m y c o l o g i c a l r e s e a r c h 1 1 0 ( 2 0 0 6 ) 346 – 356 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/mycres Metschnikowia noctiluminum sp. nov., Metschnikowia corniflorae sp. nov., and Candida chrysomelidarum sp. nov., isolated from green lacewings and beetles Nhu H. NGUYEN, Sung-Oui SUH, Cennet K. ERBIL, Meredith BLACKWELL* Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA article info abstract Article history: Fourteen yeast isolates belonging to the Metschnikowia clade were isolated from the diges- Received 6 May 2005 tive tracts of lacewings (Neuroptera: Chrysopidae), soldier beetles and leaf beetles (Coleoptera: Received in revised form Cantharidae and Chrysomelidae), and a caddisfly (Trichoptera: Hydropsychidae). The insect 21 September 2005 hosts were associated with sugary substances of plants, a typical habitat for yeasts in Accepted 3 November 2005 this clade. Based on DNA sequence comparisons and phenetic characters, the yeasts Published online 14 February 2006 were identified as Candida picachoensis, Candida pimensis, and four undescribed taxa. Among Corresponding Editor: Teun Boekhout the undescribed taxa, three yeasts were distinguished from one another and from other described taxa by nucleotide differences in the ribosomal DNA repeat, which were sufficient Keywords: to consider them as new species. Two of the novel yeast species are described as Metschni- Coleoptera kowia noctiluminum (NRRL Y-27753T) and M. corniflorae spp. nov. (NRRL Y-27750T) based in Insect-fungus interactions part on production of needle-shaped ascospores, which are found in most Metschnikowia Molecular phylogeny species. Sexual reproduction was not observed in the third new yeast, Candida chrysomeli- Neuroptera darum sp. nov. (NRRL Y-27749T). A fourth isolate, NRRL Y-27752, was not significantly dis- Yeasts tinct from Metschnikowia viticola and Candida kofuensis to be described as a new species. Phylogenetic analysis of the D1/D2 loop sequences placed M. noctiluminum within the M. viticola clade, while C. chrysomelidarum was a sister taxon of Candida rancensis. Metschnikowia corniflorae was phylogenetically distinct from other new species and fell outside of the large-spored Metschnikowia group. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Yeasts in the genus Metschnikowia and a few related Candida species are often found associated with a variety of substrates including plants, insects, aquatic arthropods and sea water (Miller & Phaff 1998; Barnett et al. 2000). Those directly associated with insect and flower communities, predominantly beetles, have been well documented by various researchers (Gimenez-Jurado et al. 2003; Hong et al. 2003; Lachance & Bowles 2002, 2004; Lachance et al. 1998a, 1998b, 2001a, 2001b, 2003, 2005). However these yeasts were isolated from the surface of the host insects. The digestive tract environment of insects is a largely unexplored habitat for many microorganisms, including yeasts and yeast-like fungi. An incredible array of previously unknown yeasts occurring in clusters throughout the yeast phylogenetic tree has been discovered in the gut of beetles and other insects (e.g. Suh & Blackwell 2004, 2005; Suh et al. 2003, 2004a, 2004b, 2005a, 2005b; Zhang et al. 2003). More recently, yeasts belonging to the Metschnikowia clade such as Metschnikowia chrysoperlae, Candida picachoensis, and Candida pimensis were isolated from the gut and egg surface of several Chrysoperla (green lacewing) * Corresponding author. E-mail address: mblackwell@lsu.edu 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2005.11.010 New yeast species from insects species (Neuroptera: Chrysopidae) collected in Arizona (Suh et al. 2004a), and Metschnikowia pulcherrima isolated from Chrysoperla rufilabris collected in Mississippi (Woolfolk & Inglis 2003). The basis of many interactions between yeasts and their insect hosts is not clear. However, many animals rely on microbial enzymes for the breakdown of plant cell wall polymers, and a variety of organisms have been implicated in these degradation processes. Regarding the use of enzymes to act on plant carbohydrates, some animals have been shown to rely on microbes to perform other functions such as fermentation to improve nutritional content of the food resources, detoxification of plant metabolites, and perhaps, even pheromone production (Martin 1987; Dowd 1989, 1991; Vega & Dowd 2005). To determine whether additional associations between gut fungi and insects exist, we sampled a broad range of insects to determine the presence or absence of yeasts in their digestive tracts. Among the yeasts isolated were several unknown species belonging within the Metschnikowia clade that were close relatives of yeasts reported to be associated with insects. Here we describe three new yeast species and discuss their host relations. Materials and methods Yeast isolation The methods for isolating yeasts from insects were described in detail previously (Suh & Blackwell 2004; Suh et al. 2004b). Adult insects were collected from several locations in Louisiana (USA) and Barro Colorado Island (Panama; Table 1). Insects were separated individually into clean containers with dampened filter paper for 3 d and submerged in 95 % ethanol for 2–3 min to disinfect the surface; disinfection was followed by a wash with 0.7 % saline. The aseptically removed gut contents and the saline wash solution was plated separately on acidified YM agar (Difco YM broth, 2 % plain agar, adjusted to pH 3.5 with hydrochloric acid) and incubated at 25
C for 3–4 d. Single yeast colonies were purified at least twice and maintained on 2 % malt extract agar (Yarrow 1998). Type strains of new species were deposited in the Agricultural Research Service Culture Collection (NRRL) and duplicates at Centraalbureau voor Schimmelcultures (CBS; Table 1). Morphological and physiological observations were performed according to established methods (Yarrow 1998; Barnett et al. 2000). Ascospore formation was determined for all isolates by first singly plating then crossing isolates with similar or identical D1/D2 sequences in all combinations on dilute (1:19) V8 agar at 17
