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Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Ascomycete yeasts are found commonly in the guts of basidioma-feeding beetles but little is known about their occurrence in the gut of other insects. In this study we isolated 95 yeasts from the gut of adult insects in five neuropteran families (Neuroptera: Corydalidae, Chrysopidae, Ascalaphidae, Mantispidae and Hemerobiidae) and a roach (Blattodea: Blattidae). Based on DNA sequence comparisons and other taxonomic characteristics, they were identified as more than 15 species of Saccharomycetes as well as occasional Cryptococcus-like basidiomycete yeasts. Yeast species such as Lachancea fermentati, Lachancea thermotolerans and Hanseniaspora vineae were isolated repeatedly from the gut of three species of corydalids, suggesting a close association of these species and their insect hosts. Among the yeasts isolated in this study 12 were identified as five novel Candida species that occurred in three phylogenetically distinct clades. Molecular phylogenetic analyses showed that Candida chauliodes sp. nov. (NRRL Y-27909T) and Candida corydali sp. nov. (NRRL Y-27910T) were sister taxa in the Candida albicans/ Lodderomyces elongisporus clade. Candida dosseyi sp. nov. (NRRL Y-27950T) and Candida blattae sp. nov. (NRRL Y-27698T) were sister taxa in the Candida intermedia clade. Candida ascalaphidarum sp. nov. (NRRL Y-27908T) fell on a basal branch in a clade containing Candida membranifaciens and many other insect-associated species. Descriptions of these novel yeast species are provided as well as discussion of their ecology in relation to their insect hosts.
Key words: green lacewings, insect-fungus associations, insect gut yeast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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, Schowalter 2000, Dudley 2002). The ecology of insects, including their interactions with symbiotic microorganisms, also may contribute to their success. In particular gut microorganisms range widely from intracellular bacteria such as Wolbachia and Buchnera (Werren 1997, Douglas 2003, Dillon and Dillon 2004) to eukaryotic microbes inhabiting the gut of New World termites and wood roaches (Keeling 2002, Kitade 2004). We are interested in the possibility that yeasts isolated from the guts of insects might produce essential amino acids, vitamins, enzymes for detoxification, degradation, and fermentation of food resources and even essential pheromone production (Dowd 1989, 1991, Vega and Dowd 2005).
Yeasts have been isolated frequently from the gut or surface of insects that feed on a variety of materials, including basidiomycete fruiting bodies, woody substrates, ephemeral flowers and nectar exudates (Kurtzman 2001, Lachance and Bowles 2002, 2004, Lachance et al 2001a, b, 2005, Rosa et al 2003, Suh et al 2003, 2004a, , Suh et al b, 2005, Teixeira et al 2003, Suh and Blackwell 2004, 2005, Pimentel et al 2005, Nguyen et al 2006). When they were discovered many of these yeasts represented novel species, some placed in entirely new clades (e.g. Suh et al 2004b, 2006). Most of the previous isolations, however, have been restricted to a small number of specific insect groups, primarily beetles (Coleoptera), various bees and wasps (Hymenoptera), flies (Diptera) and lacewings (Neuroptera). Because of the remarkable diversity of insects, we wondered whether yeasts occur regularly in the gut of other insects. Therefore we attempted to isolate yeasts from the gut and surface of insects in 13 orders (48 families), excluding those that feed directly on fungi or flowers. Although previously we discussed some yeasts associated with lacewings (Suh et al 2004a, Nguyen et al 2006) here we focus on additional isolates from other families of Neuroptera, including those that are associated with other insects in a more comprehensive study.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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). Individual insects were separated in clean containers lined with dampened filter paper and held 3 d. Methods for yeast isolation were detailed by Nguyen et al (2006) and Suh et al (2004b). Briefly, starved insects were submerged in 95% ethanol 2–3 min to disinfect the surface; this step was followed by a wash with 0.7% saline solution, and the wash liquid was plated on acidified YM agar (Difco YM broth, 2% plain agar, adjusted to pH 3.5 with HCl) to serve as negative control. Aseptically removed gut contents were plated separately on acidified YM agar 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). An attempt was made to isolate all colonies with different morphologies. Lyophilized holotype specimens and active cultures were deposited in the Agricultural Research Service Culture Collection (NRRL) and duplicate cultures at Centraalbureau voor Schimmelcultures (CBS) (TABLE I). Insect vouchers were deposited at the Louisiana State Arthropod Museum (LSAM), Baton Rouge, Louisiana. Morphological and physiological observations were performed according to Yarrow (1998) and Barnett et al (2000). Ascospore formation was determined for individual and similar isolates crossed in all combinations on dilute (1:19) V8, half strength cornmeal, and YM agars at 17 and 25 C, then observed periodically up to 2 mo.
