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Evidence that caterpillar labial saliva suppresses infectivity of potential bacterial pathogens.

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138
Musser et al.
Archives of Insect Biochemistry and Physiology 58:138–144 (2005)
Evidence That Caterpillar Labial Saliva Suppresses
Infectivity of Potential Bacterial Pathogens
Richard O. Musser,1* Hyeog S. Kwon,1 Spencer A. Williams,1 C. James White,1
Michael A. Romano,1 Scott M. Holt,1 Shay Bradbury,1 Judith K. Brown,2 and Gary W. Felton3
Salivary enzyme, glucose oxidase (GOX) from the caterpillar Helicoverpa zea, catalyzes the conversion of glucose to gluconic
acid and hydrogen peroxide. Because hydrogen peroxide has well-known antimicrobial properties, we examined whether
caterpillar labial saliva could reduce the infectivity of bacterial pathogens. We examined the effects of caterpillar saliva on the
growth of two bacteria species Serratia marcescens and Pseudomonas aeruginosa. Wells formed in LB agar contained a
solution of salivary gland extract (Sx) and glucose, GOX and glucose, Sx only, GOX only, or glucose only. After 18 h of
incubation, the diameter of cleared bacteria was measured. Wells treated with only GOX, Sx, or glucose showed no measurable area of clearing, while wells treated with GOX with glucose or Sx with glucose had considerable clearing. To determine if
saliva could provide protection to caterpillars in vivo, a surgery was performed on caterpillars that prevented the secretion of
labial saliva. Caterpillars were fed a diet containing either no added bacteria or treated with high levels of S. marcescens or P.
aeruginosa. Caterpillars that could not secrete saliva had significantly higher levels of mortality when feeding on diet treated
with either bacterium than caterpillars that could secrete saliva when feeding on equal levels of bacteria-treated diet. Our
evidence demonstrates for the first time that insect saliva in situ can provide protection against bacterial pathogens and that
the salivary enzyme GOX appears to provide the antimicrobial properties. Arch. Insect Biochem. Physiol. 58:138–144,
2005. © 2005 Wiley-Liss, Inc.
KEYWORDS: bacteria; pathogen; saliva; induced defenses
INTRODUCTION
Insect saliva has numerous functions, such as digestion, water balance, circumventing animal-host
defenses, maintenance of mouth parts, pheromone
production, pathogen transmission, anti-predator
defense, and is suspected to have anti-microbial
properties (Brough, 1983; Ribeiro, 1995; Felton
and Eichenseer, 1999). Only recently has it been
determined that herbivore saliva can suppress induced herbivore defenses of plants analogous to
saliva from blood-feeding arthropods suppressing
1
Department of Biological Sciences, Western Illinois University, Macomb, Illinois
2
Department of Plant Sciences, Center of Insect Science, University of Arizona, Tucson, Arizona
3
Department of Entomology, Pennsylvania State University, University Park, Pennsylvania
the defensive response of their vertebrate hosts
(Kahl et al., 2000; Musser et al., 2002a,b, 2005a,b;
Na and Chenzhu, 2004). Glucose oxidase (GOX),
the salivary component of the caterpillar Helicoverpa zea (Boddie), was the first characterized suppressor of induced plant defenses (Musser et al.,
2002a).
However, insect herbivore survival is not only
dependent on circumventing plant defenses, but
must also avoid the detrimental effects of insect
pathogens. Saliva of mammals and other organisms is widely known to have antimicrobial prop-
Paper presented at the 51st Annual Meeting of the Entomological Society of America, October 2003. Symposium entitled Insect Saliva: An Integrative Approach.
Contract grant sponsor: Illinois Department of Agriculture; Contract grant sponsor: University Research Council, Western Illinois University; Contract grant sponsor:
Center for Insect Science, University of Arizona.
*Correspondence to: Richard O. Musser, Department of Biological Sciences, Western Illinois University, Macomb, IL 61455. E-mail: ro-musser@wiu.edu
© 2005 Wiley-Liss, Inc.