C (Barnett et al. 2000) and observed periodically up to two months. Phylogenetic analysis Nucleic acids were extracted and purified using the procedures of previous reports (Suh & Blackwell 2004; Suh et al. 2004b). The primer sets NS1–NS8, LS1–LR5, and ITS5–ITS4 were used for PCR amplification of the rDNA repeats. Purified double-stranded PCR products served as templates for sequencing with an ABI PRISMÔ BigDye Terminator Cycle 347 sequencing kit version, 3.1 (Applied Biosystems, Foster City, CA, USA). The complete sequence of the SSU rDNA, D1/D2 region of LSU rDNA, and the ITS and 5.8 S rDNA were obtained with the primers NS1, NS2, 18H, NS5, NS8, LS1, LR3, ITS1, and ITS4 using an ABI PRISM 377 automated DNA sequencer. GenBank accession numbers for SSU and LSU rRNA gene (rDNA) sequences from this study are listed in Table 1. DNA sequences were aligned with the multialignment program Clustal X (Thompson et al. 1997), and alignments were optimized visually; the beginnings and ends of sequences were excluded from the analyses. The sequences from newly isolated yeasts were compared with sequences acquired from GenBank. Saccharomyces cerevisiae was designated as the outgroup taxon. Maximum parsimony analyses were performed using PAUP 4.0b10 (Swofford 2002). Heuristic tree searches were executed using the tree bisection–reconnection branch-swapping algorithm with random sequence analysis. Bootstrap values of the most parsimonious tree were obtained from 1000 replications. Bayesian MCMC (B-MCMC) analysis was performed with MrBayes version 3.0b4 (Huelsenbeck 2000) to estimate the probability of nodes. The analysis consisted of 500 000 generations of four chains sampling every 10 generations, and the first 50 000 generations were discarded as burn in. The remaining trees were imported into PAUP to estimate the posterior probability. Base-pair differences in a gene were counted using BLAST 2 searches (Tatusova & Madden 1999) or from a manually aligned sequence database. PCR fingerprinting The methods and conditions of DNA extraction, PCR, and electrophoresis for microsatellite-primed PCR (MSP-PCR) were performed according to the methods of Sampaio et al. (2001). Two primers, (GTG)5 and (GAG)5, were used for the analysis. RAPD methods and analysis were performed according to the methods of Su et al. (2001) with the primers OPA2, OPA8, OPA14 and OPA16. Gel patterns were read manually, put into PAUP 4.0b10 as binary codes, and analysed using UPGMA algorithm. Results Yeast isolates and new species Fourteen yeast cultures were isolated from the digestive tracts of lacewings, soldier beetles, leaf beetles, and a caddisfly (Table 1). The results from BLAST searches of LSU rDNA D1/D2 loop sequences showed that all of the new yeasts were similar to species of Metschnikowia and their anamorphs. Isolates with identical sequences were grouped by genotype: (1) IY 03-8-14-1-3-1 and three other isolates from the gut of C. rufilabris; (2) NRRL Y-27751 and NRRL Y27817, also from C. rufilabris; (3) NRRL Y-27829 isolated from an unidentified lacewing; (4) NRRL Y-27753 and IY 038-14-2-2-2 from the gut of Ceraeochrysa lineaticornis; (5) NRRL Y-27750 and NRRL Y-27816 isolated from the gut of a flower-feeding cantharid beetle, Rhaxonycha sp.; (6) NRRL Y-27749 and BG 02-7-16-002A-2-1 from chrysomelid beetles; 348 Table 1 – Metschnikowia and Candida yeasts species isolated from this study Species Strain designation CBS NRRL Metschnikowia noctiluminum 9907 Y-27753T Genotype LSU GenBank no. SSU rDNA LSU rDNA Base pair difference in D1/D2 loopa Source IY 03-8-14-2-2-1 4 AY611606 AY 611609 T IY 03-8-14-2-2-2 4 - - 0 BG 03-5-4-1-1 5 AY611607 AY611610 T BG 03-5-4-1-2 5 - - 0 BG 02-7-16-002A-1-1 6 AY520164 AY520294 T BG 02-7-16-002A-2-1 6 - - 0 Isolate NRRL Y-27752 9906 Y-27752 IY 03-6-7-2-3-2 7 AY611608 AY611611 T Gut of Hydropsyche sp. (Trichoptera: Hydropsychidae), caught at lights, USA: Louisiana: Walker. Candida picachoensis - IY 03-8-14-1-3-1 1 - - 0 Gut of Chrysoperla rufilabris (Neuroptera: Chrysopidae), caught at light, USA: Louisiana: Walker. Gut of C. rufilabris, caught at light, USA: Louisiana: Walker. Gut of C. rufilabris, caught sweeping vegetation, USA: Louisiana: Baton Rouge. Gut of C. rufilabris, caught sweeping vegetation, USA: Louisiana: Baton Rouge. Gut of C. rufilabris, caught sweeping vegetation, USA: Louisiana: Walker. Gut of C. rufilabris, caught sweeping vegetation, USA: Louisiana: Baton Rouge. - - Metschnikowia corniflorae 9905 Y-27750T - Y-27816 Candida chrysomelidarum 9904 Y-27749T - - - - IY 03-8-14-1-3-2 IY 04-1-3-1-1-1 1 1 - - 0 0 - - IY 04-1-3-1-4-1 1 - - 0 - Y-27751 IY 03-5-25-3-1-1 2 - AY611612 2 10068 Y-27817 IY 04-1-3-1-2-1 2 - AY611613 2 Candida pimensis 10069 Y-27829 IY 03-5-10-4-1-2 3 - - 0 Gut of Ceraeochrysa lineaticornis (Neuroptera: Chrysopidae), caught at lights, USA: Walker, Louisiana. Gut of C. lineaticornis, caught at lights, USA: Louisiana: Walker. Gut of Rhaxonycha sp. (Coleoptera: Cantharidae), ex flowers of Cornus drummondii, Baton Rouge, Louisiana, USA. Gut of Rhaxonycha sp., ex flowers of C. drummondii, USA: Louisiana: Baton Rouge. Gut of unidentified chrysomelid (Coleoptera: Chrysomelidae), Panama: Barro Colorado Island. Gut of unidentified chrysomelid, Panama: Barro Colorado Island. a The D1/D2 loop sequences were compared with those of the type strains of each species. The differences included gaps, insertions, and substitutions. The D1/D2 sequences of C. picachoensis and C. pimensis from this study were compared with those of the type strains of the species, NRRL Y-27607T and NRRL Y-27619T, respectively. ITS