Microorganisms associated with field-collected Chrysoperla rufilabris (Neuroptera: Chrysopidae) adults with emphasis on yeast symbionts

Biological Control 29 (2004) 155–168 www.elsevier.com/locate/ybcon

Microorganisms associated with field-collected Chrysoperla rufilabris (Neuroptera: Chrysopidae) adults with emphasis on yeast symbionts Sandra W. Woolfolk and G. Douglas Inglis* Department of Entomology and Plant Pathology, Mississippi State, MS 39762, USA Received 4 March 2003; accepted 13 June 2003

Abstract The indigenous gut microflora associated with the alimentary canal of 24 field-collected Chrysoperla rufilabris adults was examined qualitatively and quantitatively at five sample times and two locations in Mississippi. Yeasts were isolated from the diverticula of 17 insects, and there was no effect of location or sample time on the occurrence of yeasts. Metschnikowia pulcherrima was present in all of the diverticula that contained yeasts, and densities in this gut region ranged from 5.3
102 to 5.4
105 colony forming units (CFUs). Numerous yeast cells were observed in diverticula, and these cells accumulated within pronounced diverticular folds. Large numbers of Met. pulcherrima were also observed in the foregut, and to a lesser extent in the midgut and hindgut of field-collected adults. In an ancillary experiment, no yeasts were observed in Cry. rufilabris adults that had recently eclosed (ca. 24 h) in the laboratory. Thirteen of the insects were positive for filamentous fungi, but the majority of the gut regions contained < 102 CFUs. Furthermore, there was very little commonality in the taxa isolated among the sample times and locations suggesting that filamentous fungi are transients in Cry. rufilabris alimentary canals. Nineteen insects were positive for bacteria. Populations varied among the gut regions, and more bacteria were recovered from the midguts of adults at one collection site. Twenty-five aerobic bacterial taxa were isolated, and Enterobacter aerogenes was the most commonly isolated taxon. However, this bacterium was only isolated from four insects on four occasions. Other bacterial taxa were even less frequently isolated, suggesting that bacteria are likely transients. Our findings indicate that Cry. rufilabris adults may form a symbiosis with the yeast, Met. pulcherrima, but not with filamentous fungi or bacteria. This information will facilitate studies to elucidate the mechanism and impact of this interaction, and may facilitate the rearing of this important predator for use in biological control programs against pest insects. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Chrysoperla rufilabris; Gut; Alimentary canal; Metschnikowia pulcherrima; Yeasts

1. Introduction Chrysopids (i.e., lacewings) are one of the most common groups of predators used in biological control programs against insects. Among a number of species, Chrysoperla rufilabris (Burmeister) and Chr. carnea (Stephens) are the most prevalent taxa used in North America (Daane and Hagen, 2001; Henry et al., 2001; Tauber and Tauber, 1983; Tauber et al., 2000). Larvae * Corresponding author. Present address: Agriculture and AgriFood Canada, Lethbridge Research Center, 5403-1st Avenue S, Lethbridge, Canada AB T1J 4B1. Fax: 1-403-382-3156. E-mail address: [email protected] (G. Douglas Inglis).

1049-9644/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1049-9644(03)00139-7

of these species are predaceous, whereas adults mainly feed on pollen or honeydew (Principi and Canard, 1984; Ridgway and Murphy, 1984). Their effectiveness as biological control agents has been documented in cage, greenhouse, and field environments in a variety of field crops, tree crops, forests, and horticultural crops worldwide (McEwen et al., 2001). For example, releases of Chr. carnea larvae reduced leafhopper populations by more than 31% in vineyards (Daane et al., 1996), and application of Chrysopa sinica Tjeder (synonym: Chrysoperla sinica) eggs at various rates (1.5–4.5
105 eggs/ha) reduced Helicoverpa armigera (H€ ubner) populations by 67–83% in China (Wang and Nordlund, 1994). More than 130 companies in North America

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(Hunter, 1997) and 26 companies in Europe (Van Lenteren et al., 1997) produce and/or sell biological control products including lacewings. This does not include many other companies in Central and South America, Asia, and Australia. Currently sold mainly to organic gardens and greenhouses, lacewings represent an economically substantial industry. The invention of artificial diet by Cohen and Smith (1998) and Cohen (1998, 1999) has tremendously reduced the cost of rearing from $500/kg to only $6.00/kg. Reduction of rearing costs brings us closer to the goal of releasing lacewings as biological control agents in field crops. Four US companies (i.e., Beneficial Insectary, BioLogixs, Buena Biosystems, and Oregon Freeze Dry) currently hold licenses to use CohenÕs diet. Little is known about the microflora associated with Chrysoperla sp. adults. Buchner (1965) and Koch (1967) indicated that predaceous insects should not require mutualistic symbionts because their prey provides all the essential nutrients. However, the diet of many nonpredaceous Chrysoperla adults is restricted to pollen, nectar, and/or honeydew, and Hagen and Tassan (1966, 1972) and Hagen et al. (1970) postulated that lacewing adults possess yeast symbionts in their crops, which provide essential amino acids that are normally absent in their diet. They observed that ‘‘budding yeasts’’ were common within the crops of lacewing adults. Unfortunately, the yeast taxon isolated by Hagen et al. (1970) was not identified to species and was published only as Torulopsis sp., a genus that has subsequently been reduced into synonymy with a variety of anamorphic (e.g., Candida, Cryptococcus, and Rhodotorula) and teleomorphic (e.g., Debaryomyces, Metschnikowia, and Saccharomyces) genera. Johnson (1982) reported that the yeast, Candida multigemmis (Buhagiar) Meyer and Yarrow (Yarrow and Meyer) (synonyms: Candida multisgemmis, Torulopsis multigemmis), was commonly associated with the adults of several chrysopid species (i.e., Nodita occidentalis Johnson, Eremochrysa tibialis Banks, and Eremochrysa punctinervis (MacLachlan) complex, and probably Mallada perfectus Banks). Largely based on the results of these studies, it is now a commonly held concept that lacewing adults form an obligate symbiosis with yeasts (e.g., Tauber et al., 2000). Furthermore, Hagen and Tassan (1972) postulated that chrysopid adults from different locations possess different yeast taxa in their crops, and that the yeast symbionts determine the fitness of non-indigenous chrysopids. However, a detailed examination of microorganisms associated with field-collected Chrysoperla adults using modern taxonomic methods, and their importance to lacewings has not been undertaken. Currently most rearing programs utilize commercially available yeasts (e.g., Saccharomyces cerevisiae Meyen ex Hansen) as a component of the adult diet. If chrysopid adults do form a mutualistic symbiosis with specific microorganisms, it

may be possible to utilize them to enhance fecundity in rearing settings. The first step in this process is to examine the composition of the microflora, and determine whether chrysopids possess a resident microflora. Therefore, the objective of this study was to temporally examine fungi and bacteria present within the various regions of the alimentary canal of field-collected C. rufilabris lacewing adults in Mississippi (two locations), with the goal of testing the hypothesis that chrysopid adults possess a resident microflora.

