styloconica and dorsal legulae (dl) differ among L. a. astyanax (B), P. interrogationis (C), and P. rapae (D). Sensilla styloconica are dense and flattened in nonflower visitors (L. a. astyanax, P. interrogationis),
but scattered and rounded on the proboscis of P. rapae.
Journal of Morphology
12
M.S. LEHNERT ET AL.
Fig. 11. Scanning electron micrographs showing differences in overall appearance of Zone 2 between butterflies of different feeding
habits. Zone 2 of flower visitors, such as D. plexippus (A), have sensilla basiconica (bs) and nonoverlapping dorsal legulae (dl). Nonflower visitors, such as L. a. astyanax (B), have enlarged sensilla styloconica (ss) and overlapping dorsal legulae.
lies in the overlay of structural modifications for
specialized feeding habits on the generalized structural arrangement of all lepidopteran proboscises.
The fundamental organization of all lepidopteran
proboscises—a tapered, porous tube with a rough
galeal surface often bearing slender cuticular projections well depicted by Krenn and Kristensen
(2000)—reflects adaptations for capillary action
and the acquisition of liquid from surface films
and droplets, including those in floral corollas that
often provide only a trace of nectar (Monaenkova
et al., 2012; Lehnert et al., 2013; Kwauk et al.,
2014). These fundamental structural adaptations
for fluid uptake are present in the oldest extant
haustellate Lepidoptera, such as the Eriocraniidae
(Krenn and Kristensen, 2000; Monaenkova et al.,
2012) and, therefore, date to more than 100 mya
(Grimaldi and Engel, 2005). Lepidoptera that feed
from flowers must be able to acquire fluids not
only from pools, but also from droplets and films
in the corolla when nectar is in limited supply
(Monaenkova et al., 2012).
With the diversification of flowering plants, the
early Lepidoptera bearing fibrous proboscises
would have been structurally positioned to exploit
new food sources such as flowers with nectar.
Fig. 12. Scanning electron micrographs showing proboscis surface patterns and topography. A shows the longitudinal groove (lg)
on proboscises of L. a. astyanax near the dorsal legulae, with lower branches (lb) and triangular upper branches (ub). Sensilla trichodea (ts) and basiconica (bs) are in or near the longitudinal grooves. The microbump (mb) pattern on proboscises of L. a. astyanax
changes from a bumpy texture near the dorsal legulae to rows of bumps on ridges interspersed by macrovalleys. The proboscises of
D. plexippus (B) have longitudinal grooves that are narrower than those of L. a. astyanax and microbumps with microvalleys (mi)
and macrovalleys.
Journal of Morphology
STRUCTURE OF BUTTERFLY PROBOSCIS
13
Fig. 13. Dendrogram depicting agglomerative hierarchical clustering with average linkage distance of means of 13 proboscis characters for five species of butterflies. Although the dendrogram implies no ancestral or derived relationships and is not equivalent to a
cladogram, it nonetheless mirrors the topology of current phylogenetic relationships (e.g., Regier et al., 2013).
Subsequent modifications of the proboscis would
have adapted flower-visiting lepidopterans to
increased foraging efficiency from tubular flowers.
Proboscis characters unique to flower visitors,
such as a reduced sensillar brush (Krenn et al.,
2001) and tapering, are probably related to flower
entry. On the basis of these characters alone, a
species can be categorized as predominantly a
flower visitor or a nonflower visitor. Most proboscis
characters in our analysis probably are related to
the entry of fluid into the proboscis of all haustellate Lepidoptera (Monaenkova et al., 2012; Lehnert et al., 2013); therefore, the suite of characters
for acquiring fluids from droplets and wetted
surfaces swamps out other characters that aid floral entry, which are relevant only to flower visitors. Although not indicative of feeding guilds, the
set of proboscis characters used in our analysis
produces a dendrogram with a topology that
reflects the evolutionary relationships of the five
species (Regier et al., 2013; Kawahara and Breinholt, 2014), affirming the relevance of proboscis
structural characters in lepidopteran classification
(Kristensen, 1984; Krenn and Kristensen, 2004;
Kristensen et al., 2007).
The two flower-visiting species with puddlevisiting males, P. glaucus and P. rapae, did not
cluster together, nor did males differ from females
for any of the 21 evaluated proboscis features.
Puddling species use the same structural features
involving uniform principles of fluid acquisition
from wetted surfaces that are present in all haustellate Lepidoptera (Monaenkova et al., 2012). In
addition, puddling behavior, although often
reported as a male-specific behavior, has been
reported in females of P. glaucus and P. canadensis (Scriber, 1987, 2002). Hence, the apparent
absence of sexual dimorphism and lack of structural indicators in the proboscis of puddling species is not unexpected. Nonetheless, the possibility
of finer structural differences, such as the interlegular spacing, between males and females cannot
be excluded (Kwauk et al., 2014).
Selection pressures exerted by diverse floral
structure (Soltis et al., 2009; Tiple et al., 2009)
probably have resulted in the diversity of proboscis
structure among flower visitors. The best-known
relationship between flower and proboscis structure is the corresponding length of the floral tube
and the proboscis (Kunte, 2007; Krenn, 2010;
Arditti et al., 2012; Bauder et al., 2015); however,
other patterns can be found. The Papilionidae and
Nymphalidae, for example, which feed on nectar
from similar (e.g., larger) flowers (Tiple et al.,
2009), have similar dorsal legular shapes that differ from those of pierids. Future studies could
examine a potential relationship between proboscis
structure and nectar composition and viscosity.