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and Suh et al (2004b). The primer sets NS1-SS3E and LS1-LR5 were used for PCR amplification of the small subunit (SSU) and large subunit (LSU) rDNA. Purified double-stranded PCR products served as templates for sequencing with an ABI PRISMTM BigDye Terminator Cycle sequencing kit v. 3.1. Complete sequences of the SSU rDNA and D1/D2 region of LSU rDNA were obtained with primers NS1, 18H, SS3E, LS1 and LR3 using an ABI PRISM 377 automated DNA sequencer. The sequences from newly isolated yeasts were compared with sequences acquired from GenBank. These sequences from GenBank were used in analyses: Candida sp. BG 01-7-24-009A-1-1 (AY242231
[GenBank]
; AY242343
[GenBank]
), Candida sp. BG 02-7-19-003A-3-1 (AY520174
[GenBank]
; AY520304
[GenBank]
), Candida sp. BG 02-7-21-004G-1-3 (AY520187
[GenBank]
; AY520317
[GenBank]
), Candida sp. BCC 7717 (AY228490
[GenBank]
; AY228491
[GenBank]
), Candida sp. NRRL Y-27702 (AY640203
[GenBank]
; AY640205
[GenBank]
), Candida albicans (AB013586
[GenBank]
; U45776
[GenBank]
), C. ambrosiae (AY227713
[GenBank]
; AY013716
[GenBank]
), C. amphixiae (AY520197
[GenBank]
; AY520327
[GenBank]
), C. atlantica (AB013527
[GenBank]
; U45799
[GenBank]
), C. atmosphaerica (AB013526
[GenBank]
; U45779
[GenBank]
), C. arcana (AY242235
[GenBank]
; AY242347
[GenBank]
), C. bokatorum (AY426950
[GenBank]
; AY309798
[GenBank]
), C. boleticola (AB013532
[GenBank]
; U45777
[GenBank]
), C. bolitotheri (AY242142
[GenBank]
; AY242249
[GenBank]
), C. cerambycidarum (AY520169
[GenBank]
; AY520299
[GenBank]
), C. chrysomelidarum (AY520164
[GenBank]
; AY520294
[GenBank]
), C. elateridarum (AY518524
[GenBank]
; AY518530
[GenBank]
), C. ergatensis (AB013524
[GenBank]
; U45746
[GenBank]
), C. ernobii (AY227714
[GenBank]
; U94921
[GenBank]
), C. cylindracea (AB013510
[GenBank]
; U45823
[GenBank]
), C. derodonti (AY242234
[GenBank]
; AY242346
[GenBank]
), C. dubliniensis (X99399
[GenBank]
; U57685
[GenBank]
), C. fructus (AB018163
[GenBank]
; U44810
[GenBank]
), C. fukazawae (AB027756
[GenBank]
; AY313957
[GenBank]
), C. fungicola (AB027754
[GenBank]
; AY313958
[GenBank]
), C. glabrata (AY198398
[GenBank]
; U44808
[GenBank]
), C. haemulonii (AB013572
[GenBank]
; AY267823
[GenBank]
), C. homilentoma (AB018166
[GenBank]
; U45716
[GenBank]
), C. insectamans (AB013518
[GenBank]
; U45791
[GenBank]
), C. insectorum (AB013565
[GenBank]
; U45791
[GenBank]
), C. intermedia (AB013571
[GenBank]
; U44809
[GenBank]
), C. kruisii (AB013543
[GenBank]
; U45718
[GenBank]
), C. lyxosophila (AB013522
[GenBank]
; U76204
[GenBank]
), C. maltosa (D14593
[GenBank]
; U45745
[GenBank]
), C. membranifaciens (AB013551
[GenBank]
; U45792
[GenBank]
), C. mesenterica (AB013552
[GenBank]
; U45720
[GenBank]
), C. multigemmis (AB013535
[GenBank]
; U45782
[GenBank]
), C. nemodendra (AB054546
[GenBank]
; U70246
[GenBank]
), C. oleophila (AB013534
[GenBank]
; U45793
[GenBank]
), C. oregonensis (AB013545
[GenBank]
; U44815
[GenBank]
), C. ovalis (AB054552
[GenBank]
; U70248
[GenBank]
), C. palmioleophila (AB013520
[GenBank]
; U45758
[GenBank]
), C. parapsilosis (AB013588
[GenBank]
; U45754
[GenBank]
), C. peltata (AB054559
[GenBank]
; U71066
[GenBank]
), C. picachoensis (AY452053
[GenBank]
; AY452039
[GenBank]
), C. pimensis (AY452055
[GenBank]
; AY452051
[GenBank]
), C. plutei (AY520388
[GenBank]
; AY520259
[GenBank]
), C. pseudoglaebosa (AB013544
[GenBank]
; U71072
[GenBank]
), C. psychrophila (AB013528
[GenBank]
; U45813
[GenBank]
), C. pyralidae (AY488127
[GenBank]
; AY013715
[GenBank]
), C. quercitrusa (AB013579
[GenBank]
; U45831
[GenBank]
), C. rhagii (U63037
[GenBank]
; U45729
[GenBank]
), C. sagamina (AB027755
[GenBank]
; AY313959
[GenBank]
), C. sake (AB013529
[GenBank]
; AJ507662
[GenBank]
), C. shehatae var. insectosa (AB013584
[GenBank]
; U45773
[GenBank]
), C. silvicultrix (AB054561
[GenBank]
; U69879
[GenBank]
), C. sinolaborantium (AY520198
[GenBank]
; AY520328
[GenBank]
), C. sojae (AB013549
[GenBank]
; U71070
[GenBank]
), C. sophiae-reginae (AB013542
[GenBank]
; U45817
[GenBank]
), C. stellimalicola (AB054573
