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Microorganisms in the gut of beetles: evidence from molecular cloning
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 84 (2003) 226–233 www.elsevier.com/locate/yjipa
Microorganisms in the gut of beetles: evidence from molecular cloning Ning Zhang,1 Sung-Oui Suh, and Meredith Blackwell* Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA Received 17 September 2003; accepted 14 October 2003
Abstract We have regularly cultured yeasts from the gut of certain beetles in our ongoing research. In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results. Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles. Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing. Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches. Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Endosymbiosis; Biodiversity; Molecular cloning; Ribosomal DNA; Saccharomycetales; Mutualism
1. Introduction Fungi and insects often occur in common habitats, some of which are completely overlapping, as in the case of yeasts within the gut of certain beetles. We have proposed that the insect gut provides an unexplored habitat for the discovery of new yeasts and yeast-like fungi (Suh and Blackwell, 2004). Previously, the occurrence of endosymbiotic fungi was best known in species of Homoptera and four families of beetles (e.g., Blackwell and Jones, 1997; Nardon and Grenier, 1989; Noda et al., 1995; Suh et al., 2001, 2003). Certain anobiid beetles harbor species of Symbiotaphrina, yeast-like members of a poorly resolved discomycete–loculoascomycete (Pezizomycotina) clade (Jones and Blackwell, 1996; Noda and Kodama, 1996). The majority of yeast endosymbionts from the gut of beetles, however, are classified as species of true yeasts in the genus Candida (asexual Saccharomycetales) (Jones * Corresponding author. Fax: 1-225-578-2597. E-mail address: [email protected] (M. Blackwell). 1 Present address: Department of Plant Pathology, Pennsylvania State University, University Park, PA 16802, USA.
0022-2011/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2003.10.002
et al., 1999). The insects benefit from nutritional supplements provided by the endosymbiotic fungi (Hongoh and Ishikawa, 1997, 2000; Nardon and Grenier, 1989; Noda and Koizumi, 2003; Sasaki et al., 1996; Wetzel et al., 1992; Wilkinson and Ishikawa, 2001). Our ongoing study has led to the isolation of over 600 yeasts in culture from beetles in 25 families. Most of the beetles tested feed on basidiocarps (mushrooms), but some beetles feeding on other resources, including dead wood, also were included. Usually only one yeast species was isolated in culture from an individual beetle, but on rare occasions one or two additional yeasts were recovered from a beetle. The degree of host specificity of the yeasts varied depending on the beetle species. Among these isolates we estimated conservatively that there are more than 150 undescribed species based on phylogenetic analysis of the D1/D2 loop of the large subunit (LSU) ribosomal RNA gene (rDNA) sequences (Suh and Blackwell, 2004). The variable D1/D2 loop of the LSU rDNA has been determined for almost all known yeast species and comprises a dense database for the rapid identification of unidentified isolates (Kurtzman and Robnett, 1995, 1998).
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We routinely use yeast malt extract and cornmeal agars, both common synthetic yeast growth media, to isolate yeasts from the gut of beetles. We were concerned that additional yeasts might be present but were undetected because they occurred in low numbers in a mixed population with a predominate yeast or were strains that could not be grown under the cultural conditions we used. Molecular cloning techniques using specific primers to obtain polymerase chain reaction (PCR) amplified samples have been applied often to search for microorganisms in certain environments without purification or cultivation of those organisms (e.g., Berchtold et al., 1999; Renker et al., 2003). Using such molecular techniques, bacteriologists have estimated that only 1% of all bacteria can be cultured using routine techniques (Torsvik et al., 1990). The original purpose of this research was to determine if uncultivable ascomycete yeasts were present in the gut of four species of basidiocarp-feeding beetles and one wood-ingesting beetle by comparison of yeast taxa detected by cloning of gut contents and by culturing in agar medium. In addition to yeasts we also investigated the presence of other microorganisms with the surprising discovery of previously unknown parabasalid taxa. Our techniques relied on the cloning of the D1/D2 loop of the LSU rDNA, but the additional cloning of small subunit (SSU) rDNA was required in order to refine some of the identifications.
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2. Materials and methods 2.1. Beetles tested Beetle species from four families, were collected from basidiocarps (Erotylidae: Triplax sp.; Tenebrionidae: Platydema sp. and Neomida sp.; and Ciidae: Ceracis curtus) and dead wood (Passalidae: Odontotaenius disjunctus) in southern Louisiana (Table 1). The beetles were starved in a sterile petri dish for 1–2 days on wet filter paper at room temperature to reduce the possibility of undesired PCR amplification of substrate contaminates. 2.2. PCR, molecular cloning, and sequencing The gut was dissected from the beetle and crushed in 20 ll of diluted TE buffer (1/10 strength; 1 mM Tris–HCl (pH 7.5), 0.1 mM Na2 EDTA) in a 1.5 ml tube, and then the tube was placed on boiling water for 5 min. After cooling the gut mixture was centrifuged for 1 min at 14,000 rpm, and 2 ll of the diluted supernatant (1/50 dilution in sterile deionized water) was used as template in a 25 ll PCR. For amplification of LSU rDNA, the following conditions were used in the PCRs: 94 °C 3 min; 94 °C 1 min, 55 °C 1 min ()1 °C per cycle until 50 °C), 72 °C 2 min for 5 cycles, 94 °C 1 min, 50 °C 1 min, 72 °C 2 min for 30 cycles, and 72 °C 10 min. The primers used in the reactions were LS1 and LR5 (Hausner et al., 1993; Rehner and Samuels, 1995), which amplified about
Table 1 Beetle species examined in this study Beetle families/species Erotylidae Triplax sp. Tenebrionidae Platydema sp. Neomida sp.
Place of collection
Number of clones sequenceda
GenBank Accession No. of clone sequencesb
ex Pleurotus sp., LSU campus/Bluebonnet swamp, Baton Rouge, East Baton Rouge Par., LA
10 (LSU)
AY332008–AY332011 AY332024–AY332029
ex Ganoderma sp., St. Francisville, West Feliciana Par., LA ex Ganoderma sp., Hilltop Arboretum, Baton Rouge, LA ex Fomitella supina, Burden Farm, East Baton Rouge Par. LA/St. Francisville, West Feliciana Par., LA
5 (LSU)
AY332012–AY332016
22 (LSU)
AY332004–AY332007 AY332017–AY332023
AY332039–AY332049 Ciidae Ceracis curtus Passalidae Odontotaenius disjunctus
a b
ex Fomitella supina, St. Francisville, West Feliciana Par., LA
9 (LSU)
AY332050–AY332058
ex rotten log, Burden, Baton Rouge, East Baton Rouge Par./St. Francisville, West Feliciana Par., LA (including larva, and pupa)
36 (LSU)
AY332030–AY332038
7 (SSU)
AY332059–AY332092
LSU, LSU rDNA; SSU, SSU rDNA. All the sequences are from the clones of LSU rDNA, except AY332086–AY332092 from those of SSU rDNA.
