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First published online February 15, 2008
Journal of Experimental Biology 211, 671-677 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.013664
Larvae of the fall webworm, Hyphantria cunea, inhibit cyanogenesis in Prunus serotina
Department of Biological Sciences, State University of New York at Cortland, Cortland, NY 13045, USA
e-mail: fitzgerald{at}cortland.edu
Accepted 19 December 2007
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Summary |
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Key words: Hyphantria cunea, fall webworm, cyanide, Prunus serotina, black cherry, Arctiidae, foregut, pH
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INTRODUCTION |
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-hydroxynitrile
(mandelonitrile), which then dissociates spontaneously or in the presence of a
second enzyme to benzaldehyde and hydrogen cyanide (HCN). Cyanide causes
respiratory failure by interacting with the terminal oxidase in the
mitochondrial respiration chain. Although there have been numerous studies of
cyanogenesis in the leaves, fruits and seeds of black cherry [Hu and Poulton
(Hu and Poulton, 1999
) and
references therein], the fate of the cyanogenic leaf of cherry as it passes
though the digestive tract of invertebrate herbivores has been studied only in
the eastern tent caterpillar, Malacosma americanum
(Fitzgerald et al., 2002).
Tent caterpillars eclose from an overwintered egg mass just as the leaves of
black cherry are unfolding in the spring. The young leaves have the highest
cyanide potential (HCN-p) and the caterpillars prefer them to aged leaves
(Fitzgerald, 1995;
Fitzgerald et al., 2002).
Cyanogenesis occurs in the foregut of the caterpillar but the insect is immune
to the toxin. The toxin appears in the caterpillar's regurgitant and it has
been suggested that the caterpillars may seek out the most cyanogenic leaves
to enhance the defensive value of the regurgitant
(Peterson et al., 1987). The
ultimate fate of cyanide in the tent caterpillar is unknown, but significantly
decreasing quantities are found in the bolus as it passes along the digestive
tract, and the fecal pellets are largely devoid of the chemical
(Fitzgerald et al., 2002).
The fall webworm, Hyphantria cunea, is an ecological equivalent of
the eastern tent caterpillar, eclosing from eggs laid on the leaves of cherry
in early July, a month or more after the last of the tent caterpillars has
finished the feeding phase of its life cycle. Webworms feed until
mid-September, then pupate in the soil. Although previous studies indicate
that the cyanogenic potential of cherry leaves declines precipitously as they
age (Smeathers et al., 1973),
preliminary studies indicated that even the senescent leaves that mature
webworms consume in September have a significant HCN-p. The present study was
undertaken to determine the amount of the toxin in the caterpillar's diet. The
cyanide content of the caterpillar's defensive regurgitant, gut bolus, and
fecal pellets (frass) were measured to determine the fate of cyanide as it
passes though the alimentary tract of the insect. The effect of gut pH on
cyanogenesis was also assessed.
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MATERIALS AND METHODS |
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Analytical procedures
Cyanide assays
Samples were prepared for analysis using techniques modified from Brinker
and Seigler (Brinker and Seigler,
1992). Samples of leaf and insect material were ground in a micro
tissue-grinder in approximately 0.5 ml of 0.1 mol l–1
phosphate buffer (NaH2PO4.H2O, pH 6.8),
chilled in an ice bath to approximately 4°C unless otherwise noted.
Material was ground until fully homogenized, typically in 5–10 s. The
buffer containing the sample material was immediately transferred to the outer
chamber of an 18 ml Warburg flask, the center well of which was preloaded with
0.2–0.3 ml of 1 mol l–1 NaOH. The sealed flask was
placed in an oven and the material incubated at 35°C for a minimum of 18
h. HCN volatilizing from the sampled material was trapped in the center well
as CN–. At the end of the incubation period, the contents of
the center well were drawn off and stored in a sealed microcapsule at 2°C.
The rationale for this procedure and a comparison of the method with that
involving freezing samples in liquid nitrogen is given in Fitzgerald et al.
(Fitzgerald et al., 2002).
