Arthropod Structure & Development xxx (2014) 1e6
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Arthropod Structure & Development
journal homepage: www.elsevier.com/locate/asd
Mouthpart separation does not impede butterfly feeding
Matthew S. Lehnert*, Catherine P. Mulvane, Aubrey Brothers
Department of Biological Sciences, Kent State University at Stark, 6000 Frank Ave. NW, North Canton, OH 44720, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 November 2013
Accepted 13 December 2013
The functionality of butterfly mouthparts (proboscis) plays an important role in pollination systems,
which is driven by the reward of nectar. Proboscis functionality has been assumed to require action of the
sucking pump in the butterfly’s head coupled with the straw-like structure. Proper proboscis functionality, however, also is dependent on capillarity and wettability dynamics that facilitate acquisition of
liquid films from porous substrates. Due to the importance of wettability dynamics in proboscis functionality, we hypothesized that proboscides of eastern black swallowtail (Papilio polyxenes asterius Stoll)
(Papilionidae) and cabbage butterflies (Pieris rapae Linnaeus) (Pieridae) that were experimentally split
(i.e., proboscides no longer resembling a sealed straw-like tube) would retain the ability to feed.
Proboscides were split either in the drinking region (distal 6e10% of proboscis length) or approximately
50% of the proboscis length 24 h before feeding trials when butterflies were fed a red food-coloring
solution. Approximately 67% of the butterflies with proboscides split reassembled prior to the feeding
trials and all of these butterflies displayed evidence of proboscis functionality. Butterflies with proboscides that did not reassemble also demonstrated fluid uptake capabilities, thus suggesting that wild
butterflies might retain fluid uptake capabilities, even when the proboscis is partially injured.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Proboscis
Functionality
Lepidoptera
Wettability
Pollination
Fluid uptake
1. Introduction
Mouthpart functionality of fluid-feeding insects e more than
half of all known animal species (Foottit and Adler, 2009) e is an
important component of disease transmission and the stability of
insect-pollination systems (Kingsolver and Daniel, 1995). Mouthparts of fluid-feeding insects, such as butterflies and moths (Lepidoptera), might be subjected to damage while seeking mates,
searching for food, or from predator encounters. Mouthparts
rendered nonfunctional, therefore, could affect fitness (Krenn,
1997) and impact insecteflower interactions.
Most Lepidoptera have a coilable, tube-like proboscis that
transports fluids, such as nectar, sap, fruit juices, and blood (Adler,
1982) to the insect’s gut. The lepidopteran proboscis is composed of
two elongated maxillary galeae that are connected by overlapping
dorsal and interlinking ventral structures (i.e., legulae) to form a
food canal (Eastham and Eassa, 1955; Krenn et al., 2005; Krenn,
2010). The distal 5e20% of the proboscis has dorsal legulae that
are elongated and more widely spaced (Krenn et al., 2001) (i.e., the
drinking region), which facilitates fluid uptake (Lehnert et al.,
2013). The merging of the galeae into a functional proboscis takes
place after adult eclosion from the pupa, and consists of coiling and
* Corresponding author. Tel.: þ1 (330) 244 3349.
E-mail addresses: [email protected] (M.S. Lehnert), [email protected] (C.
P. Mulvane), [email protected] (A. Brothers).
uncoiling actions of the proboscis accompanied by the presence of
saliva droplets (Krenn, 1997). Proboscis assembly must occur before
sclerotization of the legular cuticle, otherwise the proboscis is putatively nonfunctional and reassembly cannot occur (Krenn, 1997).
A functional proboscis is widely considered a sealed tube that
operates similar to a drinking straw (Krenn, 2010; Bauder et al.,
2013), solely relying on the sucking pump in the head for fluid
uptake (Kingsolver and Daniel, 1995; Eberhard and Krenn, 2003);
however, recent experiments have demonstrated that aqueous
solutions can enter between dorsal interlegular spaces along the
proboscis (i.e., not a sealed tube) (Monaenkova et al., 2012) and that
a straw-like structure is not necessary for functionality (Grant et al.,
2012). The proboscis employs capillarity via interlegular spaces to
build liquid bridges in the food canal for the sucking pump to act on
when feeding from liquid films and porous substrates
(Monaenkova et al., 2012), such as rotting fruit. Fluid uptake is
further regulated by wettability dynamics (i.e., hydrophilicity and
hydrophobicity) of proboscis structures (e.g., hydrophilic dorsal
legulae, chemosensilla, and the food canal) and surface roughness
(e.g. microbumps that create an overall hydrophobic surface,
explained using the Cassie-Baxter model, Cassie and Baxter, 1944;
Lehnert et al., 2013). Based on our current understanding of the
multifaceted fluid uptake system of butterfly proboscides we hypothesized that previously assembled proboscides of two distantly
related nectar-feeding butterfly species, the eastern black swallowtail, Papilio polyxenes asterius (Papilionidae), and cabbage
1467-8039/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.asd.2013.12.005
Please cite this article in press as: Lehnert, M.S., et al., Mouthpart separation does not impede butterfly feeding, Arthropod Structure &
Development (2014), http://dx.doi.org/10.1016/j.asd.2013.12.005
2
M.S. Lehnert et al. / Arthropod Structure & Development xxx (2014) 1e6
butterfly, Pieris rapae (Pieridae), will maintain functionality
following experimental splitting of the galeae as long as both galeae
are subsequently placed in a feeding solution.
