The Journal of Published by the International Society of Eukaryotic Microbiology Protistologists

J. Eukaryot. Microbiol., 59(3), 2012 pp. 246–250 © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists DOI: 10.1111/j.1550-7408.2012.00617.x A Nuptially Transmitted Ichthyosporean Symbiont of Tenebrio molitor (Coleoptera: Tenebrionidae)

JEFFREY C. LORD,a KRIS L. HARTZERa and SRINIVAS KAMBHAMPATIb aUSDA, Agricultural Research Service, Center for Grain and Animal Health Research, 1515 College Avenue, Manhattan, Kansas, 66502, and bDepartment of Biology, The University of Texas, Tyler, Texas, 75799

ABSTRACT. The yellow mealworm, Tenebrio molitor, harbors a symbiont that has spores with a thick, laminated wall and infects the fat body and ventral nerve chord of adult and larval beetles. In adult males, there is heavy infection of the epithelial cells of the testes and between testes lobes with occasional penetration of the lobes. Spores are enveloped in the spermatophores when they are formed at the time of mating and transferred to the female’s bursa copulatrix. Infection has not been found in the ovaries. The sequence of the nuclear small subunit rDNA indicates that the symbiont is a member of the Ichthyosporea, a class of near the animal–fungi divergence. Key Words. Mealworm, Mesomycetozoa, small subunit ribosomal DNA, symbiosis.

HE yellow mealworm, Tenebrio molitor, is a pest of post-fixing in 1% (w/v) aqueous OsO4. To improve penetra- T stored grain products that are mass produced for bird tion of spores, 0.1% (v/v) dimethyl sulfoxide was added to and reptile diets. It also is commonly used as a model insect aldehyde fixatives in some cases. After dehydration through for experimentation. During examination of T. molitor in an ethanol series, tissues were embedded in Spurr’s resin search of an etiological agent responsible for high mortality in (Spurr 1969). Ultrathin sections were stained with uranyl ace- commercial cultures, refractile oval bodies were detected in the tate and lead citrate. ventral nerve chord and other tissues. The only record of a DNA collection and extraction. Spores were collected from similar body in T. molitor is a single photograph of an more than 1,000 testes dissected from T. molitor adults. The infected nerve ganglion attributed to W. Kellen and labeled testes were disrupted using a 15-ml glass tissue homogenizer Haplosporida in a guide to identification of insect pathogens with 1 M Tris-HCl buffer at pH 8. Spores were separated (Poinar and Thomas 1984). Haplosporida, as currently consti- from the disrupted tissue on a discontinuous colloidal silica tuted, are endoparasites of marine and sometimes freshwater (Ludox, Aldrich, Milwaukee, WI) or sucrose gradient centri- animals and have distinctive lidded spores (Adl et al. 2005). fuged for 6 h at 16,000 g. The spores were then disrupted Organisms similar to that found in T. molitor and previously using a Mini-Beadbeater (Biospec Products, Bartlesville, OK). considered to be insect-infecting Haplosporida have been The supernatant was collected and DNA was isolated using reclassified (Sprague 1979). Here, we establish that the organ- DNeasy (Qiagen Inc., Valencia, CA) according to the manu- ism is a member of the protozoan class Ichthyosporea, also facturer’s instructions. known as Mesomycetozoa (Mendoza et al. 2002), and describe DNA amplification and sequencing. Partial sequence of the aspects of its tissue specificity, morphology, and transmission. large subunit (LSU) rDNA was amplified using the 28x and 28ee primers (Hillis and Dixon 1991). PCR amplifications were carried out with TaKaRa Ex Taq TM (Takara Bio, Madi- MATERIALS AND METHODS son, WI) according to manufacturer’s instruction. For small Tissue preparation. Tenebrio molitor were from two com- subunit (SSU) rDNA, primer combinations from White et al. mercial sources, Nature’s Way (Ross, OH) and Connecticut (1990) (i.e. NS1-4 and NS8), Hillis and Dixon (1991) (i.e. 18e, Valley Biological Supply (Southampton, MA), and a colony 18g, 18k), and Vilgalys (http://www.biology.duke.edu/fungi/ that has been maintained at the Center for Grain and Animal mycolab/primers.htm) (i.e. SR7, SR5, SR1R, SR7R) were used Health Research, Manhattan, KS since 1970. To examine as initial amplifications, with the combination of SR5-NS3 spermatophores, which are formed during the first 30 s of providing the longest sequence. Primers for the T. molitor mating and are emptied and expelled from the bursa copula- symbiont (TMS) were designed from the resulting sequence trix within 18 h after mating (Gadzama and Happ 1974), vir- fragments. Confirmatory amplification of the SSU rDNA was gin adults were combined in a Petri dish used as a mating performed using combinations of universal and newly arena. Mating pairs were allowed to couple for ~ 1 min, after designed primers 5F-SR7, 600F-NS4, 5F-18k, 150F-600R, which the bursa copulatrix and spermatheca were removed 150F-900R, 150-1140R, 500F-900R, and 500F-1140R (5F 5′-AG from females and examined for the presence of spores. Fresh CCTGCTGTCTAAGTATAAATGGG-3′, 600F 5′-GTTGTT tissues were examined with phase contrast microscopy. Spore GCAGTTAAAAMGCTCG-3′, 600R 5′-CGAGCKTTTTAA measurements using a Vickers (York, UK) image-splitting CTGCAACAAC-3′, 900R 5′-ATTAACGCCCCCAACTAT micrometer were made on 50 spores pooled from several bee- CCC-3′, 1140R 5′-CTCCCCCCGACCCAAAAACTTTG tles. For transmission electron microscopy (TEM), testes, AT-3′, 150F 5′-CTAATACATGCCCAAAAGGG-3′, 500F 5′- nerve ganglia, and fat body were fixed in 2.5% (v/v) glutaral- CGGCTACCACTTCTACGGAGG-3′). PCR amplifications dehyde and 2% (w/v) paraformaldehyde in phosphate buffer, were carried out in 25-ll reaction volumes with 1 llor30–50 pH 7.2, for 72 h at 4 °C, then 1% (v/v) acrolein and 2.5% (v/ ng of genomic DNA according to manufacturer’s instruction v) glutaraldehyde for 2 h at room temperature followed by for e2TAK DNA Polymerase (Takara Bio, Madison, WI). Amplifications consisted of an initial denaturing period (95 °C for 1 min), 40 cycles of denaturing (95 °C for 10 s), annealing Corresponding Author: J. Lord, USDA, Agricultural Research (50 °Cor55°C for 20 s) and extension (72 °C for 1.5 min), Service, Center for Grain and Animal Health Research, 1515 Col- and a final extension period (72 °C for 1 min). lege Avenue, Manhattan, Kansas 66502 USA—Telephone number: PCR products were purified for sequencing using the Wiz- +785-776-2705; FAX number: +785-537-5584; e-mail: jeff.lor- ard® SV gel and PCR Clean-up System A9282 (Promega, [email protected] 246 LORD ET AL.—ICHTHYOSPOREAN SYMBIONTS OF TENEBRIO MOLITOR 247