and 5.8SrDNA sequences for isolates within the Metschnikowia viticola clade are DQ002494 for isolate NRRL Y-27752, DQ002495 for M. noctiluminum (Y-27753T), DQ002492 for Metschnikowia viticola (CBS 9950T), and DQ002493 for Candida kofuensis (NRRL Y-27226T). CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; NRRL, Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA; LSU, Mycology Laboratory, Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, USA; T, type strain. N. H. Nguyen et al. Gut of unidentified chrysopid, caught sweeping vegetation, USA: Louisiana: Baton Rouge. New yeast species from insects and (7) NRRL Y-27752 from a caddisfly of the genus Hydropsyche. Sometimes phenotypic differences among colonies isolated from the gut of a single host were observed. In such cases several colonies were purified in order to obtain all potential genetic diversity, and although these colonies usually were identical in all characters, NRRL Y-27753 (IY 03-8-14-2-2-1) and IY 03-8-14-2-2-2 differed in the rate of ascospore formation. Yeasts with genotypes 1 and 2 were identified as C. picachoensis and yeast with genotype 3 as C. pimensis based on the identical (or almost identical) D1/D2 loop sequences with the type of the respective species. Genotype 2 isolates differed from the type strain of C. picachoensis by only two nucleotide substitutions. On diluted V8 agar, the two isolates of genotype 2 produced pedunculate asci (Fig 1), which are characteristic of the genus Metschnikowia (Miller & Phaff 1998). However, no ascopores were observed within these structures after periodic observation from 3 d up to six months. The result was the same after repeated observations on 2 % malt agar. Based on the great similarity of D1/ D2 sequences, the two isolates were considered to be conspecific with C. picachoensis, although there were variations between these isolates and the type strain for assimilation of D-glucosamine both as a carbon and a nitrogen source (data not shown). The results from MSP-PCR also supported placement of genotype 2 isolates as C. picachoensis (Fig 2). Identical band patterns were observed for the type strain of C. picachoensis, a strain with identical D1/D2 sequence, and the two isolates of genotype 2 (lanes 1–4 in Fig 2); the patterns of the C. pimensis type and genotype 3 were clearly distinguished from those of C. picachoensis (lanes 5–6 in Fig 2). However, identical D1/D2 loop sequences and other taxonomic characters were used as criteria to place this isolate in the same species (Suh et al. 2004a). Yeasts with genotypes 4–6 were clearly distinguished from one another by sequence variation and were considered as new species. Comparisons of the D1/D2 sequences of yeasts with genotype 5 did not show a high degree of similarity with any members of Metschnikowia clade. The result of a BLAST search indicated that the closest sequence to this Fig 1 – Sterile ascus of Candida picachoensis NRRL Y-27751 on diluted V8 juice agar (1:19) at 17
C after five weeks. Bar [ 5 mm. 349 Fig 2 – Microsatellite-primed PCR (MSP-PCR) patterns of insect associated yeasts obtained with primers (GAG)5 (lanes 1–10) and (GTG)5 (lanes 11–14). Lanes 1–4, C. picachoensis (1, NRRL Y-27607T; 2, NRRL Y-27608; 3, NRRL Y-27751; 4, NRRL Y-27817); lanes 5–6, C. pimensis (5, NRRL Y-27619T; 6, NRRL Y-27829); lanes 7 and 11, M. noctiluminum NRRL Y-27753T; lanes 8 and 12, isolate NRRL Y-27752; lanes 9 and 13, C. kofuensis NRRL Y-27226T; lanes 10 and 14, M. viticola CBS 9950T. M, Molecular weight marker; C, Negative control. genotype was that of Metschnikowia krissii, but with low similarity. Yeasts with genotype 6 differed from Candida rancensis by more than 40 bp in the D1/D2 loop. Yeasts with genotype 4 were distinguished from those of the nearest genotypes (genotype 7, Candida kofuensis, and Metschnikowia viticola) by 6–8 and 22–25 nucleotide differences in the D1/D2 loop and ITS regions, respectively. MSP-PCR and RAPD patterns easily distinguished yeasts of genotype 4 from those of other genotypes (Figs 2–3). Therefore, we consider the variation of these isolates and those of genotypes 5 and 6 sufficient to be recognized as an independent species. The genotype 7 isolate, NRRL Y-27752, differed from M. viticola by 4 bp (three gaps and one substitution), from C. kofuensis by 6 bp, and from genotype 4 isolates by 6 bp in the D1/D2 loop. The ITS region was 4 bp different from M. viticola, 13 bp from C. kofuensis, and 24 bp from genotype 4 isolates. Péter et al. (2005) considered M. viticola to be a possible teleomorph of C. kofuensis based on their similar D1/D2 sequences (2 bp difference). In addition to small differences in D1/D2 loop sequences among members of this group, there are other differences such as the assimilation results for D-ribose and succinate (Table 2). Conjugation, clamydospore, or ascospore formation was not observed in cultures of the genotype 4 isolate or in crosses with C. kofuensis. It is often difficult to establish species limits among similar strains. The task is even more difficult when strains apparently reproduce only asexually and geographical distributions are poorly known. The closely related clade members may merely be different strains of a highly variable species. Additional isolates will be necessary to resolve this question, and we refer to this yeast as ‘‘isolate NRRL Y-27752’’. Therefore, here we describe the three yeasts with unique genotypes as new species in the genera Metschnikowia (isolates with genotypes 4 and 5) and Candida (isolates with genotypes 6). 350 N. H. Nguyen et al. Fig 3 – Random amplified polymorphic DNA (RAPD) patterns from primers OPA2 (lanes 1–6), OPA8 (lanes 7–12), OPA14 (lanes 13–18) and OPA16 (lanes19–24). Lanes 1, 7, 13, and 19, M. noctiluminum Y-27753T; lanes 2, 8, 14, and 20, M. noctiluminum IY 03-8-14-2-2-2. Lanes 3, 9, 15, and 21, isolate NRRL Y-27752; lanes 4, 10, 16, and 22, C. kofuensis Y-27226T; lanes 5, 11, 17, and 23, M. viticola CBS 9950T; lanes 6, 12, 18, and 24, M. viticola CBS 9949. M, Molecular weight marker; C, Negative control. Taxonomy Metschnikowia noctiluminum N.H. Nguyen, S.O. Suh, Erbil & M. Blackw. sp. nov. (Fig 4A–C) Etym.: The species epithet, noctiluminum (noc.ti’lu.mi.num) L. gen. plu. neu. n., ‘‘of the night lights,’’ refers to the attraction of the insect hosts towards the shimmering night lights of North Corbin Elementary School, Walker, LA, USA, the collection locality of the type strain. In medio liquido dextrosum et peptonum et extractum levidenis continente post 7 dies ad 25
