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Ecology

Yeasts associated with the infrabuccal pocket and colonies of the carpenter ant Camponotus vicinus¹

     Department of Wood Science and Engineering, Oregon State University, Corvallis, Oregon 97331

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

After scanning electron microscopy indicated that the infrabuccal pockets of carpenter ants (Camponotus vicinus) contained numerous yeast-like cells, yeast associations were examined in six colonies of carpenter ants from two locations in Benton County in western Oregon. Samples from the infrabuccal-pocket contents and worker ant exoskeletons, interior galleries of each colony, and detritus and soil around the colonies were plated on yeast-extract/ malt-extract agar augmented with 1 M hydrochloric acid and incubated at 25 C. Yeasts were identified on the basis of morphological characteristics and physiological attributes with the BIOLOG^® microbial identification system. Yeast populations from carpenter ant nest material and material surrounding the nest differed from those obtained from the infrabuccal pocket. Debaryomyces polymorphus was isolated more often from the infrabuccal pocket than from other material. This species has also been isolated from other ant species, but its role in colony nutrition is unknown.

Key words: Camponotus vicinus, carpenter ants, Debaryomyces polymorphus, infrabuccal chamber, yeast

	INTRODUCTION

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Although carpenter ants are important structural pests in the northern hemisphere, little is known about their relationships with microorganisms, particularly those found in their digestive tracts. Many insects, particularly xylophageous insects, have associated bacteria, fungi or protozoa that provide various nitrogenous compounds and essential vitamins to their hosts (Gusteleva 1975). Although carpenter ants are not xylophageous, all Camponotus species harbor bacterial associates in their midintestines (Buchner 1965).

The digestive biology of the carpenter ant is of interest because a filtering device anterior to the crop, the infrabuccal pocket, prevents worker ants from ingesting solid food particles (Eisner and Happ 1962). The infrabuccal pocket, a spheroidal pouch found on the ventral portion of the buccal tube (Forbes 1938), keeps the crop free of particulate matter greater than 150 µm diam (Eisner and Happ 1962). The carpenter ants thus obtain their nutrition from a liquid diet. Because the brood is fed on regurgitated food, the entire protein requirements of a colony must be supplied by hemolymph and water-soluble proteins that the ants obtain from live and dead insect prey (Ayre 1963). Carpenter ants obtain carbohydrates from aphid honeydew and from dead and dried insects, but during periods of brood production in early spring and midsummer, they feed predominantly on insect proteins.

Although the microbial communities of the alimentary tracts of some insects have been studied in great detail, almost nothing is known about the micro-organisms living in ants. Only recently have investigators looked at the microbial communities in carpenter ants. Hansen et al (1999) found gram-negative bacteria and several unidentified yeast species associated with the infrabuccal pocket of carpenter ants and suggested that they might have some digestive function. Carpenter ants might obtain free amino acids or essential vitamins from associated micro-organisms, or microorganisms in the infrabuccal chamber might be active in the extracellular digestion of large particulate matter that otherwise cannot pass into the ant’s midgut.

Few studies have examined the fungal associates of Camponotus, and none have examined yeasts. Ayre (1963) found large amounts of crop amylase in the midgut of carpenter ants and concluded that consumption of fungal hyphae might account for the presence of this enzyme. However, no research has surveyed the yeast flora associated with these ants. Yeasts also have been found in nest material of the forest ant Formica rufa, suggesting that these yeasts might play a role in colony nutrition (Golubev and Bab’eva 1972a, Smith 1944). Yeasts were found in great numbers only in active, habitable ant mounds, and this led the investigators to believe there might be a relationship between the yeasts and the ants because the yeasts appeared to be harmless. The actual relationship, if any, was never examined (Golubev and Bab’eva 1972a, b). A more recent investigation of the yeast flora of the fire ant Solenopsis invicta suggested that certain yeasts were prevalent in colonies and in larvae of this species and colonies harboring yeasts tended to be more vigorous (Ba et al 2000, Ba and Phillips 1996).

Developing a better understanding of the associations between carpenter ants and micro-organisms can help elucidate the role of Camponotus in forest ecosystems and might lead to the development of improved control strategies.