2. Materials and methods
2.1. Butterfly rearing and proboscis measurements
Eggs of P. p. asterius were obtained from a female captured in
North Canton, OH. The larvae were reared on parsley (Petroselinum
crispum) and kept in RubbermaidÒ Takealong containers. Larvae of
P. rapae were obtained from Carolina Biological Supply Company
(Burlington, NC, USA) and reared on artificial diet. Larvae and pupae
of both species of butterflies were maintained at 22 C, 61% relative
humidity (r.h.), and an L18:D6 photoperiod in an environmental
chamber (Percival Scientific, Inc., Perry, IN, USA). Adults from an F2
generation of P. p. asterius also were used for experiments. All adults
were fed a dilute honey:water solution (1:5) daily for at least three
days before feeding experiments and kept in glassine envelopes in a
refrigerator (4 C) between feeding times.
Proboscis lengths and drinking region lengths were measured to
determine possible effects on functionality between treatments. In
order to acquire proboscis measurements, butterflies were stabilized on a piece of Styrofoam and proboscides were uncoiled using
insect pins. Images of the total length of proboscides (0.78
magnification) and drinking regions (4.0 magnification) were
acquired for each butterfly with a Leica M205 C stereomicroscope
and an IC 80HD camera (Heerbrugg, Switzerland) and measured
using ImageJ software (http://rsbweb.nih.gov/ij/). The drinking
region was measured from the tip of the proboscis to a transition
point where the dorsal legulae narrow and remain similar in width
for the remainder of the proboscis length (Fig. 1A). Although
wettability dynamics of proboscides have been reported for other
butterfly species (Monaenkova et al., 2012; Lehnert et al., 2013), we
demonstrated these dynamics using the proboscis of an individual
P. p. asterius. The galeae were split and one galea (unstained) was
placed on a slide in dH2O with a coverslip and imaged using an
Olympus Confocal Microscope IX81 with DSU (Center Valley, PA,
USA) (999.6 ms exposure, 20 magnification, 30 slices, 1.60 average
depth slice, CY3 channel). The other galea was stained with Nile red
for 24 h and imaged similarly to distinguish hydrophilic and hydrophobic structures. Proboscides of P. rapae were dehydrated in an
ethanol series (80%, 90%, 100%, 24 h each), air-dried with hexamethyldisalizane, platinum sputter-coated for approximately
1 min, and imaged with a Hitachi TM3000 scanning electron microscope (Hitachi High Technologies America, Inc., Dallas, TX, USA).
2.2. Experimental feeding trials
All butterflies were fed a 20% sucrose solution and kept at room
temperature (24 C, 61% r.h.) in a netted Bug Dorm (BioQuip
Products, Rancho Dominguez, CA, USA) 24 h prior to feeding experiments. Randomly selected butterflies were prepped for the
experimental feeding trials by separating the two galeae either in
the drinking region using an insect pin or had approximately 50% of
their proboscis separated proximally starting at the tip (inset in
Fig. 1A) immediately after being fed the 20% sucrose solution.
Before proboscides were split, all butterflies had their proboscides
Fig. 1. Proboscis assembly and fluid uptake of split proboscides of butterflies. (A) Stereomicroscope image of an uncoiled proboscis of P. p. asterius displaying the galeae (ga) and
overlapping dorsal legulae (dl). The dorsal legulae are larger and more widely spaced in the drinking region; the remainder of the proboscis represents the nondrinking region. The
inset shows a proboscis of P. p. asterius split with insect pins (ip) for the red-50 treatment. (B) Photograph of a P. p. asterius obtained shortly after emergence showing the partially
assembled proboscis (separated galeae) during the assembly process. (C) SEM image of a single galea of P. rapae showing the food canal (fc) and dorsal (dl) and ventral legulae (vl)
that interlink during proboscis assembly. (D) Stereomicroscope image of the dorsal legulae of P. p. asterius in the nondrinking region; there is little overlap of the dorsal legulae. (E)
SEM image of a proboscis of P. rapae showing the overlapping dorsal legulae in the nondrinking region and microbumps (mb). The arrangement of the dorsal legulae differs between