C cellulae vegetative subglobosae aut ellipsoideae (5–8  6–9 mm), singulae vel binae; pseudohyphae non fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25
C, cremea et butyrosa. In agaro farina Zeae maydis confecto post 7 dies ad 25
C, pseudohyphae et hyphae verae non fiunt. Ascosporae fiunt in agaro V8 cremor post 14 dies ad 17
C. Glucosum fermentatur. Galactosum, maltosum, a-methyl-D-glucosidum, sucrosum, trehalosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Glucosum, galactosum (infirme), L-sorbosum, D-glucosaminum (infirme), sucrosum (lente), maltosum (lente), trehalosum (lente), a-methyl-D-glucosidum (infirme), cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, ribitolum (infirme), xylitolum (infirme), D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum, DLacidum lacticum (infirme), acidum succinicum et acidum citricum (infirme) assimilantur. D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, L-arabinitolum, galactitolum, inositolum, D-glucuronatum, methanolum, ethanolum, propane-1, 2-diolum, butano-2, 3-diolum, acidum quinicum et D-glucaratum non assimilantur. Ethylaminum, L-lysinum cadaverinum et glucosaminum (infirme) assimilantur. Kalium nitratum, natrium nitritum, creatinum, creatininum, imidazolum et D-tryptophanum non assimilantur. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum in temperatura 35
C non fiunt. Non crescit in medio 10 mg mll cycloheximido addito. Typus: USA: Louisiana: Walker, isolata a ile neuropterorum adultus (Chrysopidae: Ceraeochrysa lineaticornis), 2003, e.g. N.H. Nguyen et al. (NRRL Y-27753 – holotypus; CBS 9907 – isotypus). After 7 d growth in YM broth at 25
C cells are subglobose to ellipsoidal (5–8  6–9 mm), and occur singly, in pairs, or in chains (Fig 4A). Pseudohyphae are not present. After 7 d on YM agar at 25
C, colonies are cream and butyrous with an undulate margin. After 7 d on Dalmau plate culture on corn meal agar at 25
C, pseudohyphae or true hyphae are not present. Aerobic growth is off-white in color with an undulate margin. Asci arise from chlamydospores on dilute V8 juice agar after 14 d at 17
C. Asci are sphaeropedunculate, usually 20–40 mm in length, and containing mostly one, but sometimes two needle-shaped ascospores, after three weeks on diluted V8 agar (Fig 4B and C). Ascospores are usually stable within the ascus and are released in the presence of Cladosporium gossypiicola (unpublished observation, Fig 4C). See Table 2 for a summary of physiological characteristics. Metschnikowia corniflorae N.H. Nguyen, S.O. Suh, Erbil & M. Blackwell, sp. nov. (Fig 4D and E) Etym.: The species epithet, corniflorae (cor.ni’flor.ae) L. gen. sing. fem. n., ‘‘of Cornus flowers,’’ refers to Cornus drummondii flowers on which the host beetle was collected. In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25
C cellulae vegetative subglobosae aut ovoidae (3–6  3–6 mm), singulae vel binae; pseudohyphae non fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25
C, albida et butyrosa. In agaro farina Zeae maydis confecto post 7 dies ad 25
C, pseudohyphae et hyphae verae non fiunt. Ascosporae fiunt in agaro V8 cremor post 7 dies ad 17
C. Glucosum fermentatur. Galactosum, maltosum, a-methyl-D-glucosidum, sucrosum, trehalosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Glucosum, galactosum, L-sorbosum, New yeast species from insects 351 Table 2 – Physiological characters of the new yeast species and related taxa in this study Metschnikowia corniflorae Candida chrysomelidarum Metschnikowia noctiluminum Isolate NRRL Y-27752 Metschnikowia viticola Candida kofuensis Assimilation of carbon sources D-galactose D-glucosamine D-ribose D-xylose L-arabinose D-arabinose Sucrose Maltose Trehalose a-Methyl-D-glucoside Lactose Raffinose Glycerol Ribitol Xylitol L-arabinitol D-glucitol D-gluconate DL-lactate Succinate Citrate Ethanol 1,2-Propanediol Quinic acid þ þ þ d w w þ þ þ þ þ þ þ þ þ w þ þ w þ þ w w þ d þ þ þ d þ þ d þ þ d þ - w w d d d w þ w w þ þ w þ w - d w þ d þ d d d þ d þ þ þ w d - þ þ/w d þ þ þ þ/w þ þ þ þ þ/s þ þ þ -* þ þ -* d* þ þ þ þ þ þ þ* -* þ þ þ þ -* -* -* Assimilation of nitrogen sources Nitrite Creatine Creatinine D-glucosamine Imidazole D-tryptophan w - w - w - w - na na na na na na na Vitamin requirements w/o Thiamin w/o Pyridoxine and thiamine þ þ þ þ þ þ w w na na na na w þ - þ þ þ w - þ þ þ w - d þ þ - þ þ þ þ þ/w þ þ* -* w -* Growth At 30
C On 50 % D-glucose On 60 % D-glucose On 10 % sodium chloride On 16 % sodium chloride The following characteristics were invariable in all species compared. Fermentation of D-glucose (þ), D-galactose (-), maltose (-), a-methyl-Dglucoside (-), sucrose (-), a,a-trehalose (-), melibiose (-), lactose (-), cellobiose (-), melezitose (þ), raffinose (-), inulin (-), starch (-), D-xylose (-); assimilation of D-glucose (þ), L-sorbose (þ), L-rhamnose (-), cellobiose (þ), salicin (þ), arbutin (þ), melibiose (-), melezitose (þ), inulin (-), soluble starch (-), erythritol (-), D-mannitol (þ), galactitol (-), myo-inositol (-), D-glucono-1,5-lactone (þ*), 2-keto-D-gluconate (þ*), D-glucuronate (-*), methanol (-), butane 2,3 diol (-), D-glucarate (-), nitrate (-), ethylamine (þ), L-lysine (þ), cadaverine (þ); growth on media without (w/o) all vitamins (-), w/o myo-Inositol (þ), w/o pantothenate (þ), w/o biotin (-), w/o biotin and thiamin (-), w/o pyridoxine (þ), w/o niacin (þ), w/o p-aminobenzoic acid (þ); growth on 0.01 % cycloheximide (-), 0.1 % cycloheximide (-), growth at 35