Samples were analyzed with a Dionex Ion Chromatograph (Sunnyvale, CA, USA) with a GP 50 gradient pump and ED 40 electrochemical detector. The machine was fitted with a 4 mm AG9-HC guard column and a 250 mmx4 mm AS7 analytical column. The eluent consisted of 41 g NaC2H3O2, 5 ml ethylenediamine, and 16.5 ml of 6.0 mol l–1 NaOH per liter, delivered at a flow rate of 1.0 ml min–1. 25 µl of the NaOH center-well-solution were injected into the apparatus. CN– eluted at approximately seven minutes (Fig. 1). A standard curve with known concentrations of CN– was prepared prior to each run.
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Experimental procedures
Effect of experimental procedures on the HCN-p of leaves
Initial studies were conducted to determine if either the method of
handling leaf samples or the feeding damage to leaves inflicted by
caterpillars during the course of the studies affected the results. In mid
June 2002, five branch tips bearing 4–5 terminal leaves each were cut
from a P. serotina tree and placed individually in water-piks. The
samples were brought indoors and immediately prepared for distillation by
cutting a section (3.5±0.1 mg) from one randomly chosen leaf from each
branch. The samples were ground and distilled in Warburg flasks. A second
sample was taken from each of the previously sampled leaves after the branches
had been in the water-piks for 24 h. These samples (4.0±0.9 mg) were
prepared and distilled in the same manner as described for freshly picked
leaves. The cyanide content of the fresh leaves and leaves held in water-piks
for 24 h was determined and statistically compared.
In early September 2002, six branch tips, each bearing 4–5 terminal leaves, were cut from six different cherry trees. The branches were immediately placed in separate water-piks and a sample (3.9±0.3 mg) taken for distillation from a randomly selected leaf on each branch as described above. A fine pin was placed in the petiole of each of the sampled leaves to mark it and each branch was placed in a cage with 20 fall webworms, which were allowed to feed ad libitum overnight. The next morning at 09.00 h, a second sample (3.8±0.3 mg) was cut from each of the marked leaves and prepared for distillation. All of the marked leaves had significant feeding damage from the caterpillars. Both sets of samples were analyzed for cyanide content and compared statistically to determine if feeding altered the cyanogenic potential of the leaves.
HCN-p of leaves and the cyanide content of the regurgitant, the bolus and frass
Studies were conducted to determine the cyanide content of regurgitant, the
bolus and frass derived from leaves of known HCN-p. In 2002, six whole field
colonies were brought into the laboratory and placed separately on freshly
cut, undamaged branches of P. serotina obtained from six different
trees. The colonies were allowed to feed overnight. At 07.00 h the next
morning, a section was cut from a leaf that the caterpillars had fed on
overnight from each of the six branches. These samples were immediately
prepared for distillation as described above. At the same time, caterpillars
were selected at random from each colony and stored in a freezer at
–23°C. These caterpillars were subsequently analyzed to determine
the cyanide content of the boluses of the foregut (N=12) and midgut
(N=9). In preparing gut bolus samples for distillation, the gut
contents were dissected from frozen caterpillars on a cold table and
transferred before thawing to the tissue grinder.
A 20 µl sample of regurgitant was collected from caterpillars from each of the six colonies at 07.00 h. To collect the regurgitant, a caterpillar was quickly pulled from the nest, causing it to express a droplet from its mouth. The droplet was drawn by capillary action into a micropipette. Some caterpillars produced little or no regurgitant and it was necessary to collect droplets from several caterpillars from each colony to obtain a single 20 µl sample. Because the purpose of this study was to assess the defensive value of the droplet at the time it was produced, 20 µl samples were injected directly into microvials filled with 0.3 ml of 1 mol l–1 NaOH. Centrifuged (8422 g, 10 min) samples of this solution were then injected into the chromatograph to determine their cyanide content.