2. Materials and methods 2.1. Insect collection and sample preparation Chrysoperla adults were arbitrarily collected from Monroe county and Oktibbeha county, Mississippi using a sweep net and/or black light trap. The Monroe county collection site (longitude 88.411°W; latitude 33.740°N) consisted predominately of fields in cotton production, and comprised an area of approximately 28 km2 . The Oktibbeha county collection site was located at the Plant Science Research Farm at Mississippi State University (longitude 88.782°W; latitude 33.416°N) and consisted of an area of approximately 1.8 km2 . The primary crops at the Research Farm were cotton and soybean. Collections from Oktibbeha county were conducted on 1 October 2000 (time 1), 1 May 2001 (time 3), 30 May 2001 (time 4), and 20 July 2001 (time 5). Collections from Monroe county were conducted on 22 April 2001 (time 2), 4 May 2001 (time 3), 25 May 2001 (time 4), and 13 July 2001 (time 5). At each time and collection site, a minimum of three adults were collected. However, we were unsuccessful at collecting insects from the Monroe country site at time 1, and from the the Oktibbeha country site at time 2. Due to the logistical limitations of quantifying specific microbial taxa associated with the alimentary canals of adults, the total number of insects collected and processed were limited to 24 (i.e., 12 adults from each site). From these insects, >1390 microbial isolates were isolated and characterized. Immediately following collections, adults were placed over ice, transported to the laboratory, and stored for 24 h at 5 °C. Insects were weighed and then submerged in 1% sodium hypochlorite containing 0.01% Tween 80 (Sigma, St. Louis, MO) for 1 min, submerged in 1% sodium thiosulfate to neutralize the sodium hypochlorite, and rinsed twice with sterile distilled water. For each sample time, three adults were then chilled in sterile 50 mM phosphate buffer containing 0.01% Tween 80 (buffer-Tween). The intact alimentary canal from each adult was excised, and the gut aseptically divided into diverticulum, foregut, midgut, and hindgut (Fig. 1). Tissues for each gut region were placed in a microcentrifuge

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Fig. 1. Alimentary canal of a Chrysoperla rufilabris adult where: Px, pharynx; Es, esophagous; Cp, crop; Pv, proventriculus; Dv, diverticulum; Fb, food bolus; Mg, midgut; Mp, Malphigian tubules; Im, ileum; Rc, rectum; Rp, rectal pads; and Ex, excision points. Bar ¼ 170 lm.

tube containing chilled buffer-Tween. Following dissection, carcasses of adults were preserved according to Borror et al. (1992) to be identified to species using keys provided by Brooks (1994), Brooks and Barnard (1990), and Penny et al. (2000).

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heximide (Sigma), and media used to recover yeasts and filamentous fungi were amended with 0.1 g/L chlortetracycline HCl (Sigma) and 0.1 g/L chloramphenicol (Sigma). Bacterial cultures were incubated at 30 °C for 72 h, and yeasts and filamentous fungal cultures incubated at 25 °C for 96 h. Colonies of bacteria, yeasts, and filamentous fungi were enumerated at the dilution yielding 30–300 colonies per petri dish. A minimum of five arbitrarily selected bacterial colonies, and five yeast and filamentous fungal colonies from the appropriate dilution per treatment were transferred onto TSA slants for bacteria and SabouraudÕs dextrose agar (SDA; BBL) amended with 1% yeast extract (SDAY) slants for yeasts and filamentous fungi. Selection of the colonies was based on the colony morphology and frequency of occurrence. In addition, rare occurring but unique colonies based on colony morphology were collected. Culture slants were grown at the appropriate incubation temperature and stored at 5 °C until identified. As a result of the similar or superior efficacy of recovery of yeasts and on MEA relative to M30 and RB, analyses of yeast populations were restricted to this medium. RB was used to enumerate filamentous fungi. For bacteria, no Pseudomonas species were recovered on Pseudosel, and very few Bacillus species were recovered on PEMBA. There were no significant differences in bacterial populations on TSA and MAC among the four gut regions (data not presented). Furthermore, the majority of the bacterial isolates recovered on both media were found to be enterics. As a result, analyses were restricted to bacteria isolated on TSA. 2.3. Characterization of fungi

2.2. Enumeration and isolation of microorganisms The excised diverticulum, foregut, midgut, and hindgut for each adult were individually homogenized in the buffer-Tween using a Kontes micropestle. Homogenates were diluted in a 10-fold dilution series, and aliquots of 100 ll from each dilution were spread onto specific media. For the isolation of yeasts and filamentous fungi, the following media were used: malt extract agar (MEA; consisting of 20 g of malt extract, 20 g dextrose, 1 g peptone, and 15 g of agar in 1 L of distilled water; pH 5.6); MEA amended with 30% dextrose (M30); and medium RB (consisting of 5 g of peptone, 10 g dextrose, 20 g agar, 1 g KH2 PO4 , 0.5 g MgSO4  7 H2 O, 30 mg rose bengal, and 1 g oxgall in 1 L of distilled water). For bacteria, the following media were used: trypticase soy agar (TSA; BBL, Cockeysville, MD) for the isolation of total bacteria; MacConkey agar (MAC; BBL) for the isolation of enterics; Pseudosel (BBL) for the isolation of pseudomonads; and polymixin pyruvate egg yolk mannitol bromothymol blue agar (PEMBA; Atlas, 1993) for the isolation of bacilli. The media used to isolate bacteria were amended with 0.2 g/L cyclo-