P. p. asterius and P. rapae.
Please cite this article in press as: Lehnert, M.S., et al., Mouthpart separation does not impede butterfly feeding, Arthropod Structure &
Development (2014), http://dx.doi.org/10.1016/j.asd.2013.12.005
M.S. Lehnert et al. / Arthropod Structure & Development xxx (2014) 1e6
investigated by prodding the proboscis with an insect pin to ensure
the galeae were assembled (Fig. 1B shows a proboscis during the
assembly process after eclosion from the pupa).
Butterflies were assigned into a control group or one of three
experimental treatments and then fed solutions for 10e13 min by
manually placing only the drinking region of both galeae into a
100 ml capillary tube 24 h after experimental splitting. The diameter
of the capillary tube was larger than the width of both galeae in the
drinking region, therefore, the tube had little effect on pressing the
galeae together, thus avoiding simulating a sealed proboscis. The
control group consisted of butterflies with an unsplit proboscis fed
an aqueous 20% sucrose solution. The three treatments consisted of:
1) butterflies with unsplit proboscides fed a 20% sucrose and redfood coloring mixture, referred to hereafter as red-sucrose solution (i.e., red-unsplit treatment), 2) butterflies with proboscides
split at the drinking region fed the red-sucrose solution (i.e., red-dr
treatment), and 3) butterflies with approximately 50% of the proboscis split and fed the red-sucrose solution (i.e., red-50 treatment).
Due to the dark pigmentation of the proboscis of P. p. asterius,
feeding ability was determined by dissecting the butterflies in PBS
physiological saline solution (7.2 pH) using insect pins and dissecting scissors within 1 h after the feeding trials (butterflies were
temporarily stored in 4 C refrigerator before dissections). The crop
of the alimentary canal was isolated and the average a* color values
(a* ¼ green to red, scale 120 to 120, L*, a*, and b* color values)
were acquired using LensEye color analysis software (Lehnert et al.,
2011). Dissections of P. rapae specimens were not necessary due to
transparency of the dorsal legulae of the proboscis and the ability to
visualize fluid flow in the food canal.
2.3. Data analysis
Significant differences (p < 0.05) in proboscis length, drinking
region length, percentage of proboscis length represented by the
drinking region (i.e., percentage drinking length), and a* color values
were tested between sexes (t-test) and between treatments (analysis of variance) for P. p. asterius. Significant differences in mean a*
color values were ranked between treatments using Fisher’s least
significant difference (LSD) test. Due to differences in sample sizes
between treatments with P. rapae, an independent sample Kruskale
Wallis test was used to determine significant differences (p < 0.05)
in proboscis length, drinking region length, and percentage drinking
length. All statistical analyses were processed in SPSS Statistic 21.0.
3. Results
3.1. Evidence for proboscis reassembly
All butterflies with experimentally split proboscides were
checked for reassembly by applying pressure to the dorsal legulae
3
in previously split regions with the tip of an insect pin before being
fed. All butterflies that had the galeae separated in the drinking
region (i.e., red-dr treatment, Table 1) had proboscides that
appeared reassembled before feeding, except for one individual of
P. p. asterius, which was observed to have a fluid, likely saliva,
present between the separated galeae of the drinking region. An
individual of P. rapae died before we could check for proboscis
functionality; however, inspection revealed a reassembled drinking
region. It is unknown if reassembly of the drinking region involved
complete interlinking of the ventral legulae; only the dorsal legulae
were inspected.
Based on visual inspection, all P. p. asterius in the red-50 treatment (n ¼ 4) had reassembled proboscides however, when prodded
with an insect pin only one remained assembled, the other three
proboscides had galeae that partially separated where pressure was
applied. Three of the four P. rapae in the red-50 treatment remained
reassembled after pressure was applied with an insect pin (Table 1).
The individual without a reassembled proboscis had the galeae
almost completely separated (over 90%), indicating further separation after our experimental splitting, and one of the galeae was
coiled and appeared dry, which prevented us from being able to
insert both galeae into the capillary tube to test proboscis functionality. There were no significant differences in proboscis length
(mean s.e.m., 17.92 0.34 mm), drinking region length
(1.64 0.03 mm), or percentage drinking region (9.21 0.23%)
between treatments or sexes of P. p. asterius (n ¼ 4 per treatment)
or P. rapae (proboscis length 10.84 0.14 mm, drinking region
length 0.77 0.02 mm, percentage drinking region 7.11 0.18%;
n ¼ 12), suggesting that the differences observed between treatments were not influenced by these structural measurements
(Table 1).