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850 bp of the 50 end of LSU rDNA including the D1/D2 loop. For amplification of SSU rDNA, we performed two rounds of PCR. PCR conditions in the first round were 94 °C for 5 min, 94 °C for 30 s, 45 °C for 45 s, and 72 °C for 2 min for 35 cycles, 72 °C for 10 min, using the primers Euk18/Euk1615 (Ohkuma et al., 2000). PCR conditions for the second round were 94 °C for 5 min, 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min 30 s for 35 cycles, and 72 °C 10 min, using the primer pairs Euk325/Euk1250, Euk325/Euk924, Euk520/Euk1250, and Euk520/Euk924 (Ohkuma et al., 2000). The PCR products were purified with a SNAP UV-free gel purification kit (Invitrogen Life Technologies, Carlsbad, CA) and were cloned using a TOPO TA Cloning Kit (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturerÕs instructions. The colonies of transformed Escherichia coli were isolated from selective agar media, and about 40 or more plasmid DNAs were compared by restriction enzyme patterns of AluI, HaeIII, and Taqa I. At least two clones were selected from each DNA fragment pattern, and the cloned sequences were determined by the primers LS1 and LR5 for LSU rDNA, and T3 or T7 for SSU rDNA with an ABI PRISM BigDye Terminator Cycle sequencing kit, version 2 or 3 using an ABI PRISM 377 automated DNA sequencer (PE Applied Biosystems, Foster City, CA). 2.3. Culture of yeasts from beetles Beetles were submerged in 95% ethanol for 1–2 min to disinfect their surfaces, and were rinsed with 0.7% saline (NaCl). The rinse liquid was plated on acidified YM agar (Difco YM broth, 2% plain agar, adjusted to pH 3.5 with HCl) as a negative control. Forceps, dissecting needles, and minute insect pins were used to dissect the beetles on sterile microscope slides under a dissecting microscope. Gut segments were crushed in saline solution with a pipette tip and streaked onto the surface of acidified YM agar plates. Plates were incubated at 25 °C, and after three days single colonies were streaked for purification (Suh et al., 2003). Because sampling is destructive, comparisons between yeasts cultured and cloned from a particular beetle species are not necessarily from beetles collected on the same date or at the exact southern Louisiana locality. 2.4. Data analysis DNA sequences were aligned with sequences obtained from GenBank using the multialignment program Clustal X (Thompson et al., 1997), and the alignment was optimized visually. The closest taxa to the cloned sequences and the similarity between the two sequences were generated using Basic Local Alignment Search Tool (BLAST) searches in GenBank. Base pair
differences were counted using the Blast 2 Sequence Program (Tatusova and Madden, 1999) or from manually aligned sequences. 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.
3. Results and discussion 3.1. Sequencing and BLAST search results Based on the variation in size and restriction enzyme patterns of the inserted DNA, 5–36 representative clones were selected and sequenced from the LSU rDNA clone libraries of each beetle species (Table 1). The cloned sequences varied in size from 350 to 850 bp, and results from BLAST searches for the sequences of each clone are summarized in Table 2. Similarity searches of all clone sequences in this study revealed that none of the sequences was identical to sequences in GenBank of any known taxa of fungi or other groups of organisms, and a number of clones even had ‘‘not significant’’ sequence matches (Table 2). Our findings indicate that a much wider variety of microorganisms than realized previously are present in the beetle gut, and many new taxa may be discovered from the specialized habitat. No sequences close to those of the basidiocarps from which the beetles were collected were discovered. The LSU rDNA of the beetles themselves might have been amplified and cloned as well. Sequences of several clones from Ceracis sp. and Odontotaenius sp. were most similar to sequences from beetles, albeit with relatively low similarity scores (Table 2). If these beetle groups were better represented in GenBank, higher similarity scores would have been available, resulting in more accurate identifications. Although our initial interest was in the presence of ascomycete yeasts, cloning revealed the presence of other interesting organisms that were unexpected. We do not know the status of all of these organisms in the gut, but several possibilities exist: the organisms may inhabit the gut regularly or only occasionally but have never been detected because they have not been cultured, or, alternatively, the organisms or their diaspores may be acquired from the substrate occasionally and pass through in the gut remaining viable or not. 3.2. Yeasts in the gut of beetles In previous studies yeasts have been cultured from the gut of beetles or recovered as uncultivable cell forms purified only by ultracentrifugation (e.g., Bismanis, 1976; Noda and Omura, 1992; van der Walt, 1961). Our
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Table 2 Closest known taxa to clones sequenced in this study and their percentage similarity from BLAST searches Beetlea
Number of clones/total
Closest speciesb
Similarityc
Triplax (LSU)
4/10 6/10
Candida ambrosiae; Saccharomycetales; Ascomycota Malassezia restricta; Ustilaginomycetes; Basidiomycota
93–94% 99%
Platydema sp. (LSU)
5/5
Candida ambrosiae; Saccharomycetales; Ascomycota
93%
Neomida sp. (LSU)
4/22 6/22 1/22 1/22 1/22 4/22 2/22 3/22
Candida pyralidae; Saccharomycetales; Ascomycota Blastobotrys elegans; Saccharomycetales; Ascomycota Stephanoascus farinosus; Saccharomycetales; Ascomycota Zygozyma smithiae; Saccharomycetales; Ascomycota Cercophora newfieldiana; Pezizomycotina; Ascomycota Capronia coronata; Pezizomycotina; Ascomycota Malassezia restricta; Ustilaginomycetes; Basidiomycota Nosema sp.; Microsporidia
94% 92% 91% Not significant 97% 97% 99% Not significant
Ceracis curtus (LSU)
2/9 2/9 2/9 3/9
Capronia coronata; Pezizomycotina; Ascomycota Dissophora decumbens; Mortierellales; Zygomycota Gromia oviformis; Cercozoa Leptocarabus truncaticollis; Carabidae; Coleoptera
97% 97% Not significant <85%
Odontotaenius disjunctus (LSU)
3/36 2/36 1/36 17/36 6/36 1/36 4/36 1/36 1/36
Pichia stipitis; Saccharomycetales; Ascomycota Rhodotorula sp. CBS 8885; Sporidiobolales; Basidiomycota Chalara sp. CL157; Pezizomycotina; Ascomycota Pentatrichomonas hominis; Parabasalia Carpophilus sp. MAL-2003; Nitidulidae; Coleoptera Cruzia americana; Cosmocercoidea; Nematoda Craneopsylla minerva; Siphonaptera; Neoptera Limulus polyphemus; Xiphosura; Arthropoda Eimeria tenella; Eimeriida; Alveolata
99% 99% 99% Not significant Not significant Not significant Not significant Not significant Not significant