[GenBank]
; U84234
[GenBank]
), C. suecica (AB013554
[GenBank]
; U45732
[GenBank]
), C. taliae (AY242145
[GenBank]
; AY242254
[GenBank]
), C. tanzawaensis (AY227713
[GenBank]
; U44811
[GenBank]
), C. temnochilae (AY242232
[GenBank]
; AY242344
[GenBank]
), C. tenuis (AB013516
[GenBank]
; U45774
[GenBank]
), C. tropicalis (M55527
[GenBank]
; U45749
[GenBank]
), C. tsuchiyae (AB013506
[GenBank]
; U49064
[GenBank]
), C. viswanathii (AB013589
[GenBank]
; U45752
[GenBank]
), C. carpophila (AY227715
[GenBank]
; U45707
[GenBank]
), C. xylopsoci (AY488128
[GenBank]
; AY013719
[GenBank]
), Clavispora lusitaniae (M55526
[GenBank]
; U44817
[GenBank]
), Debaryomyces hansenii (X58053
[GenBank]
; U45808
[GenBank]
), Dekkera bruxellensis (X58052
[GenBank]
; U45738
[GenBank]
), Dipodascus tetrasperma (AB000654
[GenBank]
; U40086
[GenBank]
), Eremothecium gossypii (AE016820
[GenBank]
; AE016820
[GenBank]
), Galactomyces geotrichum (X69842
[GenBank]
; U40118
[GenBank]
), Geotrichum clavatum (AB000655
[GenBank]
; U40112
[GenBank]
), G. fermentans (AB000651
[GenBank]
; U40117
[GenBank]
), G. histeridarum (AY520227
[GenBank]
; AY520357
[GenBank]
), Hanseniaspora uvarum (X69844
[GenBank]
; U84229
[GenBank]
), H. valbyensis (AY046254
[GenBank]
; U73596
[GenBank]
), H. vineae (AY046258
[GenBank]
; U84224
[GenBank]
), Issatchenkia orientalis (AB053239
[GenBank]
; U76347
[GenBank]
), Kazachstania viticola (AY046234
[GenBank]
; AF398482
[GenBank]
), Kluyveromyces lactis (CR382124
[GenBank]
; CR382124
[GenBank]
), K. marxianus (X89524
[GenBank]
; U94924
[GenBank]
), Kodamaea laetipori (AY520270
[GenBank]
; AY520398
[GenBank]
), K. ohmeri (AY520231
[GenBank]
; AY242300
[GenBank]
), Lachancea fermentati (X77930
[GenBank]
; U84239
[GenBank]
), L. thermotolerans (X89526
[GenBank]
; U69581
[GenBank]
), L. waltii (X89527
[GenBank]
; U69582
[GenBank]
), Lodderomyces elongisporus (X78600
[GenBank]
; U45763
[GenBank]
), Magnusiomyces magnusii (AB000653
[GenBank]
; U40097
[GenBank]
), Metschnikowia australis (AB023465
[GenBank]
;U76526), M. bicuspidata (AB023466
[GenBank]
; U44822
[GenBank]
), M. chrysoperlae (AY452054
[GenBank]
; AY452047
[GenBank]
), M. corniflorae (AY611607
[GenBank]
; AY611610
[GenBank]
), M. noctiluminum (AY611606
[GenBank]
; AY611609
[GenBank]
), M. pulcherrima (AB023473
[GenBank]
; U45736
[GenBank]
), Nakaseomyces delphensis (X83823
[GenBank]
; U69576
[GenBank]
), Naumovia castellii (Z75577
[GenBank]
; AY545582
[GenBank]
), Saprochete suaveolens (AB000656
[GenBank]
; U40119
[GenBank]
), Pichia anomala (AB054562
[GenBank]
; AF330115
[GenBank]
), P. aribbica (AY553853
[GenBank]
; AY894826
[GenBank]
), P. holstii (AB018180
[GenBank]
; U75722
[GenBank]
), P. karawaiewii (AB018168
[GenBank]
; U94921
[GenBank]
), P. membranafaciens (X58055
[GenBank]
; U75725
[GenBank]
), P. methanolica (AB018181
[GenBank]
; U75523
[GenBank]
), P. nakasei (AB053235
[GenBank]
; U75728
[GenBank]
), P. nakazawae (AB054279
[GenBank]
; U45748
[GenBank]
), P. philogaea (AB054283
[GenBank]
; U45765
[GenBank]
), P. scolyti (AB054286
[GenBank]
; U45788
[GenBank]
), P. spartinae (AB054292
[GenBank]
; U45764
[GenBank]
), P. stipitis (AB054280
[GenBank]
; U45741
[GenBank]
), P. triangularis (AB013575
[GenBank]
; U45796
[GenBank]
), Saccharomyces cariocanus (AY046224
[GenBank]
; AY130342
[GenBank]
), S. cerevisiae (SCZ75578; AY048154
[GenBank]
), Saccharomycodes ludwigii (X69843
[GenBank]
; U73601
[GenBank]
), Saturnispora dispora (AB053251
[GenBank]
; U94937
[GenBank]
), Schizosaccharomyces pombe (X54866
[GenBank]
; Z19136
[GenBank]
), Starmera amethionina (AB017897
[GenBank]
; U75424
[GenBank]
), Starmerella bombicola (AB013558
[GenBank]
; U45705
[GenBank]
), Tetrapisispora phaffii (AY046245
[GenBank]
; AY048166
[GenBank]
), Torulaspora delbrueckii (AB087379
[GenBank]
; AB087393
[GenBank]
), Wickerhamiella domercqiae (AB018157
[GenBank]
; U45847
[GenBank]
), Williopsis saturnus var. mrakii (Y11318
[GenBank]
; U94929
[GenBank]
), Zygosaccharomyces rouxii (X90758
[GenBank]
; U72163
[GenBank]
), and Zygotorulaspora mrakii (AY046239
[GenBank]
; U72159
[GenBank]
).