3.2. Feeding ability of split proboscides
All individuals that had the drinking region of both galeae
inserted into the capillary tube were observed to feed (suggested by
the depletion of solution in the tube), except one individual of
P. rapae from the unsplit-red treatment. All individuals of P. rapae
with proboscides that reassembled and fed the red-sucrose solution were observed to have a red fluid traveling through the food
canal, which was visible through the semi-transparent dorsal
legulae (Fig. 2A); butterflies in the control group were observed to
have clear fluids traveling through the proboscis. Three individuals
of P. p. asterius from each of the red-unsplit and red-50 treatment
had a red droplet appear on the dorsum of the dorsal legulae at
proximal regions of the proboscis (i.e., nondrinking region) while
feeding (two simultaneous droplets on one individual); the droplets exhibited a pulsing-like motion. In addition, anti-parallel
movements of the galeae were observed on an individual of P. p.
Table 1
Measurements (mean s.e.m.) of proboscides and confirmation of mouthpart functionality of butterfly species. All butterflies in each treatment were checked for proboscis
reassembly and feeding ability after experimental splitting. The numbers of individuals that reassembled their proboscis and fed on solutions are followed by the total sample
size for that treatment in parentheses. Mouthpart functionality was determined by color quantification of the crop (a* values) or visual observation of fluids in the proboscis.
Species
Treatment
Reassembled
proboscides
Individuals
that fed
Mouthpart functionality
(* ¼ a color values)
P. p. asterius
Control
Red-unsplit
Red-dr
Red-50
4(4)
4(4)
3(4)
1(4)
4(4)
4(4)
3(4)
1(4)
31.83
53.46
48.20
48.87
P. rapae
Control
Red-unsplit
Red-dr
Red-50
2(2)
3(3)
3(3)
3(4)
2(2)
2(2)
2(2)
3(4)
Clear fluid present
Red fluid present
Red fluid present
Red fluid present
0.82**
4.16*
1.81*
2.04*
Proboscis length
(mm)
Drinking region
length (mm)
18.19
18.61
17.98
16.90
0.89
0.25
1.00
0.21
1.59
1.72
1.65
1.62
0.08
0.05
0.03
0.09
10.75
11.21
11.02
10.47
0.49
0.24
0.14
0.23
0.72
0.82
0.82
0.73
0.01
0.04
0.03
0.04
**Significant differences (p < 0.0001) between a* color values of dissected crops.
Please cite this article in press as: Lehnert, M.S., et al., Mouthpart separation does not impede butterfly feeding, Arthropod Structure &
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M.S. Lehnert et al. / Arthropod Structure & Development xxx (2014) 1e6
Fig. 2. Evidence for functionality of previously split proboscides. (A) Proboscis (pr) of a P. rapae fed the red-sucrose solution, which was visible in the food canal (fc) through the
transparent dorsal legulae. (B) Image of a section of the alimentary canal in an individual of P. p. asterius from the red-50 treatment obtained after a dissection that revealed the
engorged red fluid-filled crop (cr), midgut (mg), and hindgut (hg).
asterius from the red-50 treatment that had a reassembled
proboscis.
All P. p. asterius fed the red-sucrose solution had an enlarged
crop filled with red fluid (Fig. 2 B). Butterflies in the control treatment also had an enlarged fluid-filled crop, but it lacked a red fluid
and was clear. Color quantification of the crops revealed no significant differences in a* (red) color values between treatments
where butterflies were fed red-sucrose solution, indicating that all
butterflies retained the ability to feed. These treatments, however,
had significantly higher a* color values (i.e., were more red)
(p < 0.0001) compared to butterflies in the control group (fed sucrose solution) (Table 1). Other components of the alimentary canal, such as the midgut and hindgut also appeared red. One
individual defecated red fluid during the dissecting process.
3.3. Wettability of P. p. asterius proboscis
Confocal microscopy of a proboscis of P. p. asterius revealed
hydrophilic and hydrophobic structures (Fig. 3). The galea stained
with Nile red exposed an overall hydrophobic galea with a microbump pattern. Only the dorsal legulae and chemo- and mechanosensilla were observed on the unstained galea, likely due to
autofluorescence.