Each of the six colonies produced copious quantities of frass overnight, and samples of this material were collected from paper sheets placed under the branches after the pellets had dried, then stored at –23°C. Fifty-seven separate analyses of these dry pellets were undertaken to determine their cyanide content. The mean mass of pellets analyzed was 1.0±0.4 mg. For 24 of these analyses, the mean cyanide content of three pellets ground together was determined. For 10 of these analyses, the mean cyanide content of two pellets ground together was determined. For the remaining 23 analyses, single pellets were analyzed.
Components of the 2002 study were replicated during subsequent field seasons. Five field colonies collected in early September 2005 were brought into the laboratory and each was allowed to feed overnight on a branch of P. serotina. Branches were collected from five different trees. The next morning at 08.00 h, two leaf samples were taken from each of three trees and one each from the other two trees to determine their cyanide content. Samples were taken from near the tips of branches. Four fecal pellets (1.4±0.1 mg) that had fallen onto paper sheets placed under each colony were collected at random when dried, then analyzed individually as described above to determine their cyanide content. The procedure used in 2002 to determine the cyanide content of the defensive regurgitant was also replicated in September 2007 by collecting 10 samples (5 µl) of regurgitant from 6th and 7th instar caterpillars that had just fed.
Analysis of data derived from the above studies indicated that the webworm was inhibiting cyanogenesis, leading to additional studies in 2006 and 2007. A colony of webworms developing on P. serotina was collected from the field in late August 2006 and maintained overnight on branches in the laboratory. The next morning, following overnight feeding, 10 samples of three pellets of frass that had fallen out of the branch overnight were collected. Half of these samples were distilled in Warburg flasks. The other five samples were ground in microvials containing NaOH, then centrifuged and the supernatant analyzed for cyanide. The combined boluses of the foreguts and midguts of eight caterpillars were removed from caterpillars killed in a freezer, then dissected while still frozen on a cold table. The boluses were ground in microvials containing NaOH. The formed fecal pellets found in the rectums of seven caterpillars were removed and treated in a similar fashion. These bolus and fecal pellet samples were centrifuged (8422 g, 10 min) and the supernatant analyzed for cyanide.
To determine if the addition of β-glycosidase to frass during the distillation process would increase the yield of cyanide, seven samples of pellets were obtained from caterpillars from different colonies and ground separately with a mortar and pestle. From each of these samples, 3 µg quantities were distilled in either standard buffer or in buffer to which 5–10 units of β-glycosidase were added (Sigma-Aldrich product number G4511).
Loss of HCN to atmosphere while feeding
A study was conducted to obtain an estimate of the fraction of HCN lost to
the atmosphere while leaves were being consumed by caterpillars. Ten 6th
instar webworm caterpillars were housed in a sealed chamber (8 cm long x
2 cm diameter) with a half of a leaf of P. serotina of known mass. A
tube leading into the chamber carried an air stream to the bottom of the
chamber, and another tube at the top carried the air to a capture chamber of
the same dimensions. Air entering the capture chamber was directed by a tube
to the bottom of the chamber and bubbled through 10 ml of 1 mol
l–1 NaOH. Glass beads were used to cause the rising air to
take a circuitous route through the NaOH, increasing the capture rate. Air
exited the chamber through an opening at the top. The air flow rate was
adjusted to 2–3 ml per min. The caterpillars were allowed to feed on the
leaf for 24 h, during which the entire leaf was consumed. The NaOH was
retrieved and analyzed for cyanide as described above. At the same time as
this study was being conducted, effluent from the other half of the leaf was
collected using an identical apparatus except that the feeding chamber was
used as an extraction chamber. The extraction chamber was immersed in a water
bath at 35°C. The purpose of this was to determine the total HCN-p of the
leaf. In preparation, the leaf section was placed in the bottom of the
extraction chamber and the chamber immersed in liquid nitrogen. The frozen
leaf was then ground to a powder. Approximately 1 ml of phosphate buffer was
added and the chamber immediately sealed. An air stream, adjusted to the same
rate as that used in the chamber housing the caterpillars, was then passed
though the chamber housing the leaf and then into the capture chamber for 24
h. The process was replicated five times with leaves from different trees and
different groups of caterpillars.