Odontotaenius disjunctus (SSU)
1/7 6/7
Trichomitus batrachorum; Trichomonadidae; Parabasalia Hypotrichomonas acosta; Monocercomonadidae; Parabasalia
90% 92–94%
Highlighted taxa are true yeasts. LSU cloned from PCR samples using LSU rDNA specific primers; SSU cloned from PCR samples using SSU rDNA specific primers. b The species or isolate with the highest score in BLAST searches in July 2003. c The percentage similarity is between two fully aligned sequences. When a sequence was partially aligned and compared with reference data of low similarity, this result is indicated as ÔNot significant.Õ a
accumulating evidence contributes to these data. As we mentioned in Section 1, more than 600 yeast isolates from the gut of beetles in 25 families have been identified in our study (Suh and Blackwell, 2004; Suh et al., 2003). The beetle species listed in Table 1 have been examined repeatedly for the presence of gut yeasts from samples collected in two or more localities in the southeastern USA, and we have cultured a number of yeasts from each beetle species. Although several yeasts have been reported from some beetles, one species always predominated. The results are summarized in Table 3 but are discussed in more detail elsewhere (Suh and Blackwell, 2004; Suh et al., 2003). Because our cultural studies were optimized to recover yeasts, the presence of organisms based on culture and cloning are comparable only for yeasts in this report. The comparison of cloned sequences with those of the cultured yeasts detected several trends. Generally, the data showed that few yeast taxa are present in an individual beetle. In Neomida sp., from which four yeast sequences within Saccharomycetales were cloned, only two of the taxa were present multiple times (Table 2). A
yeast taxon near Candida pyralidae occurred four times among the 22 clones, and one near Blastobotrys elegans was present in six of the clones. The recovery of these sequences multiple times indicates that there may be a significant association between Neomida and these taxa. These findings are positively correlated with culture of a yeast sequence similar to that of C. pyralidae from the gut of Neomida sp. on a number of occasions (Table 3). The yeast related to B. elegans, however, was not detected in cultures. The closest relative of this taxon, B. elegans, is known from dust samples. Another yeast, one close to Stephanoascus farinosus, known from a basidiocarp, also was sequences from among the clones. Thus, more than 30% of the clones from Neomida sp. were highly similar to species of Stephanoascus and their Blastobotrys anamorphs; however, these yeasts have not been isolated in culture from Neomida sp. (Tables 2 and 3). The yeasts in the genera Stephanoascus and Blastobotrys usually grow well in culture, and it is not clear why they have not been isolated from the beetles. Perhaps it indicates that there is more diversity in beetle gut yeast communities than we have determined previously,
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Table 3 Gut yeasts isolated from the beetle species listed in Table 1 Beetlesa
Yeast groupsb
Number of isolates
GenBank No. of LSU rDNA
Closest known taxa
Similarity (%)
Triplax sp.
EROT1 EROT2 EROT4 EROT9 EROT10 EROT28 EROT29
4 1 1 16 3 1 1
AY242255 AY242256 AY242258 AY242263 AY242264 AY309872 AY390772
Candida kruisii Candida kruisii Candida pyralidae Candida ambrosiae Pichia bispora Candida tanzawaensis Cryptococcus diffluens
93 93 94 94 87 97 100
Platydema sp.
TENE4 TENE9
11 2
AY242244 AY242247
Candida pyralidae Stephanoascus farinosus
94 97
Neomida sp.
TENE1 TENE2 TENE3 TENE4 TENE10 TENE16
14 1 2 6 1 1
AY242241 AY242242 AY242243 AY309892 AY242248 AY242252
Candida canberraensis Cryptococcus aerius Tremella globispora Candida pyralidae Arxula adeninivorans Candida valdiviana
94 87 89 94 94 88
Ceracis curtus
CIID1 CIID2
3 3
AY242297 AY242298
Arxula adeninivorans Candida canberraensis
96 94
Odontotaenius disjunctus
PASS1 PASS5 PASS6 PASS7
24 1 2 2
AY227720 AY227722 AY390773 AY390774
Pichia stipitis Pichia stipitis Candida sp. HA167 Candida sp. HA167
99 99 84 <84
Highlighted taxa are true yeasts similar to those recovered in clone sequences shown in Table 2. Locality and date of collections may be different from those in Table 1 because of destructive sampling. b The group names are based on the insect host family and unique LSU rDNA sequence. Isolates with identical sequences in the D1/D2 region in LSU rDNA and from the same host are the same group. a
and points to a need to enlarge our sample size. The absence in the clone library of some frequently isolated yeasts (e.g., Candida canberraensis) also indicates a small sample size in the cloning experiments. The other Neomida yeast occurred once. It was closest to Zygozyma smithiae, isolated previously from ambrosia beetle frass, but sequence similarity in BLAST searches was low (not significant, Table 2). It is possible that this yeast may have come from the habitat. A single yeast taxon was found in the gut of each of the other three beetles, Triplax sp., Platydema sp., and O. disjunctus. Four of the ten clones derived from the gut of Triplax sp. were closely related (93–94% similarity over 600 bp) to Candida ambrosiae, similar to a yeast cultured often from the gut of this beetle species. Cloning of a close relative of Pichia stipitis (PASS1) from O. disjunctus is in agreement with many culture results in which the P. stipitis-like yeasts have been cultured regularly from O. disjunctus collected from Pennsylvania to southern Louisiana. In fact this is the most consistent association that we have observed in our study of yeasts in the gut of beetles (Table 3; Suh et al., 2003). Cloning from Platydema sp. indicated that another yeast (93–94% similarity for about 600 bp) near C. ambrosiae was present (Table 2). This yeast, however, has not been cultured from the beetle gut. A different yeast
not detected in cloning, a relative of C. pyralidae, was cultured often from this beetle, but was not represented in cloned sequences (Tables 2 and 3). No yeasts from C. curtus, a small ciid (<2 mm), were detected by cloning (Table 2). This finding is in contrast to the occurrence of two species of yeasts cultured from the gut of beetles (Table 3). The cultured yeasts, near Arxula adeninivorans and C. canberraensis, were associated with several beetle species in our cultural study. These results indicate that the yeasts associates of the beetles may be more varied that previously recognized. 3.3. Basidiomycete yeasts in beetles In our search for uncultivable yeasts two basidiomycete yeast species were found that had not been detected by culture methods. One of these, a clone sequence very similar to the Rhodotorula sp. CBS 8885 (Sporidiales), was found in two of thirty-six clones from the wood-ingesting passalid beetle O. disjunctus. The other basidiomycete yeasts detected in clone sequences from Triplax sp. and Neomida sp. were close to Malassezia restricta, a smut fungus relative (Ustilaginales) with only 1–2 bp difference in about 600 bp of D1/D2 sequence of the LSU rDNA. Species of Malassezia are common in human skin and tissue (Ahearn and Simmon, 1998; Gemmer et al. 2002). M. restricta and