C (-), 1 % acetic acid (-); starch formation (-); urea hydrolysis (-); diazonium blue B reaction (-). Abbreviations: þ, positive reaction; -, negative reaction; d, delayed positive reaction; w, weak positive reaction; na, not available. Data for C. kofuensis and M. viticola were from Mikata et al. (1999) and Péter et al. (2005) except for the results marked with an asterisk (*) obtained from this study. The assimilation results of D-ribose, D-xylose and ethanol for C. kofuensis differed from the original description (Mikata et al. 1999). D-glucosaminum, D-ribosum, D-xylosum (lente), L-arabinosum (infirme), D-arabinosum (infirme), sucrosum, maltosum, trehalosum, a-methyl-D-glucosidum, cellobiosum, salicinum, arbutinum, lactosum, raffinosum, melezitosum, glycerolum, ribitolum, xylitolum, L-arabinitolum (infirme), D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum, DL-acidum lacticum (infirme), acidum succinicum, acidum citricum, ethanolum (infirme), propane-1, 2-diolum (infirme) et acidum quinicum assimilantur. L-rhamnosum, melibiosum, inulinum, amylum solubile, erythritolum, galactitolum, inositolum, D-glucuronatum, methanolum, butano-2, 3-diolum, D-glucaratum et D-galactonatum non assimilantur. Ethylaminum, L-lysinum, cadaverinum et 352 N. H. Nguyen et al. Figs 4 – New yeasts isolated from insects in this study. A–C. M. noctiluminum. A Budding yeast cells of NRRL Y-27753T on YM agar at 25
C after 7 d. B Asci and ascospores of NRRL Y-27753T on diluted V8 juice agar at 17
C after two weeks. C Asci and ascospore release of IY 03-8-14-2-2-2 on diluted V8 juice agar at 17
C after three weeks. Release of ascospores was induced by culturing yeast cells with Cladosporium gossypiicola, CBS 117483 (data unpublished). D–E M. corniflorae NRRL Y-27750T. D Budding yeast cells on YM agar at 25
C after 7 d. E Ascospores on diluted V8 juice agar at 17
C after two weeks. F Budding yeast cells of C. chrysomelidarum NRRL Y-27749T on YM agar at 25
C after 7 d. All bars [ 5 mm. glucosaminum (infirme) assimilantur. Kalium nitratum, natrium nitritum, creatinum, creatininum, imidazolum et D-tryptophanum non assimilantur. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum in temperatura 30
C non fiunt. Non crescit in medio 10 mg mll cycloheximido addito. Typus: USA: Louisiana: Baton Rouge, Isolata a ile coleoptorum adultus (Cantharidae: Rhaxonycha sp.) ex floris Cornus drummondii, 2003, e.g. N.H. Nguyen et al. (NRRL Y-22750 – holotypus; CBS 9905 – isotypus). After 7 d growth in YM broth at 25
C cells are subglobose to oval (3–6  3–6 mm), and occur singly or in pairs (Fig 4D). Pseudohyphae are not present. After 7 d on YM agar at 25
C colonies are off-white to cream and butyrous with a rough surface. After 7 d on Dalmau plate culture on corn meal agar at 25
C, pseudohyphae or true hyphae are not observed. Aerobic growth is off-white with an undulating margin. Asci arise from chlamydospores on diluted V8 juice agar after 7 d at 17
C. Asci are sphaeropedunculate, usually 10–20 mm in length, and containing one to two needle-shaped ascospores (Fig 4E). Ascospores were observed in the type strain of the species, NRRL Y-27750T, but neither clamydospores nor ascospores were observed in the other strain, NRRL Y-27816. See Table 2 for a summary of physiological characteristics. Candida chrysomelidarum N.H. Nguyen, S.O. Suh, Erbil & M. Blackw., sp. nov. (Fig 4F) Etym.: The species epithet, chrysomelidarum (chry.so.me. lid’a.rum) L. gen. plu. fem. n., ‘‘of Chrysomelidae,’’ refers to the host beetle family Chrysomelidae. In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25
C cellulae vegetative ellipsoidae (4– 7  5–9 mm), singulae vel binae; pseudohyphae non fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25
C, cremea et butyrosa. In agaro farina Zeae maydis confecto post 7 dies ad 25
C, pseudohyphae et hyphae verae non fiunt. Ascosporae non fiunt. Glucosum fermentatur. Galactosum, maltosum, a-methyl-D-glucosidum, sucrosum, trehalosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Glucosum, L-sorbosum, D-glucosaminum (lente), sucrosum, maltosum, trehalosum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum (lente), ribitolum, xylitolum, D-glucitolum (lente), D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum, acidum succinicum, acidum citricum (lente) et ethanolum assimilantur. Galactosum, D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, a-methyl-D-glucosidum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, L-arabinitolum, galactitolum, inositolum, D-glucuronatum, DLacidum lacticum, methanolum, propane-1, 2-diolum, butano-2, 3-diolum, acidum quinicum et D-glucaratum non assimilantur. Ethylaminum, L-lysinum, cadaverinum et glucosaminum (lente) assimilantur. Kalium nitratum, natrium nitritum, creatinum, creatininum, imidazolum et D-tryptophanum non assimilantur. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum in temperatura 35
C non fiunt. Non crescit in medio 10 mg mll cycloheximido addito. Typus: Panama: Barro Colorado Island, isolata a ile coleopterorum adultus (Chrysomelidae), 2002, e.g. Suh et al. (NRRL Y27749 – holotypus; CBS 9904 – isotypus). After 7 d growth in YM broth at 25
C cells are ellipsoidal (4– 7  5–9 mm), and occur singly or in pairs (Fig 4F). Pseudohyphae New yeast species from insects are not present. After 7 d on YM agar at 25
C colonies are white and butyrous. After 7 d Dalmau plate culture on corn meal agar at 25