pH of leaves, regurgitant and the bolus
Approximately 0.25 g of a P. serotina leaf collected in September
was ground with a mortar and pestle in 10 ml of distilled water. The pH of the
slurry was then measured to determine the pH of the leaf tissue. Five leaves
obtained from the same tree were measured in this way. The pH of the
regurgitant was determined immediately after 6th or 7th instar caterpillars
were fed the leaves of this same tree and compared to the pH of the
regurgitant of the same caterpillars starved for 24 h. To obtain regurgitant,
a caterpillar was held between the thumb and forefinger while the opening of a
5 µl micropipette was placed against its mouthparts. This caused the
caterpillar to regurgitate and the material to be pulled into the pipette by
capillary action. The collected regurgitant was immediately injected into a 2
mm-long glass sample tube glued horizontally to the top of a 30 mm-long glass
tube of the same diameter forming a `T', the base of which was fixed to a
wooden block to secure it in an upright position. The sample tube had an
external diameter of 1.4 mm and an internal diameter of 0.9 mm. The pH of the
regurgitant was measured by inserting the pH probe into one end of the sample
tube and the reference electrode into the other. The sample tube was observed
at the low power of a dissecting microscope to facilitate the placement of the
pH electrodes. Using this procedure, it was possible to measure the pH of
samples with a volume of approximately 2 µl. Samples of regurgitant were
drawn from 22 caterpillars that had been starved for 24 h and from 22
caterpillars that had just fed to repletion.
The pH of the contents of the foregut, midgut and hindgut were measured for
6th or 7th instar caterpillars given constant access to host leaves.
Caterpillars were killed by placing them in a freezer for 15 min, then
dissected to reveal the entire extent of the gut. Hemolymph was blotted away
and the gut probed with the electrodes under a dissecting microscope. Since
the pH of the gut varies along its extent
(Dow, 1992) maximum values were
recorded for each compartment of the tract. The pH of the guts of 10
caterpillars was measured.
Effect of gut pH on cyanogenesis
The influence of pH on cyanogenesis was investigated by following the same
procedures outlined above to determine the HCN-p of leaves except that the pH
of the buffer was varied. Leaf samples were ground and distilled in pH 7, 8,
9, 10 and 11 Hydrion® buffers. Five 3–4 mg samples were cut from
adjacent sites on the same leaf, then each was ground and distilled in one of
the buffers. A total of three leaves was treated in this manner.
Rate of food intake
To obtain an index of the rate of food intake, six 6th or 7th instar
caterpillars that had been deprived of food for 24 h were weighed and then
presented with a host leaf in separate containers and allowed to feed ad
libitum at room temperature (initial mass 0.160±0.012 g). The
duration of the feeding bout of each caterpillar was determined by direct
observation. The mass gained by each caterpillar was determined immediately
after it finished feeding. In addition, five 6th or 7th instar caterpillars
were each housed with a host leaf and video recorded at 1 frame
s–1 for an average of 10.7±2.4 h to obtain a record of
the frequency and duration of feeding bouts of individuals allowed to feed
ad libitum over extended periods. Caterpillars were maintained under
a 14 h:10 h L:D photoperiod regime at room temperature. Red light was used
during the scotophase to illuminate the foraging arena.
Extent of food processing
The fecal pellets of webworms fed P. serotina leaves range from
black though brown to bright green, suggesting that the extent to which food
is processed as it transits the digestive tract varies. Pellets of these
varying colors were rehydrated and their contents compared with respect to
apparent degradation. The cyanide contents of 42 green pellets were also
compared to that of 23 dark (brown to black) pellets.
Susceptibility of caterpillars to cyanide poisoning
The susceptibility of fall webworms to cyanide poisoning was determined by
placing single caterpillars in a chamber filled with the fumes of cyanide
liberated by grinding approximately 0.5 g of the young leaves of black cherry
in 2 ml of pH 6.8 phosphate buffer
(Fitzgerald et al., 2002). The
insects were observed for one hour and symptoms of cyanide poisoning
recorded.