Yeasts were initially grouped according to morphological characteristics (e.g., size, shape, formation of pseudohyphae or hyphae, and the production of ascospores), ability to assimilate 19 carbohydrates (i.e., D -glucose, D -galactose, D -glucosamine, D -xylose, L arabinose, sucrose, maltose, a; a-trehalose, methyl-aD -glucoside, cellobiose, melibiose, lactose, raffinose, melezitose, glycerol, ribitol, xylitol, D -mannitol, and myo-inositol), staining reaction with diazonium blue-B (DBB), and ability to hydrolyze urea (Barnett et al., 1990; Lodder and Kreger-van Rij, 1967; Rippon, 1992). The ability of representative isolates to ferment glucose, galactose, maltose, sucrose, and lactose was determined according to Barnett et al. (1990). Tentative identifications of selected isolates were obtained with an automated identification system (Biolog System, Hayward, CA) according to the manufacturerÕs protocol with the exception that biomass was produced on SDAY. Based on the Biolog identifications, yeasts were assigned to five tentative taxa. To obtain final identifications, sequence data for the partial ribosomal RNA gene, including the V9 region of

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18S, the domain 1 region of 26S, and the entire 5.8S and internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) were obtained from two representative isolates per taxon group (Group I ¼ S762, S768; Group II ¼ S810, S823; Group III ¼ S1597, S1728; Group IV ¼ S821, S1740; and Group V ¼ S1585, S1717). Isolates were streaked for purity on SDA, and biomass after 48 h was suspended in nutrient broth. Spheroplasts were obtained using 200 U of lyticase (Sigma) in Sorbitol buffer at 30 °C, and genomic DNA was extracted from the spheroplasts using the DNeasy kit (Qiagen, Mississauga, ON) according to the manufacturerÕs protocol. Genomic DNA was electrophoresed in a 1% TAE–agarose gel (Invitrogen, Burlington, ON), stained with ethidium bromide and visualized under UV light. The primers, SSZ (50 -ATA ACA GGT CTG TGA TG-30 ) and LSU4 (50 -TTG TGC GCT ATC GGT CTC-30 ) were subsequently used to amplify the partial rRNA genes and ITS regions 1 and 2 (Hausner et al., 1993). The conditions for amplification were 1 cycle at 95 °C for 3 min, followed by 30 cycles of 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C, ending with an extension cycle of 10 min at 72 °C. The mixtures consisted of a total volume of 20 ll containing 1
reaction buffer, 0.2 mM dNTPs, 2 mM MgCl2 , 0.5 lM of each primer (Sigma-Genosys, Oakville, ON), and 1 U Taq polymerase (Gibco/BRL, Burlington, ON). Each PCR was performed with a total of 2 ll of genomic DNA (500 ng/ll) and the negative control consisted of optima water instead of template. The resulting PCR products (10 ll) were electrophoresed in a 2% TAE–agarose gel, and a 100 bp ladder (Promega) was used to size products. The PCR amplicons were purified using the Qiagen QIAquick kit. To obtain sequences, the primers, SSU3 (50 -GTC GTA ACA AGG GTC TCC G-30 ), LSU2 (50 -GAT ATG CTT AAG TTC AGC G-30 ), and 5.8SB (50 -TGT ACA CAC CGC CCG TC-30 ) were used with the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA). The same reaction concentrations as described above were used, but conditions for amplification were 30 cycles of 30 s at 95 °C, 15 s at 50 °C, and 4 min at 60 °C. Prior to sequencing, excess dye was removed with the Qiagen DyeEx Spin Kit. Sequences were obtained with an ABI PRISM 377 Automated DNA sequencer, and contigs were constructed using Staden (Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK). Nucleotide sequences for isolates within the five groups were aligned using the multi-alignment program ClustalW (Thompson et al., 1994), and the alignments were refined visually using GeneDoc (Nicholas and Nicholas, 1997). Sequences for 5.8S, ITS 1 and 2 from Group I and II yeasts were compared with Bullera alba var. lactis (AF444665T ), Bul. derxii (AF444405), Bul. oryzae (AF444413T ), Bul. pseudoalba (AF444399T ), Bul. sinensis (AF444468T ), Bul. unica (AF444441T ), Bulleromyces al-

bus (AF444368T ), Cryptococcus cellulolyticus (AF444442T ), Cry. laurentii (AF410468T ), Cry. luteolus (AF444323T ), Cry. skinneri (AF444305T ), Cry. victoriae (AF444469T ), and Cryptococcus sp. (AF444385; AF444386; AF444390). Agariocostilbum hyphaenes (AF444553) was used as the outgroup. Sequences for 5.8S, ITS 1 and 2 from Group III, IV, and V yeasts were compared with sequences for Clavispora lusitaniae (AF321541), Debaryomyces hansenii (AB018041), Dekkera bruxellensis (AF043501), Lipomyces tetrasporus (U82456), Metschnikowia bicuspidata (U51436), Met. hawaiiensis (U51434), Met. zobellii (U51433), Pichia anomala (AF321543), Pic. ohmeri (AF022721), Saccharomyces dairiensis (AJ229072), and Schizosaccharomyces japonicus (Z32848). Cryptococcus luteolus (AB032641) was used as the outgroup. Group III, IV, and V yeasts were subsequently compared to Metschnikowia species using sequence data for the V9 region of the 18S rRNA gene. Sequences for Candida multigemmis (AB013535), Met. agaves (AB023475), Met. australis (AB023465), Met. bicuspidata (AB023466), Met. gruessii (AB023472), Met. hawaiiensis (AB023476), Met. krissii (AB023467), Met. lunata (AB023474), Met. pulcherrima (AB023473), Met. reukaufii (AB023469), and Met. zobellii (AB023468) were used. In some instances, the Biolog system tentatively identified isolates of combined Group VI as Candida spp, Deb. hansenii, Dek. bruxellensis, or Pichia spp. For this reason, Can. torrensii (AB013540), Deb. hansenii (X58053), Dek. bruxellensis (X83814), and Pic. anomala (AB013535) were included in the analyses. Cryptococcus luteolus (AB032641) was used as the outgroup. All sequence data were analyzed using programs contained within PHYLIP (version 3.5c, Felsenstein, 1995). Phylogenetic estimates were obtained based on neighbor-joining distance method. Divergence (or distance) of each pair of sequences was calculated by DNADIST using KimuraÕs two-parameter model. The NEIGHBOR program was used to carry out the neighbor-joining method for estimating phylogenies from the distance matrices. Support for the internal branches within the resulting trees was obtained by bootstrap analysis; 1000 bootstrap replicates were generated by SEQBOOT, majority-rule consensus trees were constructed by the CONSENSE program, and the tree was visualized using TreeView (Page, 2001). The partial rRNA gene sequence including sequences for the ITS 1 and 2, and 5.8s rRNA gene have been deposited in GenBank under the accession numbers: AY301024, AY301025, and AY301026. 2.4. Characterization of bacteria and filamentous fungi Bacteria were cultured on TSA and/or sheepÕs blood agar at 30 °C and their Gram stain reactions determined at 24 h. Catalase and oxidase capability also were determined, and bacteria were characterized using the

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automated Biolog system according to the manufacturerÕs protocol. Isolates that could not be definitively identified were further characterized according to standard references (Anonymous, 1998; Baron et al., 1994; Holt et al., 1994). Filamentous fungi were identified based on conidiogenesis according to standard references (Barron, 1968; Domsch et al., 1980; Ellis, 1971). Where possible, a minimum of one representative of each microbial taxon was stored in 20% glycerol at )80 °C.