N. Zhang et al. / Journal of Invertebrate Pathology 84 (2003) 226–233
M. globosa, however, have been reported from nature as associates of soil nematodes that were detected by cloning but not in isolations (Renker et al., 2003). These fastidious, sometimes pathogenic, yeasts have a fatty acid requirement for growth, and if they are present in the insect-associated habitat, our routine use of YM agar may account for their absence in the previous isolations from more than 25 individual beetle samples each in species of Triplax and Neomida. 3.4. Filamentous fungi in beetles Only one zygomycete (Dissophora decumbens: Mortierellales) was discovered among the clone sequences. This species is found in plant liter in associations with insects. Several filamentous ascomycete clones were identified by their similarity to known sequences in GenBank. Sequences with high similarity to Capronia coronata (97%), a species of ‘‘black yeast’’ (Pezizomycotina), were found in species of both Neomida sp. and Ceracis sp., which often are collected together from the wood-decaying basidiomycete Fomitella supina. It is possible that the beetles might have acquired the fungus from the wood substrate from which C. coronata has been isolated (M€ uller et al., 1987). Other filamentous ascomycete sequences included one similar to that of Chalara sp. CL157, an asexual ascomycete (Pezizomycotina) in O. disjunctus and another from Neomida sp. near Cercophora newfieldiana, both of which may have been of incidental occurrence from the woody substrate of the basidiomycete host. Although some filamentous fungi occasionally were present in cultures, they were not isolated because our primary interest was in the yeasts.
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reproductive systems of mammals and birds (Gerbod et al., 2000). Passalid beetles ingest wood as do termites and wood roaches and might be expected to have these organisms in their gut, but they have never been reported nor were we able to detect any of the organisms in the gut of the beetles. Our interest in discovering more about the parabasalids lead us to sequence another locus, SSU rDNA, of which more sequences are available in the GenBank database. Also, the highly variable LSU rDNA could not be aligned with confidence. Phylogenetic analyses further confirmed that parabasalid symbionts were present in the gut of this wood-ingesting passalid beetle (Fig. 1). All the clones from eukaryotic SSU rDNA specific PCR products had 90% and higher similarity with parabasalid taxa (Table 2). BLAST searches showed that the closest parabasalid taxon to the clones was Hypotrichomonas acosta, a known associate of squamate reptiles and chelonians (Fig. 1; Lee, 1960; Moskowitz, 1951). In our analysis, one clone (AY332088) was in a position that was basal to a subclade of our other six clones; this clade was strongly supported in a larger clade with H. acosta. Although we used mineral oil to limit oxygen presence and used several stains to visualize such organisms, repeated examination of all regions of the large recurved
3.5. Parabasalid and other protists in passalid beetles Clone sequences nearest to those of several protist groups known to be associated with insects were present from the gut of several beetles. These included Gromia oviformis (Cercozoa) from C. curtus, Nosema sp. (Microsporidia) from Neomida sp., and Eimeria tenella; (Alveolata) from O. disjunctus, all known as insect parasites. The most unexpected finding in the study, however, was the discovery that about 50% of the 36 LSU rDNA clones from the passalid beetle, O. disjunctus, showed a relationship to parabasalid taxa, although the sequence similarity was not significantly high (Table 2). Parabasalids are anaerobic heterotrophic protists found in association with certain insects, and they are perhaps best known as members of complex symbiotic communities of bacteria and protists in the hindgut of termites and wood roaches where they degrade celluloic materials ingested by the insect hosts (Grosovsky and Margulis, 1982). Other parabasalids are free-living in sediments or parasitic in the respiratory, digestive, and
Fig. 1. Phylogenetic tree showing the relationships of the parabasalid symbiont clones from Odontotaenius disjunctus and other species of the group. The tree is one of two most parsimonious trees obtained from about 940 bp of SSU rDNA sequence data. GenBank accession nos. of reference sequences and clones are shown after names of the organisms. Tree length ¼ 1475; consistency index ¼ 0.5444; homoplasy index ¼ 0.4556; retention index ¼ 0.6040; and rescaled consistency index ¼ 0.3288. Numbers on tree branches indicate the percentages of bootstrap samplings derived from 1000 samples that supported the internal branches by 50% or more. Note the high bootstrap support for the clade comprising the passalid clone sequences and Hypotrichomonas acosta.
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passalid gut using the compound microscope failed to reveal living parabasalids. It is possible that the organisms occur in the gut wall as parasites and, therefore, would require sectioning and other special treatments for successful observation. We believe that our findings are of significance in the area of microbial-insect associations. At the broadest level unexpected complexity of beetle environments including interactions and habitats shared with a variety of internal microorganisms is revealed. This kind of basic information often is lacking for insects and presumably could have an affect on their management. Of particular interest to insect pathology is the discovery that previously undetected, possibly parasitic, parabasalids are the most common gut organisms obtained from the clones of the wood-inhabiting passalid beetles.
Acknowledgments We thank Nhu Nguyen, an undergraduate student in our laboratory, for his skillful assistance in collecting and identifying beetle samples. This study was supported by a grant from the National Science Foundation (NSF DEB-0072741 to M.B.). Ning Zhang was supported in part by a grant to the Biological Computation and Visualization Center at Louisiana State University from the Louisiana Board of Regents (BOR HEF (200005)-01) to Harold Silverman, College of Basic Sciences, Louisiana State University.