DOI: 10.1002/arch.20031
Published online in Wiley InterScience (www.interscience.wiley.com)
Archives of Insect Biochemistry and Physiology
Caterpillar Saliva Suppresses Bacterial Infectivity
erties. The mandibular salivary glands of the ant
Calomyrmex sp. (Hymenoptera: Formicidae) has
antimicrobial properties (Brough, 1983). GOX, the
major labial salivary constituent of H. zea, converts
D-glucose and oxygen to D-gluconic acid and hydrogen peroxide (H2O2). Hydrogen peroxide has
well-known antimicrobial properties. Honeybees
secrete GOX into honey, which can suppress bacterial growth (White et al., 1963) and also GOX
can prevent bacterial contamination of food products (Dobbenie et al., 1995; Massa et al., 1994). In
our preliminary qualitative experiments, we found
that salivary extract and GOX in the presence of
glucose could prevent the formation of bacterial
colonies such as Serratia marcescens, Pseudomonas
aeruginosa, and Bacillus thuringiensis streaked on LB
agar in Petri dishes. However, no other published
study we are aware of has demonstrated in situ that
insect saliva can provide protection against bacterial insect pathogens. Here we demonstrate for the
first time that H. zea caterpillar labial saliva can
provide protection against bacterial pathogens S.
marcescens or P. aeruginosa, and provide evidence
that GOX is likely the most important antimicrobial component of labial saliva.
MATERIALS AND METHODS
Caterpillar Rearing, Labial Salivary Gland
Preparations, and Bacterial Cultures
Helicoverpa zea neonates were obtained from
North Carolina State University Insectary and were
reared on a corn-based artificial diet at 33°C on a
15-h photoperiod until the 6th instar (Chippendale, 1970; Broadway and Duffey, 1986). Intact labial salivary glands were removed from actively
feeding two-day-old 6th instar H. zea and stored
in chilled distilled water in 1.5 mL micro-centrifuge tubes as described by Eichenseer et al. (1999).
Harvested salivary glands were frozen at –80°C
until used. When needed for experiments, the salivary glands were homogenized and centrifuged at
6,000g to remove tissue and cellular debris. The
supernatant salivary gland extracts were utilized for
all of the experiments, and protein concentration
February 2005
139
was determined with a Bradford assay (Bradford,
1976). For experiments that required 10 mg/ml
protein concentration of salivary gland extract, the
supernatant was lyophilized in a freeze dryer and
reconstituted in water to the desired protein concentration. Purified GOX was purchased from
Sigma-Aldrich (St. Louis, MO). The bacteria S.
marcescens and P. aeruginosa were obtained from
cultures maintained at Western Illinois University
and were maintained on Luria Bertani (LB) agar
or broth following the manufacturer’s directions
(Difco Laboratories, Becton, Dickinson and Company, Sparks, MD).
In Vitro Pour Plate Test
For our in vitro experiments, we used a pour
plate technique with 1 ml suspension of either S.
marcescens or P. aeruginosa in LB broth with an absorbance reading at 600 nm of either 0.02 or 0.06
OD mixed in 9 ml of liquid LB agar. This mixture
was poured on top of 20 ml of solidified LB agar
in a Petri dish and spread evenly across the whole
plate. After the surface solidified, four holes were
punched through the agar to form wells. These four
wells were evenly distributed across the surface and
far enough away from each other to be independent of each other. The wells were treated with 200
µl solution at a final concentration of either 1 or
10 mg/ml of salivary extract or GOX with or without 250 mg/mL glucose. We included a glucose
only treatment. In total, the two concentrations of
S. marcescens or P. aeruginosa (0.02 and 0.06 absorption at 600 nm) were exposed to nine different treatments: 1 or 10 mg/ml salivary gland extract
without glucose; 1 or 10 mg/ml GOX without glucose; 250 mg/ml glucose without salivary extract
or GOX; 1 or 10 mg/ml salivary extract with 250
mg/ml glucose; and 1 or 10 mg/ml GOX with 250
mg/ml glucose.
After the treatments were added to the wells,
the Petri dishes were incubated at 37°C for 18 h.
Then we measured the diameter of inhibited bacterial growth zone and the diameter of the well
was subtracted to provide us with an actual diameter of the bacterial clear zone. The above experi-
140
Musser et al.
ment was replicated four times and the mean of
the diameter of inhibited bacterial growth zone was
determined for each treatment and analyzed with
a Tukey-Kramer Honestly Significant Difference
(HSD) test in a one-way ANOVA with JMP (Sall
and Lehman, 1996).