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five were common to both collection sites, interaction analyses of collection time and site were restricted to these times. The data were analyzed as a factorial experiment with four levels of gut region, three levels of time, and two levels of location, and the data were analyzed separately for each microbial class. In all instances, the mixed procedure with repeated measurement applied to the gut region. To compare CFU for individual taxa in each gut region, populations for each taxon were calculated as: log10 [(frequency of isolation) (total CFU)].

2.5. Microscopy 2.7. Microorganisms associated with newly eclosed adults The alimentary canals of lacewings were dissected and observed with a Carl Zeiss Photomicroscope 2 using a Syncroscopy Digital Microscopy system with AutoMontage software. To quantify areas of the various gut regions, a TIF image of a representative alimentary canal was captured. A binary image was then obtained, and the relative areas were quantified using Image-Pro Plus software (MediaCybernetics, Carlsbad, CA). For scanning electron microscopy, alimentary canals were dissected and separated into the four regions (i.e., diverticula, foreguts, midguts, and hindguts), the tissues fixed in 2.5% glutaraldehyde, rinsed, and dehydrated in a graded ethanol series (30, 50, 70, 95, and 100%). Tissues were then cryofractured (Bozzola and Russell, 1992; Nation, 1983; Rumph and Turner, 1998) to obtain cross, longitudinal or oblique sections. Samples were then critically point dried, coated with gold–palladium, and examined with a LEO S360 scanning electron microscope at an accelerator voltage of 15 kV.

Chrysoperla rufilabris eggs were obtained from the Biological Control and Mass Rearing Research Unit (BCMRRU), USDA-ARS at Mississippi State, and larvae were maintained in the laboratory on Ephestia eggs until they pupated. Following eclosion, adults were fed sterile sugar water (1:1) for 24 h, at which point their alimentary canals were aseptically dissected and yeasts were isolated on MEA at 25 °C as described previously.

2.6. Statistical analyses The experiment was arranged as a randomized complete block design with the individuals collected at each collection time and site serving as blocks. To test for normality, predicted values were plotted against residual values, and the univariate procedure of SAS was applied. In all instances, the colony forming unit (CFU) data were log10 -transformed. For each collection site, microbial populations were analyzed as a factorial experiment with four levels of collection time and four levels of gut region. Since the same individual was used for all gut regions, the mixed procedure of Statistical Analyses System Software (SAS Institute, Inc., 1999) was used with the repeated measurement applied to the gut treatment. The appropriate error structure was determined using AkaikeÕs Information Criterion (AIC) and SchwarzÕs Bayesian Criterion (SBC), and the Kenward–Roger degree of freedom (krdf) feature was used to adjust the degrees of freedom of the error term. In conjunction with a significant F test, the least square means (lsmeans) function of SAS was used to compare means. Given that only collection times three, four, and

Fig. 2. Scanning electron micrographs of the diverticulum of fieldcollected Chrysoperla rufilabris adults: (A) note the presence of large numbers of yeasts (arrow) in between diverticular folds; (B) note yeast cells, pollen grain (arrow), and insect scales (arrow heads). Bar ¼ 10 lm.

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3. Results 3.1. Field-collected insects All of the lacewing adults utilized in the study were identified as Chr. rufilabris. There was no difference in adult weight among the insects collected from different locations (F ¼ 0:76; df ¼ 1, 12; P ¼ 0:40) or sample times (F ¼ 0:62; df ¼ 2, 12; P ¼ 0:56). Using image analysis, the relative areas of each gut regions were: 24% for the foregut; 35% for the diverticulum; 30% for the midgut; 5% for the ileum; and 5% for the rectum (Fig. 1). 3.2. Yeasts The diverticulum was very pronounced, and large numbers of yeast-like cells (i.e., cells of approximately 4.0–6.0 lm in diameter that exhibited blastic development) were consistently observed to be aggregated on the surface of and in between folds within the divertic-

ulum (Fig. 2A). In several instances, pollen grains and insect scales also were observed in diverticula (Fig. 2B). Of the 24 Cry. rufilabris adults collected, no yeasts were isolated from seven individuals (Fig. 3). In the other 17 adults, considerable numbers of yeasts were isolated from their alimentary canals, and yeast populations exceeded 103 CFU in 13 diverticula and foreguts, five midguts, and two hindguts. At the Monroe country site, populations varied (F ¼ 8:5; df ¼ 9, 7; P ¼ 0:005) among the gut regions at various times; for the diverticulum and foregut regions, less (P < 0:05) yeasts were isolated at collection time 2. At the Oktibbeha county site, there was no interaction (F ¼ 0:1; df ¼ 9, 24; P ¼ 0:24) between gut region and collection time, but both gut region (F ¼ 5:1; df ¼ 3, 24; P ¼ 0:007) and collection time (F ¼ 4:3; df ¼ 3, 8; P ¼ 0:04) alone affected yeast densities; more (P < 0:05) yeasts were recovered at time 3, and less (P < 0:05) yeasts were isolated from the hindgut. Furthermore, an interaction (i.e., only using collection times three, four, and five in

Fig. 3. Populations (log10 colony forming units (CFU)) of the yeast, Metschnikowia pulcherrima, recovered from the four regions of the alimentary canals of field-collected Chrysoperla rufilabris adults on malt extract agar. Insects were collected at different times and at two locations in Mississippi, where MC: Monroe county collection site, OC: Oktibbeha county collection site; time 1 ¼ Julian day (jd) 275 in 2000, time 2 ¼ jd 112 in 2001, time 3 ¼ jd 121 and 124 in 2001, time 4 ¼ jd 145 and 150 in 2001, and time 5 ¼ jd 194 and 201 in 2001. (A) diverticulum; (B) foregut; (C) midgut; and (D) hindgut. The asterisk indicates that collections were not made, the number associated with histogram bars is the number of adults positive for Met. pulcherrima, and the numbers in circles associated with grey histogram bars (i.e., from Oktibbeha country) represents numbers of adults positive for non-Metschnikowia yeasts. Vertical lines represent standard errors of means (n ¼ 3).