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N. Zhang et al. / Journal of Invertebrate Pathology 84 (2003) 226–233 Suh, S.-O., Marshall, C., McHugh, J.V., Blackwell, M., 2003. In the company of xylose-fermenting yeasts? Beetle radiations into wood. Mol. Ecol. 12, 3137–3146. Suh, S.-O., Noda, H., Blackwell, M., 2001. Insect symbiosis: derivation of yeast-like endosymbionts within an entomopathogenic filamentous lineage. Mol. Biol. Evol. 18, 995–1000. Swofford, D.L., 2002. PAUP. Phylogenetic Analysis Using Parsimony (* and Other Methods), Version 4.0b10. Sinauer Associates, Sunderland, MA. Tatusova, T.A., Madden, T.L., 1999. Blast 2 sequences—a new tool for comparing protein and nucleotide sequences. FEMS Microbiol. Lett. 174, 247–250. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL X windows interface: flexible strate-
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 84 (2003) 226–233 www.elsevier.com/locate/yjipa
Microorganisms in the gut of beetles: evidence from molecular cloning Ning Zhang,1 Sung-Oui Suh, and Meredith Blackwell* Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA Received 17 September 2003; accepted 14 October 2003
Abstract We have regularly cultured yeasts from the gut of certain beetles in our ongoing research. In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results. Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles. Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing. Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches. Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Endosymbiosis; Biodiversity; Molecular cloning; Ribosomal DNA; Saccharomycetales; Mutualism
1. Introduction Fungi and insects often occur in common habitats, some of which are completely overlapping, as in the case of yeasts within the gut of certain beetles. We have proposed that the insect gut provides an unexplored habitat for the discovery of new yeasts and yeast-like fungi (Suh and Blackwell, 2004). Previously, the occurrence of endosymbiotic fungi was best known in species of Homoptera and four families of beetles (e.g., Blackwell and Jones, 1997; Nardon and Grenier, 1989; Noda et al., 1995; Suh et al., 2001, 2003). Certain anobiid beetles harbor species of Symbiotaphrina, yeast-like members of a poorly resolved discomycete–loculoascomycete (Pezizomycotina) clade (Jones and Blackwell, 1996; Noda and Kodama, 1996). The majority of yeast endosymbionts from the gut of beetles, however, are classified as species of true yeasts in the genus Candida (asexual Saccharomycetales) (Jones * Corresponding author. Fax: 1-225-578-2597. E-mail address: [email protected] (M. Blackwell). 1 Present address: Department of Plant Pathology, Pennsylvania State University, University Park, PA 16802, USA.
0022-2011/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2003.10.002
et al., 1999). The insects benefit from nutritional supplements provided by the endosymbiotic fungi (Hongoh and Ishikawa, 1997, 2000; Nardon and Grenier, 1989; Noda and Koizumi, 2003; Sasaki et al., 1996; Wetzel et al., 1992; Wilkinson and Ishikawa, 2001). Our ongoing study has led to the isolation of over 600 yeasts in culture from beetles in 25 families. Most of the beetles tested feed on basidiocarps (mushrooms), but some beetles feeding on other resources, including dead wood, also were included. Usually only one yeast species was isolated in culture from an individual beetle, but on rare occasions one or two additional yeasts were recovered from a beetle. The degree of host specificity of the yeasts varied depending on the beetle species. Among these isolates we estimated conservatively that there are more than 150 undescribed species based on phylogenetic analysis of the D1/D2 loop of the large subunit (LSU) ribosomal RNA gene (rDNA) sequences (Suh and Blackwell, 2004). The variable D1/D2 loop of the LSU rDNA has been determined for almost all known yeast species and comprises a dense database for the rapid identification of unidentified isolates (Kurtzman and Robnett, 1995, 1998).
N. Zhang et al. / Journal of Invertebrate Pathology 84 (2003) 226–233
We routinely use yeast malt extract and cornmeal agars, both common synthetic yeast growth media, to isolate yeasts from the gut of beetles. We were concerned that additional yeasts might be present but were undetected because they occurred in low numbers in a mixed population with a predominate yeast or were strains that could not be grown under the cultural conditions we used. Molecular cloning techniques using specific primers to obtain polymerase chain reaction (PCR) amplified samples have been applied often to search for microorganisms in certain environments without purification or cultivation of those organisms (e.g., Berchtold et al., 1999; Renker et al., 2003). Using such molecular techniques, bacteriologists have estimated that only 1% of all bacteria can be cultured using routine techniques (Torsvik et al., 1990). The original purpose of this research was to determine if uncultivable ascomycete yeasts were present in the gut of four species of basidiocarp-feeding beetles and one wood-ingesting beetle by comparison of yeast taxa detected by cloning of gut contents and by culturing in agar medium. In addition to yeasts we also investigated the presence of other microorganisms with the surprising discovery of previously unknown parabasalid taxa. Our techniques relied on the cloning of the D1/D2 loop of the LSU rDNA, but the additional cloning of small subunit (SSU) rDNA was required in order to refine some of the identifications.
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2. Materials and methods 2.1. Beetles tested Beetle species from four families, were collected from basidiocarps (Erotylidae: Triplax sp.; Tenebrionidae: Platydema sp. and Neomida sp.; and Ciidae: Ceracis curtus) and dead wood (Passalidae: Odontotaenius disjunctus) in southern Louisiana (Table 1). The beetles were starved in a sterile petri dish for 1–2 days on wet filter paper at room temperature to reduce the possibility of undesired PCR amplification of substrate contaminates. 2.2. PCR, molecular cloning, and sequencing The gut was dissected from the beetle and crushed in 20 ll of diluted TE buffer (1/10 strength; 1 mM Tris–HCl (pH 7.5), 0.1 mM Na2 EDTA) in a 1.5 ml tube, and then the tube was placed on boiling water for 5 min. After cooling the gut mixture was centrifuged for 1 min at 14,000 rpm, and 2 ll of the diluted supernatant (1/50 dilution in sterile deionized water) was used as template in a 25 ll PCR. For amplification of LSU rDNA, the following conditions were used in the PCRs: 94 °C 3 min; 94 °C 1 min, 55 °C 1 min ()1 °C per cycle until 50 °C), 72 °C 2 min for 5 cycles, 94 °C 1 min, 50 °C 1 min, 72 °C 2 min for 30 cycles, and 72 °C 10 min. The primers used in the reactions were LS1 and LR5 (Hausner et al., 1993; Rehner and Samuels, 1995), which amplified about
Table 1 Beetle species examined in this study Beetle families/species Erotylidae Triplax sp. Tenebrionidae Platydema sp. Neomida sp.