Caterpillar Surgery for Prevention Labial
Salivary Secretions
To determine whether caterpillar labial saliva in
situ could provide protection against bacterial pathogens, a surgery was used to stop the secretion of
saliva. Our method was to remove the labial salivary glands from a small horizontal incision
through the 2nd abdominal segment (ventral side)
of the 6th instar H. zea caterpillar as described by
Musser et al. (2005a). After removing the salivary
glands, the cuticle was pinched back together with
the caudal portion overlapped by the anterior section of cuticle. The caterpillar remained lying dorsally on the tissue until the cuticle appeared sealed
and the caterpillar became active. These caterpillars could no longer secrete the labial saliva. The
control caterpillars that could secrete labial saliva,
and thus GOX, were wounded in the same manner as the ablated caterpillars; however, after making the incision on the caterpillar, the labial salivary
glands were not extracted. The caterpillars were
given 12 h to recover at room temperature before
being allowed to feed on treated diet in the experiments described below.
Bioassay to Test for Antimicrobial Effect of GOX In Situ
Caterpillars treated in one of the two above-described surgeries were allowed to feed on an artificial diet (corn based) within 12 h after the surgery.
The control diet consisted of sterile artificial diet
(autoclaved) to reduce bacterial and fungal growth.
The diet given to the caterpillars was a 1-cm3 cube
that could be eaten within 24 h and replaced with
another cube until pupation; this was done to reduce excessive and unattended bacterial and fungal growth throughout the experiment. For the
bacteria-treated diet, the caterpillars fed on the
same diet, but with the surface of the cube treated
daily with 100 µl of LB broth containing S. marcescens at an absorption of approximately 0.80 OD
read at 600 nm or P. aeruginosa with an absorption of 1.23 OD read at 600 nm with a spectrophotometer (Beckman UV/VIS, Fullerton, CA).
After this cube of diet was consumed, these caterpillars received more diet treated in the same fashion as needed. In addition, diet that appeared to
have fungal contamination was discarded even if
the cube was not completely eaten. We estimated
that each caterpillar ate approximately 200–500 µl
solution of their respective bacteria treatments. Any
caterpillars that appeared to have fed on diet with
high levels of fungal contamination were also
eliminated from the experiment. Caterpillars that
died within the first 48 to 72 h without obvious
signs of vigor and feeding were considered to have
died due to complications from surgery. Between
78 and 150 caterpillars were tested for each treatment. The final pool of "acceptable experimental"
caterpillars, based on caterpillars that survived surgery and were not feeding on diet with excessive
fungus, was 898 caterpillars. The numbers of caterpillars that died or became moths were monitored throughout the experiment. The status of
death was assigned to caterpillars that no longer
showed any signs of movement or did not successfully emerge into a moth.
Contingency Table Analysis of In Situ Bioassay Tests
In order to determine if there were differences
in the proportion of individuals surviving a given
treatment, distributions of caterpillars that emerged
versus those that failed to emerge among all treatments were compared using a contingency table
analysis (Zar, 1999). Caterpillars without salivary
glands that were fed bacteria were compared to
three groups of caterpillars: (1) caterpillars with
salivary glands surgically removed and fed a diet
without bacteria, (2) caterpillars with mock surgery (caterpillars cut open without removing the
salivary glands) and fed a diet with bacteria, and
(3) caterpillars with mock surgery and fed a diet
without bacteria. Distributions of emerged versus
Archives of Insect Biochemistry and Physiology
Caterpillar Saliva Suppresses Bacterial Infectivity
failure to emerge were compared using the Crosstabs procedure in SYSTAT Version 10.2 (SYSTAT
Software, Inc., Richmond, CA) to determine significant differences in the proportion of survivors
among the treatment conditions. If differences were
found, contingency tables were subdivided (Zar,
1999) to evaluate whether any treatment conditions were not significantly different from each
other. Those treatments were then pooled and run
against groups that were different to clearly identify the treatment groups that produced significantly fewer survivors.
RESULTS AND DISCUSSION
We performed a quantitative experiment to determine the antimicrobial properties of salivary
gland extract and specifically if GOX was the main
antimicrobial component. In this experiment, wells
were formed into agar containing either S. marcescens or P. aeruginosa. Wells that contained GOX
with glucose or salivary gland extract with glucose
had significantly higher levels of clearing than wells
treated with GOX without glucose, salivary extract
without glucose, or only glucose (see Fig. 1). In addition, treatments with GOX without glucose, with
salivary extract without glucose, or only glucose resulted in no measurable level of clearing. We determined a concentration response; the wells filled with
10 mg/mL of GOX or salivary extract with glucose
had more clearing than wells with 1 mg/mL GOX
or salivary extract with glucose. Also the antimicrobial effects of pure GOX with glucose was higher
than salivary gland extract with glucose. This trend
is likely due to GOX levels in salivary extract that
were approximately half of the pure GOX.