Statistics
Statistical analyses as detailed below were carried out with ProStat (Poly
Software International, Pearl River, NY, USA) and SigmaStat statistical
software (Systat Software Inc., Chicago, IL, USA). All values are given as the
mean ± s.e.m.
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RESULTS |
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).
HCN-p of leaves and the cyanide content of the frass, regurgitant and the bolus
Wet leaf samples collected from branches fed on by fall webworms in the
laboratory in the fall of 2002 contained 1522±234 p.p.m. cyanide. Dry
fecal pellets collected from caterpillars feeding on these same leaves
contained 2433±265 p.p.m. Wet leaf samples collected in the fall of
2005 contained 1592±276 p.p.m. cyanide. Dry fecal pellets collected
from larvae that had fed on these leaves contained 2868±552 p.p.m.
cyanide. There was no significant difference in the amount of cyanide
recovered when pellets were distilled with buffer alone or buffer to which
β-glycosidase was added (ANOVA, F=0.11, P=0.74). Fecal
pellets ground in buffer and distilled in Warburg flasks in 2006 yielded
2262±360 p.p.m. cyanide. Fecal pellets collected at the same time from
this same colony but extracted directly in NaOH yielded 117±75 p.p.m.
cyanide (Fig. 1).
Regurgitant collected in NaOH in 2002 from caterpillars following an overnight bout of feeding yielded 10±4 p.p.m. cyanide. Regurgitant collected in the same manner in 2007 from caterpillars that had just fed yielded 8±2 p.p.m. cyanide.
The foregut and midgut boluses of caterpillars distilled in buffer in 2002 contained 141±25 p.p.m. (N=12) and 137±38 p.p.m. (N=9) cyanide, respectively. Of the eight foregut and midgut boluses of caterpillars extracted directly in NaOH in 2006, two had no detectable cyanide and six had 0.4±0.1 p.p.m. Six of seven hindgut boluses extracted directly in NaOH contained 44±18 p.p.m. cyanide.
Loss of HCN to atmosphere while feeding
When caterpillars were fed leaves in enclosed chambers, 10.4±1.1% of
the HCN-p of the leaves was lost to the atmosphere. Simultaneous attack by 10
caterpillars left the partially consumed leaves with many tattered edges, and
loss to the atmosphere is likely to be largely attributable to HCN escaping
from these damaged surfaces.
pH of leaves, regurgitant and the bolus
The pH of five leaves from a P. serotina tree measured in
September was 5.9±0.1. The pH of the regurgitant of caterpillars
starved for 24 h was 12.0±0.1 while that of these same caterpillars
allowed to feed to repletion on leaves of this tree was 12.0±0.0. The
maximum pH of the bolus of caterpillars allowed to feed to repletion was
11.8±0.1 for the foregut, 11.0±0.1 for the midgut and
10.2±0.1 for the hindgut. The three values are significantly different
from each other (Kruskal-Wallis ANOVA, H=25 and SNK subtest
P>0.05). There was no significant difference between the pH of
regurgitant collected from either starved or fed caterpillars and that of the
foregut bolus (ANOVA, F=0.75, P=0.48).
Effect of gut pH on cyanogenesis
Cyanogenesis was greatest when leaf tissue was ground and distilled in
buffer at pH 7–8 (Fig.
2). Cyanogenesis declined slightly at pH 9, and markedly at pH 10,
to approximately 18% of its value at pH 7. No cyanogenesis occurred at pH
11.
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Extent of food processing
In one randomly chosen sample of 100 pellets, 61% were green and the
remainder black to dark brown. Green pellets contained significantly more
cyanide (2546±304 p.p.m.) than dark pellets (1342±370 p.p.m.)
(Mann–Whitney Rank Sum Test, T=515, P<0.001). When
rehydrated, dried pellets were shown to consist of fragments approximately
0.25 mm wide by 0.5–2 mm in length.