Place of collection
Number of clones sequenceda
GenBank Accession No. of clone sequencesb
ex Pleurotus sp., LSU campus/Bluebonnet swamp, Baton Rouge, East Baton Rouge Par., LA
10 (LSU)
AY332008–AY332011 AY332024–AY332029
ex Ganoderma sp., St. Francisville, West Feliciana Par., LA ex Ganoderma sp., Hilltop Arboretum, Baton Rouge, LA ex Fomitella supina, Burden Farm, East Baton Rouge Par. LA/St. Francisville, West Feliciana Par., LA
5 (LSU)
AY332012–AY332016
22 (LSU)
AY332004–AY332007 AY332017–AY332023
AY332039–AY332049 Ciidae Ceracis curtus Passalidae Odontotaenius disjunctus
a b
ex Fomitella supina, St. Francisville, West Feliciana Par., LA
9 (LSU)
AY332050–AY332058
ex rotten log, Burden, Baton Rouge, East Baton Rouge Par./St. Francisville, West Feliciana Par., LA (including larva, and pupa)
36 (LSU)
AY332030–AY332038
7 (SSU)
AY332059–AY332092
LSU, LSU rDNA; SSU, SSU rDNA. All the sequences are from the clones of LSU rDNA, except AY332086–AY332092 from those of SSU rDNA.
228
N. Zhang et al. / Journal of Invertebrate Pathology 84 (2003) 226–233
850 bp of the 50 end of LSU rDNA including the D1/D2 loop. For amplification of SSU rDNA, we performed two rounds of PCR. PCR conditions in the first round were 94 °C for 5 min, 94 °C for 30 s, 45 °C for 45 s, and 72 °C for 2 min for 35 cycles, 72 °C for 10 min, using the primers Euk18/Euk1615 (Ohkuma et al., 2000). PCR conditions for the second round were 94 °C for 5 min, 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min 30 s for 35 cycles, and 72 °C 10 min, using the primer pairs Euk325/Euk1250, Euk325/Euk924, Euk520/Euk1250, and Euk520/Euk924 (Ohkuma et al., 2000). The PCR products were purified with a SNAP UV-free gel purification kit (Invitrogen Life Technologies, Carlsbad, CA) and were cloned using a TOPO TA Cloning Kit (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturerÕs instructions. The colonies of transformed Escherichia coli were isolated from selective agar media, and about 40 or more plasmid DNAs were compared by restriction enzyme patterns of AluI, HaeIII, and Taqa I. At least two clones were selected from each DNA fragment pattern, and the cloned sequences were determined by the primers LS1 and LR5 for LSU rDNA, and T3 or T7 for SSU rDNA with an ABI PRISM BigDye Terminator Cycle sequencing kit, version 2 or 3 using an ABI PRISM 377 automated DNA sequencer (PE Applied Biosystems, Foster City, CA). 2.3. Culture of yeasts from beetles Beetles were submerged in 95% ethanol for 1–2 min to disinfect their surfaces, and were rinsed with 0.7% saline (NaCl). The rinse liquid was plated on acidified YM agar (Difco YM broth, 2% plain agar, adjusted to pH 3.5 with HCl) as a negative control. Forceps, dissecting needles, and minute insect pins were used to dissect the beetles on sterile microscope slides under a dissecting microscope. Gut segments were crushed in saline solution with a pipette tip and streaked onto the surface of acidified YM agar plates. Plates were incubated at 25 °C, and after three days single colonies were streaked for purification (Suh et al., 2003). Because sampling is destructive, comparisons between yeasts cultured and cloned from a particular beetle species are not necessarily from beetles collected on the same date or at the exact southern Louisiana locality. 2.4. Data analysis DNA sequences were aligned with sequences obtained from GenBank using the multialignment program Clustal X (Thompson et al., 1997), and the alignment was optimized visually. The closest taxa to the cloned sequences and the similarity between the two sequences were generated using Basic Local Alignment Search Tool (BLAST) searches in GenBank. Base pair
differences were counted using the Blast 2 Sequence Program (Tatusova and Madden, 1999) or from manually aligned sequences. 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.
3. Results and discussion 3.1. Sequencing and BLAST search results Based on the variation in size and restriction enzyme patterns of the inserted DNA, 5–36 representative clones were selected and sequenced from the LSU rDNA clone libraries of each beetle species (Table 1). The cloned sequences varied in size from 350 to 850 bp, and results from BLAST searches for the sequences of each clone are summarized in Table 2. Similarity searches of all clone sequences in this study revealed that none of the sequences was identical to sequences in GenBank of any known taxa of fungi or other groups of organisms, and a number of clones even had ‘‘not significant’’ sequence matches (Table 2). Our findings indicate that a much wider variety of microorganisms than realized previously are present in the beetle gut, and many new taxa may be discovered from the specialized habitat. No sequences close to those of the basidiocarps from which the beetles were collected were discovered. The LSU rDNA of the beetles themselves might have been amplified and cloned as well. Sequences of several clones from Ceracis sp. and Odontotaenius sp. were most similar to sequences from beetles, albeit with relatively low similarity scores (Table 2). If these beetle groups were better represented in GenBank, higher similarity scores would have been available, resulting in more accurate identifications. Although our initial interest was in the presence of ascomycete yeasts, cloning revealed the presence of other interesting organisms that were unexpected. We do not know the status of all of these organisms in the gut, but several possibilities exist: the organisms may inhabit the gut regularly or only occasionally but have never been detected because they have not been cultured, or, alternatively, the organisms or their diaspores may be acquired from the substrate occasionally and pass through in the gut remaining viable or not. 3.2. Yeasts in the gut of beetles In previous studies yeasts have been cultured from the gut of beetles or recovered as uncultivable cell forms purified only by ultracentrifugation (e.g., Bismanis, 1976; Noda and Omura, 1992; van der Walt, 1961). Our
N. Zhang et al. / Journal of Invertebrate Pathology 84 (2003) 226–233
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Table 2 Closest known taxa to clones sequenced in this study and their percentage similarity from BLAST searches Beetlea
Number of clones/total
Closest speciesb
Similarityc
Triplax (LSU)
4/10 6/10
Candida ambrosiae; Saccharomycetales; Ascomycota Malassezia restricta; Ustilaginomycetes; Basidiomycota
93–94% 99%
Platydema sp. (LSU)
5/5
Candida ambrosiae; Saccharomycetales; Ascomycota
93%
Neomida sp. (LSU)
4/22 6/22 1/22 1/22 1/22 4/22 2/22 3/22
Candida pyralidae; Saccharomycetales; Ascomycota Blastobotrys elegans; Saccharomycetales; Ascomycota Stephanoascus farinosus; Saccharomycetales; Ascomycota Zygozyma smithiae; Saccharomycetales; Ascomycota Cercophora newfieldiana; Pezizomycotina; Ascomycota Capronia coronata; Pezizomycotina; Ascomycota Malassezia restricta; Ustilaginomycetes; Basidiomycota Nosema sp.; Microsporidia
94% 92% 91% Not significant 97% 97% 99% Not significant
Ceracis curtus (LSU)
2/9 2/9 2/9 3/9
Capronia coronata; Pezizomycotina; Ascomycota Dissophora decumbens; Mortierellales; Zygomycota Gromia oviformis; Cercozoa Leptocarabus truncaticollis; Carabidae; Coleoptera