C, pseudohyphae and true hyphae are not present. Aerobic growth is off-white and smooth with a glossy surface. No ascospores are present on corn meal agar or diluted V8 juice agar after two months. See Table 2 for a summary of physiological characteristics. 353 unsupported branch basal to the large-spored Metschnikowia clade, placing it between C. kipukae and M. corniflorae (tree not shown). Identification of Metschnikowia spp. using traditional physiological methods is becoming increasingly difficult because of homogeneity as more species are discovered in this rapidly growing genus (Lachance et al. 1998b, 2003), and the use of D1/ D2 rDNA sequences is essential for rapid identification of these species. Discussion Host relations Phylogeny of the new yeast species from insects A consensus of 12 most-parsimonious trees based on sequences of the D1/D2 region of the LSU rDNA included the three new species, previously described Metschnikowia species, and related anamorphs available from GenBank (Fig 5). Multiple gene data sets will be necessary to determine the topology of the deeper branches shown as unresolved on the tree in Fig 5 (Rokas et al. 2003). The position of terminal groups, however, is assumed to indicate close relationships. The new species did not form a monophyletic group, but were placed in three subclades among other members of the Metschnikowia clade: (1) M. noctiluminum with M. viticola, C. kofuensis, and isolate NRRL Y-27752 with 100 % bootstrap support; (2) C. chrysomelidarum and C. rancensis with high statistical support and; (3) M. corniflorae basal to the large-spored Metschnikowia clade with moderate support (Fig 5). M. noctiluminum was a well-supported basal member of the M. viticola clade (A, Fig 5). The clade itself received 100 % bootstrap support. However, the position of the sister taxa were not well resolved, and as mentioned earlier, they may belong to the same taxon. C. chrysomelidarum and C. rancensis formed a statistically well-supported clade, although the position of this clade among other members of the Metschnikowia clade was not fully resolved by the comparison of D1/D2 sequences (Fig 5). From the analysis based on the combined sequences of SSU and LSU rDNA, C. chrysomelidarum also belonged to a major subclade of the Metschnikowia clade that included Metschnikowia gruessii, Metschnikowia australis, Metschnikowia bicuspidata, Metschnikowia krissii, Metschnikowia zobellii, Metschnilkowia reukaufii, Candida kofuensis, and M. viticola with comparatively high statistical support (85 % by bootstrapping and 96 % by B-MCMC analyses, data not shown). M. corniflorae was a basal member of the large-spored Metschnikowia clade and was phylogenetically distinct from all of the Metschnikowia spp. compared (Fig 5). However, its phylogenetic position within the Metschnikowia clade was questionable as there was no well-supported close sister taxon. The closest species, based on BLAST searches of the D1/D2 sequence, was M. krissii, which occurs in a different clade of the tree. Analyses of combined SSU and LSU rDNA sequences showed the species to form a branch with Metschnikowia lunata with 98 % bootstrap value (data not shown). M. corniflorae is among the few species (including M. lunata, Metschnikowia kunwiensis and Metschnikowia hibisci) that occur between the large-spored Metschnikowia clade and other Metschnikowia spp. in phylogenetic analyses (Lachance et al. 1998b). A separate analysis (with the short D1/D2 sequence of M. hibisci included) showed that M. hibisci formed an The yeasts associated with lacewings were placed into three distinct clades: (A) Metschnikowia noctiluminum with M. viticola, C. kofuensis, and isolate Y-27752 (B) Candida picachoensis and C. pimensis, and (C) M. chrysoperlae and related taxa (A, B, C in Fig 5). Although there have been reports of yeast associates of lacewings, only within the last two years were several species studied using molecular techniques (e.g. Suh et al. 2004a; Woolfolk & Inglis 2003). Suh et al. (2004a) described two new species, C. pimensis and C. picachoensis, from Chrysoperla carnea and other unidentified individuals of Chrysoperla respectively collected from Arizona. In the present study, the same yeast species also were present in C. rufilabris and in an unidentified lacewing species collected in Louisiana. Multiple collections of both species from the same lacewings in geographically distant localities indicates that these yeasts are fairly widespread and not limited to certain environments or geographical regions and the yeasts are probably not just transients but have some degree of host specificity. Conversely M. noctiluminum, isolated from a previously unsampled green lacewing species, Ceraeochrysa lineaticornis, was phylogenetically distinct from other lacewing associates (Fig 5). Although lacewings in the genera Ceraeochrysa and Chrysoperla both belong to the tribe Chrypsopini, they have different behaviour and habitat usage. The habitat differences may explain the occurrence of phylogenetically distinct yeasts in their guts. Lacewing associates from this study, M. noctiluminum, C. picachoensis and C. pimensis, were obtained from adult lacewings. Although larvae of green lacewings are generalist predators, especially effective against aphids, the adults are not predaceous and feed on nectar, pollen and aphid honeydew. More study will be necessary in order to understand the possible interactions between the yeasts and the lacewings, perhaps as suggested for other insect associated yeasts, such as those of the Pichia stipitis clade with xylose-fermenting capabilities in wood-boring beetles or detoxifying yeasts in drugstore beetles (e.g. Dowd 1989, 1991; Suh et al. 2003). M. corniflorae was isolated from the gut of adult cantharid beetles, Rhaxonycha sp., collected from flowers of the swamp dogwood, C. drummondii (Table 1). The general habitat occupied by Rhaxonycha spp. is somewhat similar to that of lacewings mentioned above. Both the larvae and adults of the beetles are predaceous on other insects, but adults also probably feed