Susceptibility of caterpillars to cyanide poisoning
All four caterpillars subjected to the fumes of cyanide succumbed to the
poison. The first indication of poisoning – regurgitation –
occurred about two minutes after the caterpillars were placed in the chamber.
After approximately 5 min, there was pronounced trembling of the thoracic
prolegs accompanied by trembling of the head, followed, a few minutes later,
by trembling of the abdominal prolegs. 10–12 min after being placed in
the chamber, the caterpillars doubled over, in some cases so far as to bring
the head into contact with the last set of abdominal prolegs. Thereafter, the
caterpillars remained motionless. The caterpillars were removed from the
chambers after one hour and observed over the next 24 h. Two showed no
recovery while the other two showed partial recovery but were permanently
injured and eventually died.
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), the trees sampled did not decline as precipitously.
Smeathers et al. reported a mean HCN-p of 2472 p.p.m. for P. serotina
leaves at the beginning of the growing season in mid-April but their data
indicate that the mean HCN-p of leaves declined to less than 1000 p.p.m. by
mid May and to less than 500 p.p.m. by September
(Smeathers et al., 1973).
Fitzgerald et al. found a mean HCN-p of 3032 p.p.m. for new, unfolding, tip
leaves collected from the Cortland area in June of 2001
(Fitzgerald et al., 2002) but
they did not measure the HCN-p of fall leaves. Peterson et al.
(Peterson et al., 1987)
reported that the tip leaves of black cherry sampled in May and June on Long
Island, NY, USA had a HCN-p of 1845 p.p.m. Santamour measured cyanide in black
cherry from June to September in Washington, DC, USA and reported a mean HCN-p
of 2644 p.p.m. (Santamour,
1998). Thus, these previous reports indicate that different
populations of P. serotina trees vary significantly in both the
maximum HCN-p and the rate at which cyanogenic potential declines as the
season progresses. Indeed, leaves collected from P. serotina trees on
2 August 2002 from nearby Tompkins County in conjunction with another study
contained only 1068±169 p.p.m. cyanide (T.D.F., personal
observation).
Wheeler and Bennington found that feeding by the larvae of Agraulis
vanillae on the sun-exposed leaves of Passiflora incarnata
resulted in a decrease in the HCN-p of the leaves, while the cyanogenic
potential of both damaged and intact shaded leaves did not differ
(Wheeler and Bennington,
2001). They attributed the decline in HCN-p in sun-exposed leaves
to an overall deterioration of the leaf and possible autotoxic effects of
released cyanide following herbivory. The present study shows that damage
inflicted on host foliage due to feeding by the webworm under laboratory
conditions did not induce a short-term localized change in the cyanogenic
potential of the plant and is consistent with the results of other studies of
the influence of herbivory on the HCN-p of leaves. Gleadow and Woodrow found
no difference in the concentration of cyanogenic glucosides in the intact and
mechanically wounded leaves of Eucalyptus cladocalyx
(Gleadow and Woodrow, 2000).
In their study of the bird cherry-oat aphid, Rhopalosiphum padi,
Leszczy
ski et al. found that sustained feeding by the insect on
Prunus padus had no significant effect on the cyanogenic glycoside
content of the leaves (Leszczyski
et al., 2003).
The present study shows that the webworm is vulnerable to cyanide poisoning
yet feeds with no apparent ill effects on cherry leaves having significant
HCN-p. Three mechanisms that enable caterpillars to feed on cyanogenic plants
without harm have been described. Rhodanese (thiosulfate sulfur transferase)
is widely distributed in insects and may be used by some species to catalyze
the conversion of HCN to thiocyanate (Conn,
1979). Long and Brattsten
(Long and Brattsten, 1982) and
Beesley et al. (Beesley et al.,
1985), however, assayed for rhodanese in 55 species of insects and
found that it occurs at much lower levels than in vertebrates and suggested
that it plays a minor role in cyanide detoxification in insects. Witthohn and
Naumann (Witthohn and Naumann,
1987) and Meyers and Ahmad
(Meyers and Ahmad, 1991)
conducted studies suggesting that detoxification of cyanide by
L-3-cyanoalanine synthase may be the primary mechanism employed by insects. In
the southern army worm, Spodoptera eridania, and the cabbage looper,
Trichoplusia ni, L-3-cyanoalanine synthase is associated with the
mitochondria (Meyers and Ahmad,
1991). Engler et al. reported that Heliconius sara
possesses an enzyme system that enables the insect to convert a cyclopentenyl
cyanogen derived from host Passiflora leaves to a compound in which a
thiol group replaces the cyanogen's nitrile group
(Engler et al., 2000). This not
only precludes the production of cyanide but also allows the released nitrogen
to be metabolized to useful compounds.