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the analysis) was observed between gut region and collection location (F ¼ 3:12; df ¼ 3, 36; P ¼ 0:038); this was attributed to larger (P 6 0:011) populations of yeasts isolated from the diverticulum relative to the midgut and hindgut from Monroe and Oktibbeha counties, and to fewer numbers (P 6 0:014) of yeasts isolated from the hindgut relative to the foregut and midgut from both collection sites. Seven hundred and fifty-two isolates of yeasts were grouped based on morphological characters, their ability to assimilate 19 carbohydrates, on their DBB stain characteristics, and ability to hydrolyze urea (i.e., urease production). Further characterization with Biolog and fermentation tests suggested that the yeasts belonged to five taxa. Based on Biolog identifications of representative isolates, the following tentative identifications were made: Group I yeasts as Cryptococcus luteolus (Saito) Skinner (90–99% identification probability); Group II yeasts as Bulleromyces albus Boekhout and Fonseca (69–98% identification probability); Group III yeasts as Dekkera bruxellensis van der Walt (10–99% identification probability); Group IV yeasts as Debaryomyces hansenii (Zopf) Lodder and Kreger-van Rij (82– 100% identification probability); and Group V yeasts as Candida multisgemmis (89–100% identification probability). The results of the DBB and urease tests supported the Biolog identifications; Group I and II yeasts exhibited basidiomycetous affinities, whereas, the other three groups possessed ascomycetous affinities (Groups III, IV, and V). The partial rRNA genes including the internal transcribed spacer regions 1 and 2, for two arbitrarily selected representatives of each group were subsequently amplified. The size of the amplicon was the same (1050 bp) for Group III, IV, and V yeasts (Fig. 4). Both of the Group II isolates produced the same size amplicon (1300 bp), but different sized PCR products were observed for the two, Group I yeasts; S762 produced a 1250 bp size amplicon, whereas, S768 produced a slightly smaller amplicon (1175 bp). DNA sequenc-

Fig. 4. Agarose gel showing the size of the PCR product for the partial ribosomal RNA gene, including the V9 region of 18S, the domain 1 region of 26S, and the entire 5.8S and internal transcribed spacer regions 1 and 2 for Metschnikowia pulcherrima and Cryptococcus spp., where lane 1: 100 base pair marker; lanes 2 (S762), 3 (S768), 4 (S810), and 6 (S823): Cryptococcus spp.; and lanes 5 (S821), 7 (S1585), 8 (S1597), 9 (S1717), 10 (S1728), and 11 (S1740): Met. pulcherrima. The control sample (i.e., no template added) was negative.

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ing revealed that Group III, IV, and V yeasts possessed identical sequences to each other, and these yeasts were deemed to be the same taxon. Hereafter, they are referred to as combined Group VI. The two isolates of Group II yeast tested also possessed identical sequence to each other and were considered to be the same taxon. In contrast, considerable polymorphism (63% similarity) were observed between the two isolates of Group I yeasts, and these isolates likely represented two taxa. Phylogenetic analysis of the ITS 1, 5.8S, and ITS 2 regions indicated that S762 (Group I) formed a clade (100% bootstrap support) with an unidentified strain of Cryptococcus sp. (CBS 8355; AF444385) isolated from a flower of Acanthaceae in Angra dos Reis, Brazil (Scorzetti et al., 2002) (Fig. 5). This clade was most closely related to Cryptococcus victoriae Montes et al. (AF444469). Furthermore, the assimilation and fermentation properties for S762 were consistent with Cry. victoriae (CBS, 2000). Of the carbohydrates we tested, the only discrepancy that was observed with the published results for Cry. victoriae was negative assimilation of xylitol by S762. Given the similarities in the sequence of ITS 1 and 2, and assimilation and fermentation characteristics, S762 was identified as Cry. victoriae. Although the other Group I yeast (S768) tested was identical to S762 in its assimilation and fermentation profile, they did not form a phylogenetic grouping. Contrary, it formed a distinct clade (100% bootstrap support) with Cry. luteolus (AF444323), possessing 98% similarity with this taxon. Based on the sequence data, S768 was deemed to be Cry. luteolus. The sequence data corresponded to the assimilation and fermentation profiles. Group II yeasts did not group closely to known isolates of basidiomycetous yeasts based on ITS regions of rDNA. The closest taxon to S823 was Bullera oryzae Nakase and Suzuki, but this yeast generally forms orange, butyrous colonies (Barnett et al., 1990), whereas S823 formed cream to orangish, mucoid colonies. Although the assimilation and fermentation properties of Bul. oryzae were consistent with S823 (all exhibited positive assimilation of D -galactose, D -glucosamine, D xylose, L -arabinose, sucrose, maltose, a-trehalose, methyl-a-D -glucoside, cellobiose, raffinose, melezitose, ribitol, xylitol, D -mannitol, and myo-inositol; negative assimilation of glycerol; and no fermentation of D -glucose, D -galactose, maltose, sucrose, and lactose), there was only 63% similarity in the ITS regions between S823 and Bul. oryzae (AF444413). Given that S823 formed a distinct clade (100% bootstrap support) from Bul. oryzae, and it also grouped loosely with Cryptococcus species, it was not possible to assign Group II yeasts isolated from Chr. rufilabris to either genus. Hereafter, this yeast is referred to as an unidentified basidiomycetous yeast. Phylogenetic analysis of combined Group VI yeasts based on the ITS 1, 5.8S, and ITS 2 regions indicated that

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Fig. 5. A dendrogram based on a majority rule consensus tree obtained from analysing the partial sequence of the internal transcribed spacer regions 1 and 2 and 5.8S rRNA dataset with the NEIGHBOR programme (neighbor-joining option) showing DNA sequence relatedness for selected basidiomycetous yeasts (Cryptococcus, Bullera, and Bulleromyces species). Isolates represented by ‘‘S’’ were isolated from the alimentary canal of Chrysoperla rufilabris adults in Mississippi, and taxa followed by ‘‘T’’ represent type specimens. The outgroup used in the analysis was Agaricostilbrum hyphaenes. The bar represents 0.1 nucleotide substitutions per base, and numbers at selected nodes indicate support for the internal branches within the resulting trees obtained by bootstrap analysis (1000 times). The single asterisk indicates a bootstrap value of 666, and the double asterisk a value of 1000.