on nectar and/or pollen (Arnett et al. 2002). Phylogenetic analysis placed the yeast among species that have been reported from studies of yeast communities of flowers with insect vectors (Lachance et al. 1998b, 1999, 2001b, 2005). We do not know if the beetles merely serve as dispersers for M. corniflorae or if a more specific interaction occurs between them. 354 N. H. Nguyen et al. Fig 5 – A consensus of 12 most-parsimonious trees obtained from sequence analysis of the LSU rDNA D1/D2 region. Reference sequences of Metschnikowia spp. were selected to show the position of the three new species (bold type) and isolate NRRL Y-27752 based on sequence comparisons of SSU and LSU rDNA. Saccharomyces cerevisiae was used as the outgroup taxon. Tree length [ 1193; CI [ 0.4342; homoplasy index [ 0.5658; RI [ 0.6832; rescaled CI [ 0.2967. Numbers on the branches (i.e. 100/100) are results of statistical analyses. The number to the left of the slash indicates bootstrap support value above 50 % in 1000 replicates with parsimony analysis. The number to the right of the slash represents probability nodes with Bayesian analysis. Arrow indicates the large-spored Metschnikowia (LSM) clade. A, B, and C, are clades with lacewing associated yeasts; *, genotypes with identical D1/D2 sequences to the type strain shown on the tree. New yeast species from insects C. chrysomelidarum was isolated only from the gut of adult chrysomelid beetles from Panama, while its closest relative, Candida rancensis, was isolated from rotting logs of Laurelia sempervirens in Chile (Ramı́rez & González 1984). The majority of chrysomelid beetle larvae are phytophagous, while the adults feed on foliage and sometimes flower parts including pollen (Arnett et al. 2002). Isolate NRRL Y-27752, isolated from an adult caddisfly, Hydropsyche sp., was closely related to C. kofuensis and M. viticola from grapes in Japan and Hungary, respectively (Mikata et al. 1999; Péter et al. 2005). The larvae of Hydropsyche sp. are aquatic and omnivorous, feeding on organic material that flows through their silken nets; the adults, however, sometimes feed on nectar and honeydew as is likely in this case (Malicky 1989). As we have reviewed above, the insects in this study occupied varied habitats, but in general most were associated with plants. The life histories of the insects vary in feeding guild with larvae being mostly predaceous and adults often feeding on or otherwise associated with flowers. The adult insect hosts almost certainly are important dispersers of the yeasts, but the possibility of additional interactions among the yeasts and insects cannot be excluded. The yeasts reported here are both taxonomically and physiologically different from most strains we have previously studied from beetles feeding on a variety of basidiomata (Suh & Blackwell 2004; Suh et al. 2004b) or wood (Suh et al. 2003). These results are to be expected based on the differences in the biology of the hosts and the nutritional resources available to the yeasts. Acknowledgements We thank Cletus Kurtzman (NRRL) and Teun Boekhout and Vincent Robert (CBS) for providing reference yeast cultures and for placing the yeast isolates from this study in culture collections under their care. We also thank Catherine Tauber and Michael Ferro for identifying the lacewing and caddisfly hosts, respectively, and John Morse for references on adult caddisfly biology. We acknowledge Glenda Newman, principal, North Corbin Elementary School, Walker, LA, USA, for permission to collect at the school. Some collections were made with Joseph V. McHugh at the Smithsonian Tropical Research Institution, Barro Colorado Research Station, Panama, and we thank him and the Smithsonian staff for logistical support in obtaining accommodations and permits. The staff of the M. D. Socolofsky Sciences Microscopy Facility, Louisiana State University, graciously assisted with microscopic expertise during this study. The GenBank public database is also acknowledged. This research was supported by the National Science Foundation, Biodiversity Surveys and Inventories Program (DEB-0072741 and DEB-0417180), including REU Supplements, and a Howard Hughes Medical Institute grant through the Undergraduate Biological Sciences Education Program to Louisiana State University. We also acknowledge the use of the DNA sequencing facility supported by NSF Multiuser Equipment Grant (DBI-0400797) to Robb Brumfield. 355 references Arnett Jr RH, Thomas MC, Skelley PE, Frand JH, Thomas MC, 2002. American Beetles, Volume II: Polyphaga: Scarabaeoidea through Curculionoidea. CRC Press, Boca Raton. Barnett JA, Payne RW, Yarrow D, 2000. Yeasts: Characteristics and Identification, 3rd edn. Cambridge University Press, Cambridge. Dowd PF, 1989. In situ production of hydrolytic detoxifying enzymes by symbiotic yeasts in the cigarette beetle (Coleoptera: Anobiidae). Journal of Economic Entomology 82: 396–400. Dowd PF, 1991. Symbiont-mediated detoxification in insect herbivores. In: Barbosa P, Krischik VA, Jones CG (eds), Microbial Mediation of Plant–Herbivore Interactions. John Wiley & Sons, New York, pp. 411–440. Gimenez-Jurado G, Kurtzman CP, Starmer WT, Spencer-Martins I, 2003. Metschnikowia vanudenii sp. nov. and Metschnikowia lachancei sp. nov., from flowers and associated insects in North America. International Journal of Systematic and Evolutionary Microbiology 53: 1665–1670. Hong SG, Bae KS, Herzberg M, Titze A, Lachance MA, 2003. Candida kunwiensis sp. nov., a yeast associated with flowers and bumblebees. International Journal of Systematic and Evolutionary Microbiology 53: 367–372. Huelsenbeck JP, 2000. MrBayes: Bayesian inference of phylogeny Distributed by the author. Department of Biology, University of Rochester. Lachance M-A, Bowles JM, 2002. Metschnikowia arizonensis and Metschnikowia dekortorum, two new large-spored yeast species associated