None of these previously described mechanisms accounts for the ability of the webworm to feed on cherry. Two interrelated factors enable the webworm to process cyanogenic leaves without succumbing to the toxin: high foregut alkalinity and a capacious foregut. Cyanogenesis in crushed leaves of P. serotina is strongly inhibited at pH 10 and completely inhibited at pH 11 (Fig. 2). The maintenance of gut alkalinity in excess of these values in the presence of the bolus largely suppresses cyanogenesis. This is supported by the relatively small quantities of cyanide recovered from NaOH extracts of regurgitant and the boluses of the fore and midguts compared to the amounts in the ingested leaf. Moreover, frass extracted directly in NaOH yielded only 5% of the cyanide obtained from distilled frass, indicating that the cyanogen survives gut transit (Fig. 1). The presence of significantly greater quantities of cyanide in the hindgut bolus compared with the anterior gut compartments is consistent with the lower pH of this compartment.
Foregut capacity coupled with a relative low rate of ingestion may enable
the webworm to maintain an alkaline gut in the presence of the bolus. The
foregut of the webworm constitutes 49% of the total length of the alimentary
tract, proportionately the longest of any of 33 species of caterpillars
measured by Grant (Grant,
2006); mean=19%. By contrast, the foregut of the eastern tent
caterpillar constitutes 21% of the total gut length
(Grant, 2006). Following a
single bout of feeding, the foregut of the tent caterpillar is fully packed
with leaf fragments to the extent that the bolus strains its capacity
(Snodgrass, 1922). The pH of
the foregut bolus of the tent caterpillar is between 6 and 7 (T.D.F.,
unpublished data) and both the regurgitant
(Peterson et al., 1987) and
the foregut bolus exude the strong odor of benzaldehyde and contain levels of
cyanide consistent with unrestrained cyanogenesis
(Fitzgerald et al., 2002). The
present study shows that during the 6th and 7th larval stadia, webworms
alternate brief periods of feeding and rest. Despite the near constant intake
of food, observations of starved caterpillars allowed to feed to repletion
indicate that the amount ingested during these feeding bouts is small. Thus,
the capacious foregut of the caterpillars is much more loosely packed with
food than that of the tent caterpillar, the bolus is watery and it bears no
trace odor of benzaldehyde. The production of large quantities of green fecal
pellets also indicates rapid passage of the bolus through the webworm's
alimentary tract. The presence of nearly twice as much cyanide in green
pellets than in dark pellets suggests that the former move through the gut
more rapidly and may be less subjected to digestive processes that deplete the
cyanogen or its enzymes.
There are few data regarding the amount of cyanide occurring in the fecal
pellets of insects that feed on cyanogenic plants. Fitzgerald et al.
(Fitzgerald et al., 2002)
found that the frass of eastern tent caterpillars fed the highly cyanogenic
young leaves of P. serotina contained 63–85 p.p.m. cyanide.
Alonso-Amelot et al. determined that the frass of Helioconius erato
and Spodoptera frugiperda fed the cyanogenic plant Passiflora
capsularis contained an average of approximately 500 and 1000 p.p.m.,
respectively, and that, as in the case of the tent caterpillar, most of the
cyanide did not survive gut transit
(Alonso-Amelot et al., 2006).