Fig. 6. Needle-shaped ascospore (arrow) of Metschnikowia pulcherrima (S1740). Bar ¼ 10 lm.

these yeasts were most closely related to yeasts in the genera, Metschnikowia and Clavispora (data not presented). Furthermore, these yeasts were observed to form needle-shaped ascospores (Fig. 6) which is characteristic of some species of Metschnikowia (Barnett et al., 1990). Due to the lack of adequate ITS sequence data for this genus, the phylogenetic relationship of combined Group VI isolates (S821, S1585, S1597, S1728, and S1740) was compared to published sequences for the V9 region of the 18S rRNA for Metschnikowia species (296 bp). Based on the V9 region, Group VI yeasts were found to be most closely related to Met. pulcherrima Pitt and Miller, with 98% bootstrap support for this clade using the neighborjoining method (Fig. 7). The assimilation and fermentation profiles between Group VI yeasts and Met. pulcherrima supported this relationship (Table 1). Despite possessing 98% similarity, Group VI yeasts formed a distinct clade (100% bootstrap support) from Met. pulcherrima, suggesting that these yeasts may prove to be a new species. However, given the similarity that we observed in physiological characteristics and in sequence of the V9 region of the 18S rRNA gene, we have assigned Group VI yeasts to Met. pulcherrima, and use this name hereafter. Candida multigemmis, the primary taxon re-

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Fig. 7. A dendrogram based on a majority rule consensus tree obtained from analysing the partial sequence of the 18S rRNA gene (V9 region) with the NEIGHBOR programme (neighbor-joining option) showing DNA sequence relatedness for selected ascomycetous yeasts. Isolates represented by ‘‘S’’ were isolated from the alimentary canal of Chrysoperla rufilabris adults in Mississippi. The outgroup used in the analysis was Cryptococcus luteolus. The bar represents 0.05 nucleotide substitutions per base, and numbers at selected nodes indicate support for the internal branches within the resulting trees obtained by bootstrap analysis (1000 times). The single asterisk indicates a bootstrap value of 432.

ported from non-Chrysoperla chrysopids by Johnson (1982), did not group closely with the Met. pulcherrima isolates from Mississippi. Of the 752 yeasts that were isolated from field-collected Chr. rufilabris, the vast majority (669 isolates or 89%) were Met. pulcherrima (Fig. 3). In contrast, Cry. victoriae (i.e., Group I), Cry. luteolus (i.e., Group II), and the unidentified basidiomycetous yeast (i.e., Group II) comprised 11% of the isolates recovered, and these yeasts were isolated in relatively small numbers from the foreguts, midguts and hindguts of adults collected at Oktibbeha county at time 1. All of the diverticula that were positive for yeasts (n ¼ 17), contained Met. pulcherrima. Only one foregut (n ¼ 16), midgut (n ¼ 16), and hindgut (n ¼ 6) that contained yeasts was negative for Met. pulcherrima; in gut regions negative for Met. pulcherrima, small populations ( 6102 CFU) of yeasts were isolated. 3.3. Filamentous fungi Filamentous fungi CFU were isolated from the alimentary canals of 13 Cry. rufilabris adults (n ¼ 24). In most instances, numbers of CFU were small ( 6102 ). However, the hindgut of one individual collected from the Oktibbeha site at time 5 contained 104 CFU. There was no effect of either gut region (F ¼ 1:56; df ¼ 3, 10; P ¼ 0:2600), location (F ¼ 0:18; df ¼ 1, 12; P ¼ 0:68), or time (F ¼ 0:28; df ¼ 2, 12; P ¼ 0:76) on isolation fre-

quency. Six taxa were isolated, and the most commonly isolated species was Fusarium moniliforme Sheldon. However, Aspergillus niger van Tieghem, Cladosporium cladosporioides (Fresen.) De Vries, Trichoderma reesei Simons, Penicillium sp., and other Fusarium species also were recovered. None of these taxa were consistently isolated from the adults at different sample times and locations. 3.4. Bacteria Bacteria were isolated from 19 of 24 adults (Fig. 8). Populations exceeding 103 CFU were observed in only three diverticula, foreguts, and hindguts, but in 12 midguts. Numbers of bacteria in the various gut regions differed (F ¼ 36:5; df ¼ 9, 7; P < 0:001) across the four collection times at the Monroe county collection. The interaction was attributed to the larger (P 6 0:02) bacterial populations isolated from the midgut at collection times 2, 3, and 4 but not 5. At the Oktibbeha county location, neither gut region (F ¼ 0:8; df ¼ 3, 24; P ¼ 0:53), sample time (F ¼ 0:4; df ¼ 3, 8; P ¼ 0:74), or the interaction between the two variables (F ¼ 1:5; df ¼ 9, 24; P ¼ 0:21) was significant. Using collection times in common to both the Monroe and Oktibbeha county collection sites, an interaction was observed between gut region and collection location (F ¼ 3:5; df ¼ 3, 36; P ¼ 0:03), but not between gut region and

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Table 1 Comparison of Metschnikowia pulcherrima (Barnett et al., 1990; CBS, 2000) and combined Group VI yeasts isolated from Chrysoperla rufilabris adults in Mississippi Characteristic

Met. pulcherrima

S821

S1585

S1597

S1728

S1740

Sucrose Maltose a; a-Trehalose Methyl-a-D -glucoside Cellobiose Melibiose Lactose Raffinose Melezitose Glycerol Ribitol Xylitol D -mannitol myo-Inositol

+a + +,) +, Dc ) + + + + + ) ) ) + + +, D + + )

+ + + + ) + + + + ) ) ) ) + + + + + )

+ + )b + ) + + + + ) ) ) ) + + + + + )

+ + + D ) + + + + ) ) ) ) + + + + + )

+ + + D ) + + + + ) ) ) ) + + + + + )

+ + ) D ) + + + + ) ) ) ) + + + + + )

Fermentation D -glucose Maltose Lactose D -galactose Sucrose Needle-shaped ascospores