Bacterial clearing was less at the higher bacteria
concentrations for GOX with glucose and salivary
gland extract with glucose. Also it is likely that there
is relative effectiveness of caterpillar saliva against
different species of bacteria. We determined that
both GOX with glucose and salivary extract with
glucose generally had slightly more effective clearing against S. marcescens than P. aeruginosa in this
experiment. These experiments provide evidence
that the combination of salivary gland extract that
February 2005
141
naturally contains high levels of GOX with glucose
was necessary for bacterial clearing and that the
caterpillar salivary enzyme GOX was likely the most
important antimicrobial factor in salivary gland
extract.
The saliva from H. zea caterpillars forms H2O2
in the presence of glucose due to GOX activity
(Eichenseer et al., 1999; Musser et al., 2002a,
2005a). Therefore, we suspect that H2O2 production from GOX activity is responsible for the bacterial clearing. However, findings from a similar
experiment by Massa et al. (2001) showed that the
addition of catalase, which rapidly converts H2O2
to water and oxygen, does not inhibit the antimicrobial properties of GOX. The authors concluded
that GOX production of gluconic acid, which lowered the pH, was primarily responsible for the
antimicrobial properties. We performed some preliminary qualitative experiments showing that catalase activity does not inhibit the antimicrobial
properties of salivary gland extracts. Similarly,
lyzosyme does not appear to provide much antimicrobial activity in salivary gland extract because
the presence of glucose was always needed to provide substantial bacterial clearing.
There was some mortality due to the surgeries.
However, our results consistently and conclusively
show that caterpillars that could not secret labial
saliva, regardless of treatment, had significantly
higher levels of mortality (over 50%) when feeding on diet containing S. marcescens (X2 = 8.14, P
= 0.043) or P. aeruginosa (X2 = 22.79, P = 0.0004)
in comparison to caterpillars that could secrete labial saliva (see Fig. 2). In addition, mortality was
not significantly different between caterpillars that
could secrete labial saliva and those that could not
secrete labial saliva on diets not treated with bacteria (X2 = 29.79, P = 0.8660 (S. marcescens); X2 =
1.60, P = 0.500 (P. aeruginosa)). We are not aware
of any studies that have demonstrated that caterpillar saliva in situ can suppress bacterial pathogens. Our study provides evidence that caterpillar
saliva in situ can provide protection against bacterial pathogens and that the salivary enzyme GOX
appears to be an important antimicrobial component. Future studies should include a field study
142
Musser et al.
Fig. 1. Average diameter clearing of LB media coated with
the bacterium S. marcescens (A) and P. aeruginosa (B) and
treated with salivary gland extract or GOX. Treatments
where mean standards error bars did not overlap were significantly different at P < 0.05.
to determine the importance of antimicrobial properties of saliva in a field ecological setting and
whether salivary enzymes provide some protection
against B. thuringiensis. Plant pathogen defenses
appear to be stimulated by insect saliva; caterpillar saliva with GOX and beetle regurgitant with
RNase can stimulate plant pathogen defenses
(Musser et al., 2002a,b). Therefore, GOX may not
only stimulate plant immune-like responses, but
may also directly kill potential bacterial plant
pathogens such as P. aeruginosa and protect the host
plant as a food source for the caterpillar. In addition, we know that GOX in the caterpillar salivary
enzyme can suppress inducible anti-herbivore defenses of plants (Musser et al., 2002a). Several species of caterpillars have GOX present in the labial
Archives of Insect Biochemistry and Physiology
Caterpillar Saliva Suppresses Bacterial Infectivity
143
Fig. 2. Average percentage survival of H.
zea caterpillars that fed on artificial diet
coated with either bacterium S. marcescens
(A) or P. aeruginosa (B) or no bacteria
(A,B). "Mock"-treated caterpillars were
wounded, but their labial salivary glands
remained intact; “ablated”-treated caterpillars had their salivary glands removed
through surgery. Different letters represent
statistically significant differences between treatments at P < 0.05.
salivary glands, which may prove to be an important multifunctional facet in the survival and fitness of caterpillars.
ACKNOWLEDGMENTS
We thank the Illinois Department of Agriculture for funding through a Sustainable Agriculture
Grant. We thank Dr. Vicki L. Chandler at the University of Arizona for providing space in her lab
for some of the experiments performed, and we
thank the Musser Lab members and faculty and
staff at Western Illinois University for their support. Special thanks goes to Drs. Ken Keudell and
Jack Huang of Western Illinois University for bacterial cultures, suggestions, and the use of the their
lab facilities.
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Archives of Insect Biochemistry and Physiology
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