97% 97% Not significant <85%
Odontotaenius disjunctus (LSU)
3/36 2/36 1/36 17/36 6/36 1/36 4/36 1/36 1/36
Pichia stipitis; Saccharomycetales; Ascomycota Rhodotorula sp. CBS 8885; Sporidiobolales; Basidiomycota Chalara sp. CL157; Pezizomycotina; Ascomycota Pentatrichomonas hominis; Parabasalia Carpophilus sp. MAL-2003; Nitidulidae; Coleoptera Cruzia americana; Cosmocercoidea; Nematoda Craneopsylla minerva; Siphonaptera; Neoptera Limulus polyphemus; Xiphosura; Arthropoda Eimeria tenella; Eimeriida; Alveolata
99% 99% 99% Not significant Not significant Not significant Not significant Not significant Not significant
Odontotaenius disjunctus (SSU)
1/7 6/7
Trichomitus batrachorum; Trichomonadidae; Parabasalia Hypotrichomonas acosta; Monocercomonadidae; Parabasalia
90% 92–94%
Highlighted taxa are true yeasts. LSU cloned from PCR samples using LSU rDNA specific primers; SSU cloned from PCR samples using SSU rDNA specific primers. b The species or isolate with the highest score in BLAST searches in July 2003. c The percentage similarity is between two fully aligned sequences. When a sequence was partially aligned and compared with reference data of low similarity, this result is indicated as ÔNot significant.Õ a
accumulating evidence contributes to these data. As we mentioned in Section 1, more than 600 yeast isolates from the gut of beetles in 25 families have been identified in our study (Suh and Blackwell, 2004; Suh et al., 2003). The beetle species listed in Table 1 have been examined repeatedly for the presence of gut yeasts from samples collected in two or more localities in the southeastern USA, and we have cultured a number of yeasts from each beetle species. Although several yeasts have been reported from some beetles, one species always predominated. The results are summarized in Table 3 but are discussed in more detail elsewhere (Suh and Blackwell, 2004; Suh et al., 2003). Because our cultural studies were optimized to recover yeasts, the presence of organisms based on culture and cloning are comparable only for yeasts in this report. The comparison of cloned sequences with those of the cultured yeasts detected several trends. Generally, the data showed that few yeast taxa are present in an individual beetle. In Neomida sp., from which four yeast sequences within Saccharomycetales were cloned, only two of the taxa were present multiple times (Table 2). A
yeast taxon near Candida pyralidae occurred four times among the 22 clones, and one near Blastobotrys elegans was present in six of the clones. The recovery of these sequences multiple times indicates that there may be a significant association between Neomida and these taxa. These findings are positively correlated with culture of a yeast sequence similar to that of C. pyralidae from the gut of Neomida sp. on a number of occasions (Table 3). The yeast related to B. elegans, however, was not detected in cultures. The closest relative of this taxon, B. elegans, is known from dust samples. Another yeast, one close to Stephanoascus farinosus, known from a basidiocarp, also was sequences from among the clones. Thus, more than 30% of the clones from Neomida sp. were highly similar to species of Stephanoascus and their Blastobotrys anamorphs; however, these yeasts have not been isolated in culture from Neomida sp. (Tables 2 and 3). The yeasts in the genera Stephanoascus and Blastobotrys usually grow well in culture, and it is not clear why they have not been isolated from the beetles. Perhaps it indicates that there is more diversity in beetle gut yeast communities than we have determined previously,
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Table 3 Gut yeasts isolated from the beetle species listed in Table 1 Beetlesa
Yeast groupsb
Number of isolates
GenBank No. of LSU rDNA
Closest known taxa
Similarity (%)
Triplax sp.
EROT1 EROT2 EROT4 EROT9 EROT10 EROT28 EROT29
4 1 1 16 3 1 1
AY242255 AY242256 AY242258 AY242263 AY242264 AY309872 AY390772
Candida kruisii Candida kruisii Candida pyralidae Candida ambrosiae Pichia bispora Candida tanzawaensis Cryptococcus diffluens
93 93 94 94 87 97 100
Platydema sp.
TENE4 TENE9
11 2
AY242244 AY242247
Candida pyralidae Stephanoascus farinosus
94 97
Neomida sp.
TENE1 TENE2 TENE3 TENE4 TENE10 TENE16
14 1 2 6 1 1
AY242241 AY242242 AY242243 AY309892 AY242248 AY242252
Candida canberraensis Cryptococcus aerius Tremella globispora Candida pyralidae Arxula adeninivorans Candida valdiviana
94 87 89 94 94 88
Ceracis curtus
CIID1 CIID2
3 3
AY242297 AY242298
Arxula adeninivorans Candida canberraensis
96 94
Odontotaenius disjunctus
PASS1 PASS5 PASS6 PASS7
24 1 2 2
AY227720 AY227722 AY390773 AY390774
Pichia stipitis Pichia stipitis Candida sp. HA167 Candida sp. HA167
99 99 84 <84
Highlighted taxa are true yeasts similar to those recovered in clone sequences shown in Table 2. Locality and date of collections may be different from those in Table 1 because of destructive sampling. b The group names are based on the insect host family and unique LSU rDNA sequence. Isolates with identical sequences in the D1/D2 region in LSU rDNA and from the same host are the same group. a
and points to a need to enlarge our sample size. The absence in the clone library of some frequently isolated yeasts (e.g., Candida canberraensis) also indicates a small sample size in the cloning experiments. The other Neomida yeast occurred once. It was closest to Zygozyma smithiae, isolated previously from ambrosia beetle frass, but sequence similarity in BLAST searches was low (not significant, Table 2). It is possible that this yeast may have come from the habitat. A single yeast taxon was found in the gut of each of the other three beetles, Triplax sp., Platydema sp., and O. disjunctus. Four of the ten clones derived from the gut of Triplax sp. were closely related (93–94% similarity over 600 bp) to Candida ambrosiae, similar to a yeast cultured often from the gut of this beetle species. Cloning of a close relative of Pichia stipitis (PASS1) from O. disjunctus is in agreement with many culture results in which the P. stipitis-like yeasts have been cultured regularly from O. disjunctus collected from Pennsylvania to southern Louisiana. In fact this is the most consistent association that we have observed in our study of yeasts in the gut of beetles (Table 3; Suh et al., 2003). Cloning from Platydema sp. indicated that another yeast (93–94% similarity for about 600 bp) near C. ambrosiae was present (Table 2). This yeast, however, has not been cultured from the beetle gut. A different yeast