DNA base pair differences were counted with BLAST 2 (Tatusova and Madden 1999) or from a manually aligned sequence database. DNA sequences initially were aligned with the multi-alignment program Clustal X (Thompson et al 1997) and optimized visually; all missing regions (e.g. beginnings and ends) were excluded from analyses. Maximum parsimony analyses were performed with PAUP 4.0b10 (Swofford 2002). Heuristic tree searches were executed with the tree bisection-reconnection branch-swapping algorithm with random sequence analysis. Bootstrap values of the most parsimonious tree were obtained from 1000 replications. The tree was obtained from a combined dataset of SSU and LSU rDNA sequences of representative species in Saccharomycotina based on the availability of both gene sequences. Branches containing new species were separately re-analyzed to include more aligned DNA characters and subsequently superimposed to form a single tree (FIG. 1). Schizosaccharomyces pombe was designated as outgroup taxon for all analyses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Rapid identification with the D1/D2 sequences indicated that 82 isolates were ascomycete yeasts in class Saccharomycetes and the remaining 13 were basidiomycete yeasts, mostly identified as Cryptococcus spp. From the sequence comparisons of the D1/D2 region the majority of ascomycete yeasts were similar to Lachancea fermentati, L. thermotolerans, Hanseniaspora vineae, Saccharomyces cerevisiae, S. cariocanus, Torulaspora delbrueckii, C. neerlandica, C. membranifaciens, Pichia guilliermondii, C. fermentati, C. quercitrusa, Saccharomycopsis fermentans, Kodamaea ohmeri, Pichia galeiformis, C. tropicalis, Ambroziozyma monospora, Metschnikowia chrysoperlae and Saprochete gigas (TABLE I
). Because these yeasts differed by only 0–2 base pairs in the D1/D2 region from the type strains of each species, they were considered conspecific using the phenetic standard proposed by Kurtzman and Robnett (1998). The six isolates near Candida neerlandica differed by three bp in the D1/D2 region along with 10 clear assimilation differences from the type strain (TABLES I and II). They are being studied in detail and will not be discussed further in this paper. Identification of yeasts near Saprochete gigas was based on the similarity of ITS sequences reported by Hoog and Smith (2004).
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, II). Some isolates formed hyphae, and all reproduced asexually by budding. Ascospores and asci were not observed in any of these species cultured alone or in crosses, thus placing them within the limits of Candida, a known polyphyletic grouping. The new species are described in at least three distinct clades as Candida chauliodes, Candida corydali, Candida ascalaphidarum, Candida dosseyi and Candida blattae. |
TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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-methyl-D-glucosidum, ,-trehalosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Glucosum, galactosum, L-sorbosum, D-glucosaminum, D-xylosum, L-arabinosum, trehalosum, glycerolum, ribitolum, xylitolum, D-glucitolum, D-mannitolum, D-glucono-1, 5-lactonum, 2-keto-D-gluconatum, D-gluconatum, acidum succinicum, acidum citricum et ethanolum assimilantur. D-ribosum, D-arabinosum, L-rhamnosum, sucrosum, maltosum, -methyl-D-glucosidum, cellobiosum, salicinum, arbutinum, melibiosum, lactosum, raffinosum, melezitosum, inulinum, amylum solubile, erythritolum, L-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, 1, 2-propanodiolum, 2, 3-butanodiolum, acidum quinicum et D-glucaratum non assimilantur. Ethylaminum, L-lysinum et cadaverinum assimilantur. Kalium nitratum, natrium nitritum, creatinum, creatininum, glucosaminum, imidazolum et D-tryptophanum non assimilantur. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum in 30 C, at non 35 C. In medio 10 µg mL–1 cycloheximido addito crestcit, nec 100 µg mL–l. HOLOTYPUS. NRRL Y-27909T (=CBS 10157) designat stirpem typicum. Isolata superficio neuropterum Chauliodes pectinicornis, Walker, Livingston Parish, Louisiana, USA, depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA.
After 7 d growth in YM broth at 25 C cells are mostly globose to some ellipsoidal (3–6 x 3–7 µm), and occur singly, in pairs, or clusters (FIG. 2). True hyphae and pseudohyphae are not present. After 7 d on YM agar at 25 C, colony surface is smooth, cream, butyrous with an undulate margin. After 7 d on Dalmau plate culture on cornmeal agar at 25 C true hyphae are present (FIG. 3) but pseudohyphae are not present. Aerobic growth is off white and the colony has a smooth surface. Colony margin is smooth with occasional filamentous growth. Asci are not produced under the conditions tested. (See TABLE II for a summary of physiological characteristics.)
Type strain. – NRRL Y-27909T (= CBS 10157) is preserved as a lyophilized preparation in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. The type strain, represented by a single isolate, was taken from the surface of a male fishfly, Chauliodes pectinicornis, at night under lights in Walker, Livingston Parish, Louisiana, USA.
Etymology. – The species epithet, chauliodes (L. plu. fem. adj.), refers to the genus of the host insect, Chauliodes pectinicornis.
Candida corydali N.H. Nguyen, S.-O. Suh et M. Blackwell sp. nov. FIG. 4
In medio liquido dextrosum et peptonum et extractum levidenis continente post 7 d ad 25 C cellulae vegetativae ellipsoidae aut globosae et subglobosae (3–7 x 3–8 µm), singulae, binae et racemae; pseudohyphae non fiunt. Cultura in agaro extramalti et faecis continente post 7 d ad 25 C, cremea et butyrosa, superfices teres. In agaro farina Zeae maydis confecto post 7 d ad 25 C, pseudohyphae et hyphae verae non fiunt. Ascosporae non fiunt. Glucosum, galactosum (lente), maltosum (infirme), ,-trehalosum (lente) fermentantur. -Methyl-D-glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Glucosum, galactosum, L-sorbosum, D-glucosaminum, D-ribosum (lente), D-xylosum (lente), L-arabinosum, sucrosum, maltosum, trehalosum, -methyl-D-glucosidum, salicinum, arbutinum, melezitosum, glycerolum, ribitolum, xylitolum, L-arabinitolum (infirme), D-glucitolum, D-mannitolum, D-glucono-1, 5-lactonum, 2-keto- D-gluconatum, D-gluconatum, DL-acidum lacticum (infirme), acidum succinicum, acidum citricum et ethanolum assimilantur. D-arabinosum, L-rhamnosum, cellobiosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, galactitolum, inositolum, D-glucuronatum, methanolum, 1,2-propanodiolum, 2,3-butanodiolum, acidum quinicum et D-glucaratum non assimilantur. Ethylaminum, L-lysinum et cadaverinum assimilantur. Kalium nitratum, natrium nitritum, creatinum, creatininum, glucosaminum, imidazolum et D-tryptophanum non assimilantur. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum in 30 C, at non 35 C. In medio 10 µg mL–1 cycloheximido addito infirme crestcit, nec 100 µg mL–l.