In a preliminary investigation, we used scanning electron microscopy (SEM) to examine the contents of the infrabuccal pockets of carpenter ants and found reticulated cells similar to those that Golubev and Bab’eva (1972a) identified as Debaryomyces polymorphus. This finding led us to examine during 1 yr in two locations in western Oregon yeasts associated with C. vicinus. The results of both the SEM work and the yeast survey are presented here.

	MATERIALS AND METHODS

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Microscopy. – Infrabuccal pocket contents of six Camponotus vicinus workers from one colony in MacDonald-Dunn Research Forest near Corvallis, Oregon, were examined by SEM. Ant heads were removed, and the dorsal portion of each head was dissected to expose the infrabuccal chamber. The dissected heads were prefixed for 1 hr at room temperature in a solution containing 0.1 M cacodylate (pH 7.3), 2% paraformaldehyde, 2.5% glutaraldehyde, 1% sucrose and 0.16 M CaCl₂, and dehydrated in a graded ethanol series (M. Nesson pers comm). The heads were critical-point dried, and the exposed infrabuccal chambers were ruptured for observation of microorganisms. The specimens were sputter-coated with gold palladium and examined with an AMR 1000A SEM (accelerating voltage 20 kV, tilt angle 30°, working distance 12 mm).

Yeast isolation from colonies and workers. – Six Camponotus vicinus colonies located within 30 km of Corvallis, Oregon, were sampled in Jan, Jul and Oct 2000 for the presence of yeasts. We collected colonies from two locations, one in MacDonald-Dunn Research Forest and the other from a farm 16 km west of Philomath. Ant colonies were found in logs and under detritus in open areas at both locations. The infrabuccal pockets of 10 medium-size workers from each colony were sampled after the ants had been examined for surface microorganisms. The ants first were placed in a sterile test tube with 10 mL of sterile PBS; the tube was shaken 1 min with the ants in the solution and then diluted 5x with phosphate buffer solution (PBS) (Hansen et al 1999). Twenty-five µL of this extract was plated on yeast-extract/ malt-extract agar augmented with 1 M hydrochloric acid (7 mL/L), one plate per dilution, and incubated at 25–27 C and examined every 2 d during a 14 d period for evidence of microbial growth. This procedure was performed for three groups of 10 ants per colony.

Infrabuccal cavity contents were obtained by removing the heads of the 10 PBS-washed ants and placing them in 0.05% hypochlorite solution for 60 s to sterilize the exoskeleton. Surface-sterilized heads were dissected under a compound microscope in a laminar flow hood to minimize contamination. The contents of the excised infrabuccal chambers were placed in 190 µL of buffered sodium phosphate solution. The solution was homogenized in a vortex mixer, and a 6x dilution was performed by placing 50 µL aliquots from the 200 µL dilution onto six plates of the aforementioned medium.

The interior galleries of each colony were opened with flame-sterilized chisels. One 5 g sample of nest material (substrate) was removed from each colony with sterile chisels and forceps. One sample of frass was collected from under or next to each colony, along with a sample of the top 50 mm of soil (2–3 g) taken 1.0 m from each nest. Nest, frass and soil samples were placed in presterilized glass Petri plates and taken to the lab. The 5-g nest material sample was placed in 10 mL of sterile PBS solution and shaken on a Kleeco^® kinetic pulverizer for 30 s. The resulting solution was diluted 8x, and 25 µL aliquots were spread on plates of the acid medium and observed as above. The 2–3 g samples of frass or soil were mixed in 10 mL of sterile PBS for 5 min (Golubev and Bab’eva 1972a). The suspension was diluted 8x with sterile buffer solution, and 50 µL aliquots were plated on the acid medium (Ba and Phillips 1996). For all samples, the plates were incubated at 25–27 C and observed at 2 d intervals for evidence of microbial growth.

Yeast identification. – Yeasts were identified based on physiological attributes by using the BIOLOG^® microbial identification system (Biolog Inc., Hayward, California). The Biolog YT Microplate^® contains 96 wells that provide 94 biochemical tests to identify the yeast by its metabolic pattern. Each well tests the organism’s ability to assimilate or oxidize a carbon source. The redox dye tetrazolium violet is used in some wells to calorimetrically indicate carbon-source oxidation. Assimilation in other wells is indicated by an increase in turbidity (Biolog 1999). Wells initially are colorless, but if the compound in the well is assimilated and there is an increase in respiration, the cells reduce the tetrazolium dye, producing a purple color, or increased cell growth increases the turbidity of the suspension in the well (Biolog 1999).