4. Discussion
This study revealed two unreported components of proboscis
functionality: 1) butterflies with previously split proboscides can
retain the ability to feed, at least under laboratory conditions, and
2) butterflies might be able to partly reassemble their proboscis
when split. We propose that proboscides of P. p. asterius that
remained partially split after experimental splitting were able to
still feed due to the wettability dynamics (Fig. 3, Lehnert et al.,
2013). The hydrophobic galeae might assist in channeling fluids
to the hydrophilic food canal, which combined with the horizontal
positioning of the proboscides in these studies, could have facilitated the movement of liquids to areas of the proboscis that
remained together where stable liquid bridges could be formed for
the sucking pump to act on (Monaenkova et al., 2012). Butterflies
with proboscides that reassembled might retain the ability to form
stable liquid bridges for fluid uptake, including the regions where
splitting had occurred. These findings could be of importance in
studies that use the butterfly proboscis as a model for the development of microfluidic devices (Tsai et al., 2011).
This study indicated that proboscides of P. rapae are more likely
to reassemble than those of P. p. asterius. Given the structural differences of the dorsal legulae between species, and their method of
overlapping, we propose that the dorsal legulae might play an
important role in proboscis reassembly (Fig. 1D,E). The zipper-like
arrangement of dorsal legulae of P. rapae, which is lacking in the
arrangement of dorsal legulae of P. p. asterius, could facilitate
reassembly. Complete reassembly of the proboscis would require
the hook-shaped ventral legulae (Fig. 1C) to interlink (Krenn, 1997),
which was not examined in this study. Various factors might influence the partial reassembly of proboscides including the presence of saliva, which contains lubricative and enzymatic properties
in insects (Terra, 1990), but requires further analysis in butterflies
(Tokarev et al., 2013). Our observations here warrant further study
of reassembly mechanisms of butterfly proboscides.
Ancient lepidopteran lineages, such as the Eriocraniidae
(Grimaldi and Engel, 2005; Krenn, 2010), possess a short proboscis
with a structural arrangement that would support capillarity (e.g.,
interlegular spaces) (Monaenkova et al., 2012). Observations of
Eriocraniidae have indicated that the galeae become separated
while feeding on water (Kristensen, 1968), which suggests that
capillarity and wettability might play an essential role in fluid
Please cite this article in press as: Lehnert, M.S., et al., Mouthpart separation does not impede butterfly feeding, Arthropod Structure &
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Fig. 3. Confocal microscopy images of structures and overall wettability of proboscides of P. p. asterius. (A) An unstained galea showing the food canal (fc), dorsal legulae (dl),
chemosensilla (cs), and ventral legulae (vl). (B) An unstained galea (ga) shows the autofluorescence of the dorsal legulae and mechanosensilla (ms). (C) The other galea of the same
proboscis shown in (B), but stained with Nile red, reveals the microbump (mb) pattern and overall hydrophobic galea (chemosensilla, cs, also have autofluorescence). All confocal
images were acquired of the drinking region of the proboscis.
uptake processes in these Lepidoptera. Capillary action and
wettability have adaptive value when feeding from exposed liquid
films (Monaenkova et al., 2012; Lehnert et al., 2013), which were
likely present before the availability of pools of nectar associated
with the radiation of the Angiosperms; the coilable proboscis of
Lepidoptera originated during the mid-Mesozoic (Labandeira,
2010). Retaining these physical properties for fluid uptake in
more derived Lepidoptera facilitate feeding on liquid films (e.g.,
rotting fruit) (Monaenkova et al., 2012; Lehnert et al., 2013), and are
beneficial if the proboscis is injured, as indicated in this study.
This study suggests that wild butterflies with split or injured
proboscides might be able to retain some functionality. It is unclear
if butterflies with split proboscides have fluid uptake rates that
significantly deviate from those with fully assembled proboscides
however, this is an aspect of proboscis functionality worth
exploring in future studies. A split proboscis might be a common
occurrence in wild individuals. Lab-reared butterflies, for instance,
often do not properly assemble their proboscis, possibly due to dry
ambient conditions or an obstruction, such as a leg or maxillary
palp, preventing assembly (personal observations). Injured
mouthparts could be a common occurrence in other fluid-feeding
insects important in pollination systems, such as flies (Diptera)
and bees and wasps (Hymenoptera) (Barth, 1991; Willmer, 2011),
where capillarity and wettability also might play an intricate role in
fluid uptake.
Acknowledgments
We thank Meredith Jenkins, Eric Brown, and Valerie Kramer for
assisting in colony rearing, and Peter H. Adler, Shorook Attar, and
Richard Harper for reading an early version of the manuscript. We
also thank the Clemson Microscope Facility for assistance with SEM
imaging. This work was partially supported by a National Science
Foundation [EFRI 0937985].
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Please cite this article in press as: Lehnert, M.S., et al., Mouthpart separation does not impede butterfly feeding, Arthropod Structure &
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