HOLOTYPUS. NRRL Y-27910T (=CBS 10158) designat stirpem typicum. Isolata in superficio neuropterum Corydalus cornutus, Walker, Livingston Parish, Louisiana, USA, depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA.
After 7 d growth in YM broth at 25 C cells are globose to ellipsoidal (3–7 x 3–8 µm) and occur singly, in pairs, or in small clusters (FIG. 4). Neither pseudohyphae nor true hyphae are present. After 7 d on YM agar at 25 C colonies are beige, butyrous with a smooth surface and margin. After 7 d on Dalmau plate culture on cornmeal agar at 25 C pseudohyphae and true hyphae are not present. Aerobic growth is off white with an undulate colony margin. Ascospores are not produced under the conditions tested. (See TABLE II for a summary of physiological characteristics.)
Type strain. – NRRL Y-27910T (=CBS 10158) is preserved as a lyophilized preparation in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. The type strain was isolated from the surface of a female Corydalus cornutus (Neuroptera: Corydalidae), at night under lights in Walker, Livingston Parish, Louisiana, USA.
Etymology. – The species epithet, corydali (L. gen. sing. masc. n.), "of Corydalus," refers to the genus of the host insect, Corydalus cornutus.
Candida ascalaphidarum N.H. Nguyen, S.-O. Suh et M. Blackwell sp. nov. FIGS. 5–6
In medio liquido dextrosum et peptonum et extractum levidenis continente post 7 d ad 25 C cellulae vegetativae globosae aut ellipsoideae (3–5 x 3–6 µm), singulae vel racemae. Hyphae verae et pseudohyphae non fiunt. Cultura in agaro extramalti et faecis continente post 7 d ad 25 C, cremea et butyrosa; superfices teres et aridus in media. In agaro farina Zeae maydis confecto post 7 d ad 25 C, hyphae verae fiunt sed pseudohyphae non fiunt. Ascosporae non fiunt. Glucosum, galactosum, maltosum, sucrosum (lente), et ,-trehalosum (lente) fermentantur. -Methyl-D-glucosidum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Glucosum, galactosum, D-glucosaminum, D-xylosum, L-arabinosum (lente), D-arabinosum (lente), sucrosum, maltosum, trehalosum, -methyl-D-glucosidum, L-arabinitolum (lente), cellobiosum, arbutinum, melezitosum, glycerolum, erythritolum, ribitolum, xylitolum, D-glucitolum, D-mannitolum, D-glucono-1, 5-lactonum, D-gluconatum (lente), acidum succinicum, acidum citricum, ethanolum et 1,2-propanodiolum (lente) assimilantur. L-sorbosum, D-ribosum, L-rhamnosum, salicinum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, galactitolum, inositolum, 2-keto-D-gluconatum, D-glucuronatum, DL-acidum lacticum, methanolum, 2,3-butanodiolum, acidum quinicum et D-glucaratum non assimilantur. Ethylaminum, L-lysinum et cadaverinum assimilantur. Kalium nitratum, natrium nitritum, creatinum, creatininum, glucosaminum, imidazolum et D-tryptophanum non assimilantur. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum in 35 C, at non 40 C. In medio 10 µg mL–l cycloheximido addito crestcit, nec 100 µg mL–l.
HOLOTYPUS. NRRL Y-27908T (= CBS 10156), designat stirpem typicum. Isolata in ile neuropterorum Ululodes macleayanus, Walker, Livingston Parish, Louisiana, USA, depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA.
After 7 d growth in YM broth at 25 C cells are mostly ellipsoidal to oval with some globose (3–5 x 3–6 µm), occurring singly, in pairs, or clusters (FIG. 5). True hyphae and pseudohyphae are not present. After 7 d on YM agar at 25 C, colony surface appears in two layers, the outer is smooth, cream and butyrous while the inner layer is white, slightly folded and dry. After 7 d on Dalmau plate culture on cornmeal agar at 25 C true hyphae are present (FIG. 6) but pseudohyphae are not present. Aerobic growth is off white and smooth. Colony margin is smooth with occasional filamentous growth. Asci are not produced under the conditions tested. (See TABLE II for a summary of physiological characteristics.)
Type strain. – NRRL Y-27908T (= CBS 10156) is preserved as a lyophilized preparation in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. The type strain was isolated from the gut of a male owlfly, Ululodes macleayanus, 2003 by N.H. Nguyen, at night under lights in Walker, Livingston Parish, Louisiana, USA.
Etymology. – The species epithet, ascalaphidarum (L. gen. fem. n.), "of Ascalaphidae," refers to the family of the host insect.