Unknown yeast isolates were grown on Biolog Universal Yeast^® agar for 48 h at 26 C, streaked with a sterile swab and suspended in 12 mL of sterile water. The turbidity of the solution was adjusted to 47% transmittance by using a spectrophotometer. Each unknown yeast was placed in suspension, and three Biolog YT Microplates^® were inoculated with 100 µl of the suspension and incubated at 26 C; they were checked on Biolog’s Microlog^® software at 24, 48 and 72 h. These intervals let a particular metabolic pattern form that then was interpreted by the software.

Yeast isolates from all sampled sites were compared to determine whether any species were found consistently at all sites and locations. We also wanted to see whether any of the yeasts we had identified were associated with species of Formica and Solenopsis.

Preliminary study of yeast in ant nutrition. – In preliminary trials to assess the effects of the presence of the Debaryomyces polymorphus on workers, Camponotus vicinus worker ants were collected from a single colony and exposed to 39 C for 48 h. This time/temperature combination was shown in previous tests to eliminate microflora from the infrabuccal cavity (Mankowski 2001). Groups of eight workers and 15 second to third instar larvae were placed in individual Petri dishes and fed a basal diet consisting of various amino acids and inorganic salts (Mankowski 2001). Cells of D. polymorphus were prepared by growing yeast in 125 ml of vitamin-free media (Barnett 1990) on a rotary shaker for 5 d at room temperature. The resulting culture was filtered through a 0.22 µm membrane; the collected yeast cells were rinsed with sterile distilled water and resuspended in 20 ml of distilled water. Approximately 100 µl of the mixture then was applied to each worker in half of the chambers so that the workers would ingest yeast cells during normal grooming. The workers were weighed before and after 12 wk of exposure at 20–23 C. Each treatment (basal media alone or amended with yeast cells) was tested on eight plates, each containing eight workers plus the larvae. The data were subjected to an analysis of variance (ANOVA), then the variables were analyzed with general linear models using least square means to compare any significant means (SAS 2002).

	RESULTS

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Microscopy. – Bacteria and yeast cells were abundant in the infrabuccal pocket of Camponotus vicinus (FIG. 1). Cells with exterior reticulations similar to those found by Golubev and Bab’eva (1972b), but with smaller dimensions than those found in nests of Formica rufa, were prevalent in the pockets. Micro-organisms were not restricted to any one area of the pocket but were scattered throughout the food pellet in the chamber.

View larger version (119K): [in this window] [in a new window]   FIG. 1. Interior of an infrabuccal pocket: (A) an assortment of cells, and (B) a higher magnification, showing yeast-like cells.

View larger version (36K): [in this window] [in a new window]   FIG. 2. (A) Effect of season on frequency of the yeast Debaryomyces polymorphus in six carpenter ant colonies. (B) Occurrence of the yeast D. polymorphus from six carpenter ant colonies over 1 y (M = McDonald-Dunn Forest; P = Philomath).

We isolated several species of Bulleromyces from the soil as well, as Fellomyces fuzhouensis, Rhodotorula glutinus and Debaryomyces hansenii from substrate, frass and soil. Candida edax was isolated from all samples, but it occurred more often in the substrate and soil than on the ants. Candida ergastensis was found in the infrabuccal pocket, substrate and frass. Cryptococcus laurentii was isolated only once from the infrabuccal pocket and once from the soil.

Filobasidiella neoformis, Zygoascus hellenicus, Debaryomyces maramus, Rhodotorula aurantiaca, R. pustula and Sporidiobolus pararoseus A were found less frequently than other species, particularly D. polymorphus and P. guilliermondii, in substrate, frass and soil.

	DISCUSSION

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The limited isolations from the ant exoskeleton most likely reflect ant grooming behavior. Many ant species have metapleural glands that contain antimicrobial substances used to keep colonies free of micro-organisms. However, the metapleural glands of Camponotus are atrophied and it is not known how these ants keep themselves clean except by grooming (Wilson and Holldobler 1990). Because ants sometimes were covered with colony material or frass during collection, the few isolates found on the exoskeleton might be the result of contamination during sampling.