Which Proboscis Characters Are Indicators
of Feeding Habits?
Lepidoptera that feed from porous substrates,
such as decaying fruits, have a brushy proboscis
formed of dense rows of elongated sensilla styloconica (Knopp and Krenn, 2003; Molleman et al.,
2005). Species without a brushy proboscis, by
Journal of Morphology
14
M.S. LEHNERT ET AL.
default, typically are recognized as flower visitors,
although scattered sensilla styloconica can be
present (Krenn et al., 2001; Petr and Stewart,
2004). Nonflower visitors have unique proboscis
characters in addition to a dense sensillar brush,
such as dorsal legulae extended to the tip of the
proboscis and wider, lower branches of dorsal legulae in the proximal region (Zone 1). For flower visitors, the opposite conditions hold. The presence of
Zone 3 in the flower visitors might have adaptive
value in facilitating the proboscis to enter narrow
floral corollas, but requires further study. Proboscis structure of nonflower visitors might provide
increased hydrophilic surface area and capillarity
for fluid uptake from porous substrates (Lehnert
et al., 2013).
The dense rows of sensilla styloconica and dorsal legulae that extend to the apex, might hinder
the proboscis from entering floral tubes by increasing friction or drag in the corolla. Serrations on
the dorsal legulae of L. a. astyanax could impede
floral entry, but might assist in fluid uptake by
scraping surfaces of rotting fruit. All proboscises
that we studied taper distally, especially in flower
visitors, which could enhance floral entry and
access to nectar (Krenn et al., 2001), particularly
in combination with a distalmost region of the proboscis (Zone 3) free of dorsal legulae.
Which Proboscis Characters Are Poor
Indicators of Feeding Habits?
A longer Zone 2 has been associated with
nonflower-visiting nymphalids (Krenn et al.,
2001). Although a longer Zone 2 might increase
the surface area of the proboscis applied to wetted
surfaces, such as rotting fruit, (Knopp and Krenn,
2003; Molleman et al., 2005; Lehnert et al., 2013),
the nonflower visitors in our study do not have a
proportionally longer Zone 2 than the other
butterflies.
We showed that changes in the width of the
upper and lower branches along the proboscis,
which are associated with the structural distinctiveness of the drinking region (Lehnert et al.,
2013; Kramer et al., 2015), are species specific and
that only the upper branches of dorsal legulae
enlarge from Zone 1 to Zone 2 in our studied species. The structural origins of the upper branches
of dorsal legulae in Zone 2 differ among species,
but their consistent presence across species suggests functional importance and similar selection
pressures. The grooves between the upper and
lower branches of the dorsal legulae of P. rapae
might help channel fluids to the food canal.
Grooves in dorsal legulae with overlapping
arrangements are found in many species including
Cryphia muralis (Noctuidae), Scrobipalpa costella
(Gelechiidae) (Faucheux, 2013), Archaeoprepona
Journal of Morphology
demophon (Nymphalidae) (Krenn, 2010), and V.
cardui (Nymphalidae) (Kwauk et al., 2014).
No evidence suggests that legular hand switches
or microbump patterns indicate feeding guilds.
Hand switches, especially the zipper-like hand
switches of P. rapae, might increase proboscis flexibility or minimize galeal separation during feeding (Lehnert et al. 2014). Zipper-like hand
switches also are found in P. icarus (Lycaenidae)
(Krenn et al., 2005). Microbumps and valleys
might facilitate channeling of fluids to regions of
the proboscis where uptake can occur (Lehnert
et al., 2013) and aid proboscis flexibility while
feeding (Krenn et al., 2005) and coiling (Krenn,
1990; Krenn et al., 2005; Lehnert et al. 2015). The
presence of surface sculpturing on the proboscis
might represent a general character for feeding
from droplets and films; pool feeding would not
require external channeling of fluid.
CONCLUSIONS
The proboscis of haustellate Lepidoptera is fundamentally adapted for fluid uptake from droplets
and surface films. Its overall structure does not
unequivocally permit species to be assigned to
flower-visiting versus nonflower-visiting groups or
to a puddle-visiting group. Specific characters of
the proboscis, however, such as the presence or
absence of a dense sensillar brush, allow most species to be assigned to a feeding guild. Characters
related to floral entry, such as increased tapering
of the proboscis, characterize flower visitors.
We suggest that the diversity of proboscis structure among flower visitors reflects the diversity of
floral structure. Flower-visiting nymphalids
exhibit greater structural variation of the proboscis than do nonflower-visiting nymphalids (Krenn
et al., 2001). Proboscis length, in general, varies
more among the flower-visiting Lepidoptera than
it does among nonflower visitors (Kunte, 2007). On
the contrary, feeding from wetted surfaces, such as
rotting fruit, involves less structural variation,
owing to greater uniformity of the porous nature
of the substrates. Thus, no structural indicators
for puddling species or between genders in species
with puddling males were apparent. The adaptive
value of other structural attributes of the proboscis, such as the zipper-like handedness of dorsal
legulae, require further exploration.
ACKNOWLEDGMENTS
We thank the Clemson University Electron
Microscopy Laboratory for assistance with scanning electron microscopy, Ms. Bennie M. Saylor for
photographs of the adult butterflies, Angela H.
Newman for illustrations of the proboscises, and
Prof. Harald W. Krenn for insightful comments on
the manuscript. This is Technical Contribution No.
STRUCTURE OF BUTTERFLY PROBOSCIS
6369 of the Clemson University Experiment Station, under project number SC-1700433.
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