Candida dosseyi N.H. Nguyen, S.-O. Suh et M. Blackwell sp. nov. FIGS. 7–8
In medio liquido dextrosum et peptonum et extractum levidenis continente post 7 d ad 25 C cellulae vegetativae ellipsoidae, globosae et subglobosae (3–7 x 5–7 µm), singulae, binae et racemae parvae. Hyphae verae et pseudohyphae non fiunt. Cultura in agaro extramalti et faecis continente post 7 d ad 25 C, pineceus candidus, butyrosa, superfices teres. In agaro farina Zeae maydis confecto post 7 d ad 25 C, margina ciliata, superfices teres. Pseudohyphae hyphae fiunt sed hyphae verae non fiunt. Ascosporae non fiunt. Glucosum, galactosum, maltosum -methyl-D-glucosidum (lente), sucrosum, ,-trehalosum, cellobiosum (lente), melezitosum (lente), raffinosum et inulinum fermentantur. Melibiosum, lactosum, amylum solubile et D-xylosum non fermentantur. Glucosum, galac-tosum, D-glucosaminum, D-ribosum (lente), D-xylosum, D-arabinosum, L-rhamnosum, sucrosum, maltosum, trehalo-sum, -methyl-D-glucosidum, cellobiosum, salicinum, arbutinum, raffinosum, melezitosum, inulinum, glycerolum (lente), ribitolum, xylitolum, D-glucitolum, D-mannitolum, D-glucono-1, 5-lactonum, 2-keto-D-gluconatum, D-gluconatum, DL-acidum lacticum (infirme), acidum succinicum, acidum citricum, ethanolum, 1,2-propanodiolum (lente), et acidum quinicum assimilantur. L-sorbosum, L-arabinitolum, melibiosum, lactosum, amylum solubile, erythritolum, L-arabinosum, galactitolum, inositolum, D-glucuronatum, methanolum, 2,3-butanodiolum et D-glucaratum non assimilantur. Ethylaminum, L-lysinum et cadaverinum assimilantur. Kalium nitratum, natrium nitritum, creatinum, creatininum, glucosaminum, imidazolum et D-tryptophanum non assimilantur. Biotinum externum ad crescentiam necessarium est. Amylum non formatur. Diazonium caerulian B non respondens. Augmentum in 35 C at non 40 C. In medio 10 µg mL–l cycloheximido addito crestcit, nec 100 µg mL–l.
HOLOTYPUS. NRRL Y-27950T (=CBS 10313), designat stirpem typicum. Isolata in ile neuropterorum Corydalus cornutus, 2005, leg. A. Dossey, Port Hammock, Levy County, Florida, USA, et isolata ab N.H. Nguyen, et depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA.
After 7 d growth in YM broth at 25 C cells are globose to ellipsoidal (3–7 x 5–7 µm), and occur singly, in pairs, or in small clusters (FIG. 7). Pseudohyphae are present but not true hyphae. After 7 d on YM agar at 25 C, colonies are beige and slightly pinkish, butyrous with smooth surface and an undulate margin. After 7 d on Dalmau plate culture on cornmeal agar at 25 C pseudohyphae are present (FIG. 8) but true hyphae are not present. Aerobic growth is off white with a filamentous colony margin. Ascospores are not produced under the conditions tested. (See TABLE II for a summary of physiological characteristics.)
Type strain. – NRRL Y-27950T (=CBS 10313) is preserved as a lyophilized preparation in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. The type strain was isolated from the gut of a female Corydalus cornutus caught at night under lights in Port Hammock, Levy County, Florida, USA.
Etymology. – The species epithet, dosseyi (L. gen. masc. adj.), honors Aaron Dossey, who provided Florida insect specimens sampled in this study.
Candida blattae N.H. Nguyen, S.O. Suh et M. Blackwell sp. nov. FIGS. 9–10
In medio liquido dextrosum et peptonum et extractum levidenis continente post 7 d ad 25 C cellulae vegetativae globosae, ellipsoidae, aut subglobosae (3–5 x 4–6 µm), singulae, racemae, et catenae. Pseudohyphae fiunt sed hyphae verae non fiunt. Cultura in agaro extramalti et faecis continente post 7 d ad 25 C, alba, butyrosa, superfices teres. In agaro farina Zeae maydis confecto post 7 d ad 25 C, margina ciliata, superfices teres. Hyphae vera et pseudohyphae hyphae fiunt. Ascosporae non fiunt. Glucosum, galactosum (lente), maltosum (lente), sucrosum (lente), ,-trehalosum, cellobiosum (lente), melezitosum (lente), raffinosum et inulinum fermentantur. -Methyl-D-glucosidum, melibiosum, lactosum, amylum solubile et D-xylosum non fermentantur.
Glucosum, galactosum, L-sorbosum, D-glucosaminum, D-xylosum, D-arabinosum (lente), L-rhamnosum, sucrosum, maltosum, trehalosum, -methyl-D-glucosidum, cellobiosum, salicinum, arbutinum, raffinosum, melezitosum, inulinum, glycerolum, ribitolum, xylitolum, D-glucitolum, D-mannitolum, D-glucono-1, 5-lactonum, 2-keto-D-gluconatum, D-gluconatum, DL-acidum lacticum (infirme), acidum succinicum, acidum citricum, ethanolum, 1,2-propanodiolum (infirme) et acidum quinicum assimilantur. D-ribosum, L-arabinitolum, melibiosum, lactosum, amylum solubile, erythritolum, L-arabinosum, galactitolum, inositolum, D-glucuronatum, methanolum, 2,3-butanodiolum et D-glucaratum non assimilantur. Ethylaminum, L-lysinum et cadaverinum, glucosaminum et D-tryptophanum assimilantur. Kalium nitratum, natrium nitritum, creatinum, creatininum et imidazolum non assimilantur. Biotinum externum ad crescentiam necessarium est. Amylum non formatur. Diazonium caerulian B non respondens. Augmentum lente in 35 C at non 40 C. In medio 10 µg mL–l cycloheximido addito crestcit, nec 100 µg mL–l.