+ ) ) ), D ) +

+ ) ) ) ) +

+, Wd ) ) ) ) +

) ) ) ) ) +

+ +, W ) +, W ) +

+ ) ) ) ) +

Assimilation D -glucose D -galactose D -glucosamine D -xylose L -arabinose

a

Positive reaction. Negative reaction. c Delayed reaction. d Weak reaction. b

collection time (F ¼ 1:6; df ¼ 6, 36; P ¼ 0:19), or collection time and collection location (F ¼ 0:08; df ¼ 2, 12; P ¼ 0:92). The significant interaction observed between gut region and collection location was attributed to the substantially larger populations (P < 0:001) of bacteria isolated from the midguts of adults collected from Monroe county. Twenty-five taxa of bacteria were recovered from the alimentary canal of Chr. rufilabris adults, including: Bacillus cereus Frankland and Frankland, Brevibacterium otitidis Pascual et al., Brevibacterium sp. Carnobacterium gallinarum Collins et al., Deinococcus proteolyticus (Kobatake et al.) Brooks and Murray, Enterobacter aerogenes Hormaeche and Edwards, Ent. cloacae (Jordan) Hormaeche and Edwards, Ent. cancerogenus (Urosevic) Dickey and Zumoff, Enterobacter sp., Enterococcus casseliflavus (Vaughan et al.) Collins et al., Ent. cecorum (Devriese et al.) Williams et al., Ent. faecium (Orla-Jensen) Schleifer and Kilpper-Balz, Enterococcus sp., Klebsiella planticola Bagley et al., Kle. terrigena Izard et al., Klebsiella sp., Lactococcus garviae (Collins et al.) Schleifer et al., Leuconostoc lactis Garvie, Pediococcus pentosaceus Mees, Pediococcus sp., Rahnella

aquatilis Izard et al., Serratia marcescens Bizio, Staphylococcus lentus (Kloos et al.) Schleifer et al., Sta. simulans Kloos and Schleifer, and Streptococcus suis (Elliott) Kilpper-Balz and Schleifer. Enterobacter aerogenes was the most prevalent taxon recovered, but considerable variability in isolation frequency was observed among the different collection times and the two collection sites. For example, this bacterium was only isolated from the diverticula (time 4) and midguts (time 3 and 4) of adults collected from Monroe country. In contrast, Ent. aerogenes was isolated from all four gut regions of insects collected in Oktibbeha country, but only from insects collected at sample time 3 and 5. Enterobacter cloacae also was isolated from all four gut regions, but only from insects collected at Monroe county at time 5. The remaining taxa were infrequently isolated. 3.5. Microorganisms associated with newly eclosed adults No yeasts were recovered from any of the 24-h-old adults that had eclosed from laboratory reared Chr. rufilabris larvae.

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Fig. 8. Populations of bacteria (log10 colony forming units (CFU)) recovered from the four gut regions of the alimentary canals of field-collected Chrysoperla rufilabris adults on trypticase soy agar. Insects were collected at different times and at two locations in Mississippi, where MC: Monroe county collection site, OC: Oktibbeha county collection site; time 1 ¼ Julian day (jd) 275 in 2000, time 2 ¼ jd 112 in 2001, time 3 ¼ jd 121 and 124 in 2001, time 4 ¼ jd 145 and 150 in 2001, and time 5 ¼ jd 194 and 201 in 2001. (A) diverticulum; (B) foregut; (C) midgut; and (D) hindgut. The asterisk indicates that collections were not made, and the number associated with vertical bars represents the number of adults positive for bacteria. Vertical lines represent standard errors of means (n ¼ 3).

4. Discussion A number of reports have indicated that yeasts are present in diverticula of chrysopid adults (Hagen and Tassan, 1966, 1972; Hagen et al., 1970; Johnson, 1982). To test the hypothesis that chrysopid adults possess a resident microflora within their alimentary canals, we isolated microorganisms from the diverticula, foreguts, midguts, and hindguts of Chr. rufilabris adults collected on four dates at two disparate field locations in Mississippi. Independent of the collection time and location, large populations of the yeast, Metschnkowia pulcherrima, were observed in the diverticula and foregut regions of the alimentary canal in the majority of the adults (17 of 24) that we collected. Furthermore, large numbers of yeast cells were observed in the diverticulum, and cells tended to accumulate within prominent folds within this region of the alimentary canal. In contrast to yeasts, no taxon of bacteria or filamentous fungi were consistently isolated from any of the gut regions at the different sample times and locations. The high frequency that

Met. pulcherrima was associated with field-collected adults suggests that it is a resident of the alimentary canal of Cry. rufilabris in Mississippi. However, Met. pulcherrima was not isolated from seven (29%) of the adults sampled. In all seven instances, no yeasts were recovered from the diverticula. One possibility to explain this observation is that adults obtain Met. pulcherrima from the environment and that the adults that we processed on these occasions were newly eclosed and therefore, they had not had an opportunity to obtain yeasts. Hagen et al. (1970) suggested that Chr. carnea obtain yeasts from the environment based on the observation that larvae were devoid of yeasts. However, we have frequently isolated yeasts from the alimentary canals of field-collected Chr. rufilabris larvae in Mississippi (unpublished data). Therefore, to determine whether adults possess yeasts in their alimentary canals upon eclosion from pupae, we attempted to isolate yeasts from the alimentary canals of recently emerged adults. In no instance did we isolated yeasts from these individuals supporting Hagen et al.Õs (1970) conclusion that

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Chr. rufilabris adults acquire yeasts from the environment, and that they are not transmitted vertically. The genus, Metschnikowia, is ubiquitous in nature. In particular, Met. pulcherrima had been isolated from fruits, surfaces of flowers, and nectar (e.g., common milkweed) (CBS, 2000; Eisikowitch et al., 1989, 1990). Although yeasts are commonly associated with the alimentary canals of insects (Ba and Phillips, 1996; Bismanis, 1976), Met. pulcherrima does not commonly form associations with insects. However, it has been isolated from bees (Barnett et al., 1990), it has been found (i.e., reported as Candida pulcherrima (Lindner)) in the feces from larvae of the fruit maggot, Laspeyresia pomonella (Linn
e) (Listemann, 1988), and Gibson and Hunter (2002) recently isolated Met. pulcherrima from laboratory-reared Chrysoperla comanche Banks adults in Arizona. Hagen et al. (1970) reported that a yeast, identified as Torulopsis sp., was prominent in the diverticula of Chr. carnea adults. A number of anamorphic Torulopsis species have been reduced into synonomy with Met. pulcherrima including Torulopsis burgeffiana, Torulopsis dattila var. rohrbachense, Torulopsis pulcherrima f. acolorata, Torulopsis pulcherrima var. pulcherrima, Torulopsis pulcherrima var. rubra, and Torulopsis pulcherrima var. variabilis (CBS, 2000). Unfortunately Hagen et al. (1970) did not identify the yeast associated with Chr. carnea adults to species, and no specimens were deposited in a culture collection to our knowledge. Johnson (1982) isolated the yeast, Candida multigemmis (synonym ¼ Torulopsis multigemmis), from the adults of several chrysopid species, but this yeast was not isolated from Chr. rufilabris adults in Mississippi. To obtain information on the relative importance of various gut regions in harboring microorganisms, we quantified and identified microorganisms in the four regions of the alimentary canal: the diverticulum, foregut, midgut, and hindgut. The frequency of occurrence of microbial taxa can provide evidence on the contribution that these microorganisms make to the insect. In particular, we wished to determine if microbiological evidence supports HagenÕs (1986) supposition that yeasts located in the esophageal diverticulum provide adults with essential amino acids that are not readily obtained in their diet components. We observed substantially larger populations of yeasts in the foregut and diverticulum relative to the midgut and hindgut regions. However, the various regions varied in volume and this confounded direct comparisons of population densities. Unfortunately, given the small size of the gut tissues and liquid/hemolymph associated with these tissues following dissection, it was not possible to accurately weigh the regions prior to conducting microbiological assessments. To address the different volumes of the four gut regions, we used image analysis of an image of an intact alimentary canal from a Chr. rufilabris adult. After adjusting for area, populations of yeasts averaged 1.6
103