with floricolous beetles. FEMS Yeast Research 2: 81–86. Lachance M-A, Bowles JM, 2004. Metschnikowia similis sp. nov. and Metschnikowia colocasiae sp. nov., two ascomycetous yeasts isolated from Conotelus spp. (Coleoptera: Nitidulidae) in Costa Rica. Studies in Mycology 50: 69–76. Lachance M-A, Bowles JM, Kwon S, Marinoni G, Starmer WT, Janzen DH, 2001a. Metschnikowia lochheadii and Metschnikowia drosophilae, two new yeast species isolated from insects associated with flowers. Canadian Journal of Microbiology 47: 103–109. Lachance M-A, Bowles JM, Starmer WT, 2003. Metschnikowia santaceciliae, Candida hawaiiana, and Candida kipukae, three new yeast species associated with insects of tropical morning glory. FEMS Yeast Research 3: 97–103. Lachance M-A, Bowles JM, Starmer WT, Barker JSF, 1999. Kodamaea kakaduensis and Candida tolerans, two new ascomycetous yeast species from Australian hibiscus flowers. Canadian Journal of Microbiology 45: 172–177. Lachance M-A, Ewing CP, Bowles JM, Starmer WT, 2005. Metschnikowia hamakuensis sp. nov, Metschnikowia kamakouana sp. nov., and Metschnikowia mauinuiana sp. nov., three endemic yeasts from Hawaiian nitidulid beetles. International Journal of Systematic and Evolutionary Microbiology 55: 1369–1377. Lachance M-A, Rosa CA, Starmer WT, Bowles JM, 1998a. Candida ipomoeae, a new yeast species related to large-spored Metschnikowia species. Canadian Journal of Microbiology 44: 718–722. Lachance M-A, Rosa CA, Starmer WT, Schlag-Edler B, Barker JSF, Bowles JM, 1998b. Metschnikowia continentalis var. borealis, Metschnikowia continentalis var. continentalis, and Metschnikowia hibisci, new heterothallic haploid yeasts from ephemeral flowers and associated insects. Canadian Journal of Microbiology 44: 279–288. Lachance M-A, Starmer WT, Rosa CA, Bowles JM, Barker JSF, Janzen DH, 2001b. Biogeography of the yeasts of ephemeral flowers and their insects. FEMS Yeast Research 1: 1–8. Malicky H, 1989. Feeding of adult caddisflies. Trichoptera Newsletter 16: 18. 356 Martin M, 1987. Invertebrate–Microbe Interactions. Cornell University Press, Ithaca. Mikata K, Ueda-Nishimura K, Goto S, Kurtzman CP, Suzuki M, Yarrow D, Nakase T, 1999. Reidentification of yeast strains deposited as Candida agrestis, with a description of Candida kofuensis sp. nov. Microbiology & Culture Collection 15: 49–57. Miller MW, Phaff HJ, 1998. Metschnikowia Kamiensky. In: Kurtzman CP, Fell JW (eds), The Yeasts: a Taxonomic Study, 4th edn. Elsevier, Amsterdam, pp. 256–267. Péter G, Tornai-Lehoczki J, Suzuki M, Dlauchy D, 2005. Metschnikowia viticola sp. nov., a new yeast species from grape. Antonie van Leeuwenhoek 88: 155–160. Ramı́rez C, González A, 1984. Two new amycelial Candida isolated from decayed wood in the evergreen rainy Valdivian forest of southern Chile. Mycopathologia 88: 99–103. Rokas A, Williams BL, King N, Carroll SB, 2003. Genome-scale approaches to resolving incongruence in molecular phylogenies. Nature 425: 798–804. Sampaio JP, Gadanho M, Santos S, Duarte F, Pais C, Fonseca A, Fell JW, 2001. Polyphasic taxonomy of the genus Rhodosporidium: R. kratochvilovae and related anamorphic species. International Journal of Systematic and Evolutionary Microbiology 51: 687–697. Su G, Suh S-O, Schneider RW, Russin JS, 2001. Host specialization in the charcoal rot fungus, Macrophomina phaseolina. Phytopathology 91: 120–126. Suh S-O, Blackwell M, 2004. Three new beetle-associated yeasts in the Pichia guilliermondii clade. FEMS Yeast Research 5: 87–95. Suh S-O, Blackwell M, 2005. The beetle gut as a habitat for new species of yeasts. In: Vega FE, Blackwell M (eds), Insect Fungal Associations: Ecology and Evolution. Oxford University Press, New York, pp. 244–256. Suh S-O, Gibson CM, Blackwell M, 2004a. Metschnikowia chrysoperlae sp. nov., Candida picachoensis sp. nov. and Candida pimensis sp. nov., isolated from the green lacewings Chrysoperla comanche and Chrysoperla carnea (Neuroptera: Chrysopidae). International Journal of Systematic and Evolutionary Microbiology 54: 1883–1890. N. H. Nguyen et al. Suh S-O, Marshall CJ, McHugh JV, Blackwell M, 2003. Wood ingestion by passalid beetles in the presence of xylosefermenting gut yeasts. Molecular Ecology 12: 3137–3145. Suh S-O, McHugh JV, Blackwell M, 2004b. Expansion of the Candida tanzawaensis yeast clade: 16 new Candida species from basidiocarp-feeding beetles. International Journal of Systematic and Evolutionary Microbiology 54: 2409–2429. Suh S-O, McHugh JV, Pollock DD, Blackwell M, 2005a. The beetle gut: a hyperdiverse source of novel yeasts. Mycological Research 109: 261–265. Suh S-O, Nguyen NH, Blackwell M, 2005b. Nine new Candida species near C. membranifaciens isolated from insects. Mycological Research 109: 1045–1056. Swofford DL, 2002. PAUP. Phylogenetic Analysis Using Parsimony (*and Other Methods), *version 4.0b10. Sinauer Associates, Sunderland, Massachusetts. Tatusova TA, Madden TL, 1999. Blast 2 sequencesda new tool for comparing protein and nucleotide sequences. FEMS Microbiology Letters 174: 247–250. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG, 1997. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882. Vega FE, Dowd PF, 2005. The role of yeasts as insect endosymbionts. In: Vega FE, Blackwell M (eds), Insect Fungal Associations: Ecology and Evolution. Oxford University Press, New York, pp. 211–243. Woolfolk SW, Inglis GD, 2003. Microorganisms associated with field-collected Chrysoperla rufilabris (Neuroptera: Chrysopidae) adults with emphasis on yeast symbionts. Biological Control 29: 155–168. Yarrow D, 1998. Methods for the isolation, maintenance and identification of yeasts. In: Kurtzman CP, Fell JW (eds), The Yeasts: a Taxonomic Study, 4th edn. Elsevier, Amsterdam, pp. 77–100. Zhang N, Suh S-O, Blackwell M, 2003. Microorganisms in the gut of beetles: evidence from molecular cloning. Journal of Invertebrate Pathology 84: 226–233.