Although the frass of the webworm contains relatively large quantities of
cyanide compared with these species, the present study indicates that a
significant proportion of the HCN-p of the leaf is not recoverable as cyanide
in the caterpillar's frass. P. serotina leaves collected in late
summer lost an average of 58% of their mass when oven dried. The aeration
study showed that an average of 10.5% of the HCN-p of the leaf is lost to the
atmosphere as the caterpillars feed. Schroeder and Malmer found that the
webworm feeding on P. serotina had an assimilation of efficiency of
38% (Schroeder and Malmer,
1980). When loss of mass due to assimilation and drying and loss
of HCN to the atmosphere are considered collectively, dry fecal pellets of
webworms would be expected to have approximately 3.5 times as much cyanide per
unit mass as the leaf tissue they ingest if all of the ingested cyanogen were
transferred to the frass. The frass of caterpillars fed leaves collected in
2002 with a mean HCN-p of 1522 p.p.m. had 45.6% of the expected value while
those fed leaves collected in 2005 with a mean HCN-p of 1592 p.p.m. had 51.5%.
Although some cyanogensis occurs in the hindgut, this loss may be largely
attributable to the destruction of prunasin since the addition of
β-glucosidase to the Erlenmeyer flasks during the distillation process
did not significantly increase the yield of cyanide.
Alkalinization of the midgut in larval Lepidoptera is due to the secretion
of K+ in the gut lumen by epithelial goblet cells
(Dow, 1984;
Dow, 1992;
Moffett and Koch, 1992). As
Dow (Dow, 1992) noted, the
development of microprobes for measuring pH revealed that the midguts of
caterpillars are markedly more alkaline than earlier reports suggested. Thus,
while Berenbaum's (Berenbaum,
1980) survey of the literature indicated that the midgut pH of 60
species of caterpillars ranged from 7.0 to 10.3, with the majority <9.0,
measurements with microelectrodes showed that caterpillar midguts have the
highest pH values of any biological system. Dow (Dow, 1981) recorded a peak pH
of 12.0 in the midgut fluids of the death's head hawkmoth, Acherontia
atropos, while Schultz and Lechowicz recorded pH values as high as 12.4
in the midgut of the gypsy moth, Lymantria dispar
(Schultz and Lechowicz, 1986).
Since the midgut is the site of digestion, interest in the effect of gut pH on
the bolus has focused almost exclusively on this compartment of the alimentary
tract and there are few data on the pH of the foregut. pH values reported for
the webworm in the present study appear to be the highest yet recorded for the
regurgitant and foregut fluids of any insect. As in this study, Krishnan et
al. found that pH of the foregut of the last instar larva of the beetle
Leptinotarsa decemlineata (7.0) exceeded that of the midgut
(5.4–6.3) (Krishnan et al.,
2007) but did not speculate on the physiological basis for this.
In the webworm, lower midgut alkalinity may be a consequence of both the
digestive process and food packing, although neither of these possibilities
was explored in the present study.
It has been proposed that the high midgut pH of caterpillars may allow them
to tolerate plant allelochemicals that can interfere with digestion
(Berenbaum, 1980;
Govenor et al., 1997) and
facilitate the extraction of nutrients from plant material
(Felton and Duffey, 1991). In
the webworm, the maintenance of a highly alkaline foregut environment inhibits
cyanogenesis at the point where food particles first enter the caterpillar's
body and allows the caterpillar to feed on an otherwise toxic plant. However,
with 400 recorded host plant species
(Wagner, 2005), the webworm
has one of the widest host ranges of any caterpillar, and the significance of
high foregut alkalinity in this broader context is unknown. In itself, it is
unlikely to account for the insects' broad food base. Schultz and Lechowicz
(Schultz and Lechowicz, 1986)
and Appel and Maines (Appel and Maines,
1995) reported a mean pH of 7.8 for the empty foregut of the
caterpillars of L. dispar, a generalist caterpillar with an even
larger host range than the webworm
(Wagner, 2005).
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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  K. Phillips FALL WEBWORMS DICE WITH DEATH J. Exp. Biol., March 1, 2008; 211(5): iii - iii. [Full Text] [PDF]
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