Several yeast species isolated from the infrabuccal pocket—D. polymorphus, P. guillermondii, C. edax and C. ergastensis—also have been found associated with wood-boring insects and their frass (Barnett et al 1990). Pichia guilliermondii also has been found in floral pollen, where it produces enzymes that aid in the digestion of pollen by honey bees (Gilliam 1997). In addition, D. polymorphus has been isolated from the mounds of active Formica rufa colonies (Golubev and Bab’eva 1972b). In our study, this yeast frequently was isolated from substrate, the infrabuccal pocket and frass but rarely was found in the soil outside the colonies, suggesting that its development was related to favorable colony conditions.

Ba et al (2000) found D. hansenii, P. guilliermondii, Candida famata and Cryptococcus terreus in the brood chamber soil in mounds of the imported red fire ant Solenopsis invicta. Candida species have been isolated from adult hemolymph and larval guts of S. invicta (Ba and Phillips 1996). We found D. hansenii on substrate but never isolated it from the ants themselves. Even though D. hansenii is reported from a wide variety of habitats, it is not known to occur on or in wood (Barnett et al 1990). Many Cryptococcus species commonly occur in the soil, and it was interesting to find them associated with the infrabuccal pocket and ant galleries (Barnett et al 1990).

Although Candida edax was isolated predominantly from soil, this species commonly is associated with insect frass and wood, as are many species of Rhodotorula (Barnett et al 1990). Rhodotorula pustula is commonly found on fruit, and the significance of its presence in the ant colonies is unknown.

Debaryomyces polymorphus was isolated from substrate material, frass under the colonies and once from outside the colonies. It was the most common infrabuccal pocket yeast in this study, found throughout the year in each colony surveyed (FIGS. 2A and 2B), but it was more frequent in July and October than in January. It also was found consistently in substrate material and frass of the colonies during the former 2 mo. It is of interest to note that D. polymorphus was found in the infrabuccal pocket in January when ants are not feeding. This finding suggests that the fungus was retained in the infrabuccal pocket. Ants generally do not retain infrabuccal material for long (Eisner and Happ 1962). Debaryomyces polymorphus was isolated once in January and once in October from soil in different locations but was found in all colonies sampled. It appeared to be more common in colonies collected from McDonald-Dunn Research Forest than in those from Philomath (FIG. 2B).

Debaryomyces species also are found in the intestines of xylophageous beetles, where they comprise 50–85% of the microflora (Gusteleva 1975). Gusteleva (1975) showed that these yeasts were active producers of B vitamins—biotin, thiamin, pyridoxine—and nicotinic and pantothenic acids, which they can synthesize more intensively than can bacteria associated with xylophageous insects. Ba and Phillips (1996) found that fire ant colonies with yeasts weighed more than those without, suggesting that these organisms aided in nutrition. In another study, Ba et al (1995) found that yeasts Candida parapsilosis and Yarrowia lipolytica synthesized ergosterol and zymosteroil in the larvae of Solenopsis. Yeasts can aid in the breakdown of toxins, aiding in the diets of certain fruit flies (Starmer et al 1986), and can produce chemicals that modify insect behavior (Leufven and Nehls 1986). In a preliminary study examining the effects of live Debaryomyces polymorphus on Camponotus vicinus, the addition of yeast cells to colonies fed the basal diet resulted in significantly higher worker weights than in colonies receiving only the basal diet (average weights of 0.11 and 0.13 mg, respectively; P = 0.005). The close relationship between yeasts, wood-inhabiting insects and other ant species, as well as the common presence of D. polymorphus in colonies of C. vicinus, implies that carpenter ants may use yeast as a source of nutrients or enzymes that aid in the metabolism of dietary components in the infrabuccal pocket. Further studies of these possible interactions are under way.

	FOOTNOTES

 
Accepted for publication September 8, 2003.

¹ This is Paper 3503 of the Forest Research Laboratory, Oregon State University, Corvallis.

² Corresponding author. E-mail: morrellj{at}frl.orst.edu

	LITERATURE CITED

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