not detected in cloning, a relative of C. pyralidae, was cultured often from this beetle, but was not represented in cloned sequences (Tables 2 and 3). No yeasts from C. curtus, a small ciid (<2 mm), were detected by cloning (Table 2). This finding is in contrast to the occurrence of two species of yeasts cultured from the gut of beetles (Table 3). The cultured yeasts, near Arxula adeninivorans and C. canberraensis, were associated with several beetle species in our cultural study. These results indicate that the yeasts associates of the beetles may be more varied that previously recognized. 3.3. Basidiomycete yeasts in beetles In our search for uncultivable yeasts two basidiomycete yeast species were found that had not been detected by culture methods. One of these, a clone sequence very similar to the Rhodotorula sp. CBS 8885 (Sporidiales), was found in two of thirty-six clones from the wood-ingesting passalid beetle O. disjunctus. The other basidiomycete yeasts detected in clone sequences from Triplax sp. and Neomida sp. were close to Malassezia restricta, a smut fungus relative (Ustilaginales) with only 1–2 bp difference in about 600 bp of D1/D2 sequence of the LSU rDNA. Species of Malassezia are common in human skin and tissue (Ahearn and Simmon, 1998; Gemmer et al. 2002). M. restricta and
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M. globosa, however, have been reported from nature as associates of soil nematodes that were detected by cloning but not in isolations (Renker et al., 2003). These fastidious, sometimes pathogenic, yeasts have a fatty acid requirement for growth, and if they are present in the insect-associated habitat, our routine use of YM agar may account for their absence in the previous isolations from more than 25 individual beetle samples each in species of Triplax and Neomida. 3.4. Filamentous fungi in beetles Only one zygomycete (Dissophora decumbens: Mortierellales) was discovered among the clone sequences. This species is found in plant liter in associations with insects. Several filamentous ascomycete clones were identified by their similarity to known sequences in GenBank. Sequences with high similarity to Capronia coronata (97%), a species of ‘‘black yeast’’ (Pezizomycotina), were found in species of both Neomida sp. and Ceracis sp., which often are collected together from the wood-decaying basidiomycete Fomitella supina. It is possible that the beetles might have acquired the fungus from the wood substrate from which C. coronata has been isolated (M€ uller et al., 1987). Other filamentous ascomycete sequences included one similar to that of Chalara sp. CL157, an asexual ascomycete (Pezizomycotina) in O. disjunctus and another from Neomida sp. near Cercophora newfieldiana, both of which may have been of incidental occurrence from the woody substrate of the basidiomycete host. Although some filamentous fungi occasionally were present in cultures, they were not isolated because our primary interest was in the yeasts.
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reproductive systems of mammals and birds (Gerbod et al., 2000). Passalid beetles ingest wood as do termites and wood roaches and might be expected to have these organisms in their gut, but they have never been reported nor were we able to detect any of the organisms in the gut of the beetles. Our interest in discovering more about the parabasalids lead us to sequence another locus, SSU rDNA, of which more sequences are available in the GenBank database. Also, the highly variable LSU rDNA could not be aligned with confidence. Phylogenetic analyses further confirmed that parabasalid symbionts were present in the gut of this wood-ingesting passalid beetle (Fig. 1). All the clones from eukaryotic SSU rDNA specific PCR products had 90% and higher similarity with parabasalid taxa (Table 2). BLAST searches showed that the closest parabasalid taxon to the clones was Hypotrichomonas acosta, a known associate of squamate reptiles and chelonians (Fig. 1; Lee, 1960; Moskowitz, 1951). In our analysis, one clone (AY332088) was in a position that was basal to a subclade of our other six clones; this clade was strongly supported in a larger clade with H. acosta. Although we used mineral oil to limit oxygen presence and used several stains to visualize such organisms, repeated examination of all regions of the large recurved
3.5. Parabasalid and other protists in passalid beetles Clone sequences nearest to those of several protist groups known to be associated with insects were present from the gut of several beetles. These included Gromia oviformis (Cercozoa) from C. curtus, Nosema sp. (Microsporidia) from Neomida sp., and Eimeria tenella; (Alveolata) from O. disjunctus, all known as insect parasites. The most unexpected finding in the study, however, was the discovery that about 50% of the 36 LSU rDNA clones from the passalid beetle, O. disjunctus, showed a relationship to parabasalid taxa, although the sequence similarity was not significantly high (Table 2). Parabasalids are anaerobic heterotrophic protists found in association with certain insects, and they are perhaps best known as members of complex symbiotic communities of bacteria and protists in the hindgut of termites and wood roaches where they degrade celluloic materials ingested by the insect hosts (Grosovsky and Margulis, 1982). Other parabasalids are free-living in sediments or parasitic in the respiratory, digestive, and
Fig. 1. Phylogenetic tree showing the relationships of the parabasalid symbiont clones from Odontotaenius disjunctus and other species of the group. The tree is one of two most parsimonious trees obtained from about 940 bp of SSU rDNA sequence data. GenBank accession nos. of reference sequences and clones are shown after names of the organisms. Tree length ¼ 1475; consistency index ¼ 0.5444; homoplasy index ¼ 0.4556; retention index ¼ 0.6040; and rescaled consistency index ¼ 0.3288. Numbers on tree branches indicate the percentages of bootstrap samplings derived from 1000 samples that supported the internal branches by 50% or more. Note the high bootstrap support for the clade comprising the passalid clone sequences and Hypotrichomonas acosta.
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passalid gut using the compound microscope failed to reveal living parabasalids. It is possible that the organisms occur in the gut wall as parasites and, therefore, would require sectioning and other special treatments for successful observation. We believe that our findings are of significance in the area of microbial-insect associations. At the broadest level unexpected complexity of beetle environments including interactions and habitats shared with a variety of internal microorganisms is revealed. This kind of basic information often is lacking for insects and presumably could have an affect on their management. Of particular interest to insect pathology is the discovery that previously undetected, possibly parasitic, parabasalids are the most common gut organisms obtained from the clones of the wood-inhabiting passalid beetles.
Acknowledgments We thank Nhu Nguyen, an undergraduate student in our laboratory, for his skillful assistance in collecting and identifying beetle samples. This study was supported by a grant from the National Science Foundation (NSF DEB-0072741 to M.B.). Ning Zhang was supported in part by a grant to the Biological Computation and Visualization Center at Louisiana State University from the Louisiana Board of Regents (BOR HEF (200005)-01) to Harold Silverman, College of Basic Sciences, Louisiana State University.
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