HOLOTYPUS. Y-27698T (= CBS 9871) designat stirpem typicum. Isolata in ile blattae, 2002, at Barro Colorado Island, Panama, et depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA.
After 7 d growth in YM broth at 25 C cells are globose to ellipsoidal (3–5 x 4–6 µm), and occur singly, in pairs or in small clusters and chains (FIG. 9). Pseudohyphae are present (FIG. 10). After 7 d on YM agar at 25 C, colonies are off white, butyrous with smooth surface and an undulate margin. After 7 d on Dalmau plate culture on cornmeal agar at 25 C both pseudohyphae (FIG. 10) and true hyphae are present. Aerobic growth is off white with a filamentous colony margin. Ascospores are not produced under the conditions tested. (See TABLE II for a summary of physiological characteristics.)
Type strain. – Y-27698T (= CBS 9871) is preserved as a lyophilized preparation in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. The type strain was isolated from the gut of an unidentified cockroach at Barro Colorado Island, Panama.
Etymology. – The species epithet, blattae (L. gen. fem. n.), is from the Greek word blatta, "roach," the source of the type strain of this species.
Isolates of new species.— –
Candida chauliodes was represented by a single isolate (NRRL Y-27909) while others were isolated on at least two occasions (TABLE I). There was no D1/D2 sequence variation among the strains in each new species except in C. blattae, in which the strains differed at two base pairs (TABLE I). BLAST with the D1/D2 sequences showed that C. chauliodes and C. corydali were most similar to sequences of Candida sp. ST-18, isolated from Thai-land and Candida sp. BG 02-7-21-004G-1-3 from Panama in GenBank. Candida ascalaphidarum showed the highest similarity to C. palmioleophila in the D1/D2 sequence, while C. dosseyi and C. blattae were most closely related to Candida sp. ST-211, also isolated from Thailand. However the new species were clearly distinguished from the most similar sequences by nine or more base pairs.
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DISCUSSION |
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). The novel species were placed within three clades (A–C), only one of which has relatively high bootstrap support of 90% (clade A). Within the C. albicans clade (A), C. chauliodes and C. corydali were sister taxa and their grouping was well supported by bootstrap analysis (99%). The two species were strongly supported as members of a monophyletic group with Candida sp. ST-18 and Candida sp. BG 02-7-21-004G-1-3 based on analysis of D1/D2 sequences (data not shown). Sequence comparisons also indicated that the two novel species are most closely related to Candida sp. ST-18.
The phylogenetic position of Candida ascalaphidarum is unclear and no closely related sister taxon was identified. Although it was placed basal to the Candida membranifaciens clade (B), the position was not supported statistically. Furthermore C. palmioleophila was the sequence most similar to C. ascalaphidarum, differing by 27 bp in the D1/D2 region but not grouping together in the phylogenetic tree (FIG. 1). Candida dosseyi and Candida blattae were placed as sister species within the Candida intermedia clade (C) with strong bootstrap support. The two novel taxa formed a monophyletic group with Candida sp. ST-211 with 100% bootstrap support based on D1/D2 sequences (tree not shown).
The assimilation and fermentation capabilities of the yeasts described here are generally different from many of the yeasts associated with mushroom or wood-ingesting beetles (Suh et al 2003, 2004b). Physiological profiles of the novel species also vary substantially from the closest described species and from each other (TABLE II), and these traits may be useful in designing taxonomic keys to species. However DNA sequence data provide a faster and more effective way to identify an unknown yeast.
Insect hosts and yeasts.— –
The order Neuroptera includes primitive holometabolous insects that possess membranous yet strongly veined wings. Species from five of the more common neuropteran families present in the southeastern USA were chosen for this study. These include the green lacewings (Chrysopidae), which are well known in biological control studies, fishflies and dobsonflies (Corydalidae), owl-flies (Ascalaphidae), mantisflies (Mantispidae) and brown lacewings (Hemerobiidae). As adults Corydalidae and Chrysopidae feed mostly on various saprobic substances while Ascalaphidae, Mantispidae and Hemerobiidae are predaceous. In contrast the larvae of all five families are predators (Tauber 1991).
Corydalidae..
Many of the yeasts isolated in this study were associated with the family Corydalidae, of which we sampled adults of three species, Corydalus cornutus, Chauliodes rastricornis and Chauliodes pectinicornis (TABLE I). The larva of the dobsonfly, Corydalus cornutus, is a well known, large insect of aquatic systems. It spends its larval stages in the water of rocky-bottomed streams where it preys on larvae of blackflies (Diptera: Simuliidae) and net spinning caddisflies (Trichoptera: Hydropsychidae) (Stewart et al 1973). In contrast the adult has not received extensive study and its ecology is poorly known. The two Chauliodes species also have biological characteristics similar to Corydalus cornutus. The feeding habits of these short-lived adults have been reported only from laboratory experiments where they lap up water or sweet liquid substances. Adults of all three species have been reported to visit traps baited with a mixture of bananas, brown sugar, molasses and beer (Parfin 1952).
Representatives of the four novel yeast species described here were isolated from the three adult corydalids mentioned above, although not consistently (TABLE I). Six species within the "Saccharomyces complex" were found in the gut of these corydalids. Interestingly, three of these six species (L. thermotolerans, L. fermentati and H. vineae) were isolated repeatedly from several localities throughout the three years of the study (TABLE I).