CFU in the foregut, 3.7
103 CFU in the diverticulum, 2.0
102 CFU in the midgut, and 8.3
101 CFU in the hindgut (i.e., ileum). On average, the density of yeasts in the diverticulum was 2, 18, and 44 times greater than in the foregut, midgut, and hindgut of Chr. rufilabris adults, respectively. We utilized the dilution-plate method to recover the microorganisms. This is the conventional technique used to isolate microorganisms from the environment, including insect guts. However, the vast majority of microorganisms are not capable of growth in culture (Amman et al., 1994), and the dilution-plate method will not detect these microorganisms. Even if microorganisms are capable of growing on microbiological media, another major limitation of the dilution-plate method is that rare-occurring or poor growing isolates will most likely go undetected. Furthermore, this method favors the isolation of single-celled microorganisms and propagules, and provides a very conservative estimate of biomass for filamentous forms. We made every attempt to isolate rare-occurring isolates. In addition to Met. pulcherrima, we isolated two basidiomycetous taxa of yeasts; Cry. luteolus and Cry. victoriae. Cryptococcus luteolus has been isolated from air, leaves of tropical plants, and acidic sludge, whereas, Cry. victoriae was isolated from soil and flowering plants of Heleborus foetidus (Barnett et al., 1990; CBS, 2000). If the mechanism of ingestion and transfer of yeast cells to the diverticulum is nonspecific, it seems reasonable to conclude that Cryptococcus yeasts were present in the same environment where the adults obtained Met. pulcherrima, and they were ingested simultaneously. In most instances, relatively small populations of bacteria were recovered from the alimentary canals of Chr. rufilabris adults. Most bacteria were in the family Enterobacteriaceae, and the two most common taxa recovered were Ent. aerogenes and Ent. cloacae. Many members of the Enterobacteriaceae are common in the environment occurring in fresh water, soil, sewage, plants, vegetables, and animals including insect guts (Holt et al., 1994). Their cosmopolitan nature would allow them to be easily acquired by chrysopid adults from the environment. Given the infrequency of occurrence of specific taxa in the various gut regions of adults collected from various locations at different times, we conclude that ‘‘culturable’’ bacteria are transients within the alimentary canals of Chr. rufilabris adults. Most taxa of filamentous fungi recovered from Chr. rufilabris in Mississippi were common phytopathogens and/or residents of the phyllosphere. Fus. moniliforme was the most common taxon isolated; interestingly, this fungal taxon was reported as one of the most common soil-borne pathogens involved in the cotton seedling disease complex in the southeast of the United States (Killebrew, 1999). In addition, this taxon is also commonly associated with corn (Lucas et al., 1985; Stevens,

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1925). Similarly to bacteria, the low isolation frequency of filamentous fungi indicates that they are not residents of Chr. rufilabris guts. To facilitate integrated pest management with chrysopids, we conducted an in-depth examination of the microflora associated with field-collected Chr. rufilabris adults in Mississippi to ascertain the possibility of symbiotic relationships with bacteria and/or fungi. Our results indicated that the yeast, Met. pulcherrima, frequently occurred in the alimentary canals of Chr. rufilabris adults, with the largest populations of this yeast occurring in the cuticular lined diverticulum and foregut. The high frequency of isolation of Met. pulcherrima across different locations and collection times implicate this yeast as a potential symbiont of Chr. rufilabris adults. Hagen and Tassan (1972) postulated that chrysopid adults from different locations possess different yeast taxa in their crops, and that the yeast symbionts determine the fitness of non-indigenous chrysopids. This possibility has yet to be tested, but our research indicates that specific yeasts may be a prerequisite for the success of biological control programs using Chrysoperla species. Given the predominance of Met. pulcherrima in the alimentary canals of field-collected Chr. rufilabris adults in Mississippi, this yeast may be a better choice as a food component in rearing programs. Inoculating adults with Met. pulcherrima may have positive effects on a number of fitness parameters, including fecundity. This alone would greatly facilitate the use of predators as efficacious alternative to chemical insecticides for managing insect pests. Studies to elucidate the role of yeast taxa on Chrysoperla adults are required, and our findings facilitate such studies.

Acknowledgments We are indebted to the following people: Allen Cohen (Biological Control and Mass Rearing Research Unit (BCMRRU), USDA-ARS, Mississippi State) for his advice on the planning and completion of the experiments, for providing Chr. rufilabris eggs, and for providing a critical review of the manuscript; David Cross (Department of Entomology and Plant Pathology (EPP), Mississippi State University (MSU)) for his assistance with the field collection of lacewing adults from Monroe County, Mississippi; Terry Schiefer (EPPMSU) for his assistance on the identification of lacewing adults; Farid BalaÕa (Forage and Waste Management Unit, USDA-ARS, Mississippi State) and Brent Selinger (Department of Biological Sciences, University of Lethbridge, AB) for allowing access to the Automated Biolog system for yeasts; Kathaleen House and Kaarina Benkel (Agriculture & Agri-Food Canada (AAFC), Lethbridge, AB) for their assistance with the sequencing

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of 18S rDNA; Byron Lee (AAFC, Lethbridge, AB) for quantifying the alimentary canal areas; Randy Clear (Canadian Grain Commission, Winnipeg, MB) for identifying species of Fusarium; and Jay Yanke (AAFC, Lethbridge, AB), Grant Duke (AAFC, Lethbridge, AB), and Eric Riddick (BCMRRU, USDA-ARS, Mississippi State) for conducting critical reviews of the manuscript. This research was funded by Departmental Graduate Fellowship to SWW, and is MAFES contribution [J10371].

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