In general L. thermotolerans and L. fermentati have been isolated from sweet, fermenting substrates around the world. Conversely Hanseniaspora vineae also has a widespread distribution but is generally known from soil and spoiled food (Barnett et al 2000). The repeated isolation of these yeasts suggests that they are closely associated with the corydalids collected in this study. It is interesting to note that these yeasts have worldwide distributions, perhaps due to insects dispersing them widely (Lachance et al 2001b), inoculating yeast spawn to fresh saccharine substrates and spoiling them (Stratford et al 2002). Insects such as Drosophila and small beetles probably are the first to inoculate fresh substrates, but the corydalid vectors likely contribute to dispersion of the yeasts.
Isolation of Saccharomyces cerevisiae from corydalids is of interest. Although renowned as an industrial yeast, little is known about its occurrence in nature. Literature reports of natural occurrences of these yeasts are sparse and they are not often isolated, even in complex communities containing many other species. For example Sniegowski et al (2002) found 10 isolates out of 79 with enrichment cultures for Saccharomyces species associated with oak trees and surrounding soil. Morais et al (1992) found three isolates out of 962 while sampling yeasts that occur with Drosophila communities in Brazil. Naumov et al (1998) found only two isolates from slime fluxes of oak. Similarly we obtained only two isolates within a 4 d period in the gut of two different corydalid hosts. The frequency of isolation based on the previous studies mentioned above and our current data indicates that the species does not occur abundantly in natural habitats. The inconsistent isolation of this species and others that were found only once or twice in this study (TABLE I) suggests that they may not have been closely associated with neuropteran insects but perhaps were only transients acquired during feeding.
Observations of the gut morphology in all three species of corydalids from which yeasts were isolated showed five finger-like projections (diverticulae) that apparently have not been reported previously. These diverticulae occur in the foregut region behind the crop. Such pouches in the gut of insects, including chrysopids, often serve for temporary storage of food (Tang and Ward 1998) in which yeasts, bacteria or other obligate microorganisms reside (Bismanis 1976, Stamopoulos and Tzanetakis 1988, Marchini et al 2002, Woolfolk et al 2004). However we did not determine whether yeasts were present in the pouches, but if they are localized there this may account for the consistent associations observed between corydalid neuropterans and certain yeasts.
Chrysopidae..
Green lacewing larvae are predaceous on small insects, including aphids, but as adults they feed on sweet substances such as nectar, insect honeydew and pollen (Hagen et al 1970). Yeasts have not been isolated from larvae, but they often occur in the gut of adults. Yeasts in the genus Metschnikowia were found to be associated with lacewings (e.g. Metschnikowia noctiluminum and Metschnikowia chrysoperlae) (Woolfolk and Inglis 2003, Suh et al 2004a, Nguyen et al 2006). Their related anamorphs, Candida picachoensis and Candida pimensis, were described from green lacewing gut isolates collected in Arizona and subsequently found in Louisiana (Suh et al 2004a, Nguyen et al 2006). Metschnikowia chrysoperlae was found only in Arizona and not in Louisiana. Woolfolk and Inglis (2003) reported M. pulcherrima-like yeasts in the gut of Chrysoperla rufilabris, and Suh et al (2004a) classified the species as M. chrysoperlae based on sequence comparisons and ascospore formation. In this study M. chrysoperlae and M. pulcherrima also were isolated from Corydalus cornutus collected from Florida, although each yeast species was collected respectively only once from the gut and the body surface of different insect individuals (TABLE I). These data taken together indicate that the association of green lacewings and yeasts in the Metschnikowia clade is probably widespread across the southern USA.
Unlike the corydalid gut there have been several reports of adult lacewing gut ultrastructure showing many yeast cells within diverticulae, which take up considerable space within the body cavity (Woolfolk et al 2004). Interestingly it was noted that lacewings that fed on sugary substances have a much larger tracheal trunk around the diverticulum, a modification suggested as necessary to satisfy the oxygen demand of resident yeasts (Canard et al 1990, Gibson and Hunter 2005). Direct observation of yeast cells, isolation in culture, and gut structural modifications indicate that yeasts make up some of the underappreciated normal biota of the adult green lacewing gut.
Other insects..
Other neuropteran insects sampled including owlflies, mantisflies and brown lacewings (Ascalaphidae, Mantispidae and Hemerobiidae respectively) are predaceous in both larval and the adult stages. Few yeasts were recovered from these insects, and these were mostly basidiomycete yeasts, which might be environmental contaminants. Generally predaceous insects we have sampled previously (e.g. tiger beetles, carabid beetles and reduviid bugs) did not contain gut yeasts, and this trend continued in this study. For example yeasts were not isolated from the guts of predaceous lacewing larvae but they were present in the guts of adults that feed on plant materials. The yeasts isolated from owlflies were found in the gut of other insects (TABLE I), suggesting that these might be contaminants resulting from predation on insects. In addition the gut of adult owlflies did not contain any well defined pouches or projections such as those found in the corydalids and chrysopids. We have observed a similar trend in fungus-feeding beetles, in which the absence of specialized gut pouches sometimes corresponds with a lack of yeast/insect specificity. Such observations deserve additional study.
Several generalizations can be made based on our findings: (i) The novel species described here were not isolated in high frequency, and they probably are not specifically associated with their hosts; (ii) three species of yeasts from the "Saccharomyces complex" were isolated frequently from the three insect species sampled in the family Corydalidae; (iii) green lacewing adults seem to be connected more often with species of Metschnikowia than with other yeasts; and (iv) adult predaceous insects such as owlflies, mantisflies and brown lacewings were not closely associated with yeasts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Current address: Department of Plant and Microbial Biology, University of California, Berkeley, California 94720. 
2 Current address: American Type Culture Collection, Mycology Collection, 10801 University Boulevard, Manassas, Virginia 20110.
3 Corresponding author. E-mail: mblackwell{at}lsu.edu
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