Madison, WI). Samples were submitted to the Kansas State University Sequencing and Genotyping Facility or SeqWright DNA Technology Services (Houston, TX) for sequencing. Sequence alignment and phylogenetic inference. We per- formed a nucleotide BLAST search in the NCBI database with the 1,284 bp SSU rRNA to identify homologous regions of closely related organisms. Of the large number of highly similar sequences (e value << eÀ100) we chose 22 sequences with sequence identify of  80%, all of which are ichthyospo- reans. The list of taxa included in the phylogenetic analysis and the corresponding GenBank numbers are shown on Fig. 5. The choanoflagellate Diaphanoeca grandis was chosen as an outgroup based on Steenkamp et al. (2006). A FASTA file consisting of the outgroup and 23 ingroup sequences was imported into Geneious (BioMatters Ltd., Auckland, New Zealand). The sequences were aligned using MAFFT (algorithm=Auto; Scoring matrix 200 PAM/k=2; gap open penalty = 1.53; offset value=0.123). Phylogeny was inferred from the aligned sequences using the Geneious plug- ins for Bayesian (MrBayes; Huelsenbeck and Ronquist 2001), maximum likelihood, and parsimony (PAUP* 4b10; Swofford 2002) analyses. The parameters for MrBayes were set at: total chain length 200,000; burn-in 20,000; subsample frequency 200. (The Jukes-cantor and HKY models gave identical trees.) The general time reversible, GTR, substitution model was utilized along with the inverse gamma rate variation model with four gamma categories. The branch lengths were uncon- strained. For maximum likelihood, we utilized Modeltest using AIC and heuristic search in PAUP*; the starting tree was based on neighbor-joining. The maximum parsimony tree was bootstrapped for 10,000 replicates using the FastStep algo- rithm with a random seed in PAUP*. In all analyses, the char- acters were weighted equally and gaps were treated as missing.

RESULTS Fig. 1–2. Ichthyosporean symbiont of Tenebrio molitor in fresh Occurrence. We examined T. molitor from five commercial preparations under phase contrast. 1. Spore cluster (sc) at the edge of producers and from our own laboratory culture. The beetles a thoracic nerve ganglion (g). 2. Spores (s) scattered in spermatophore from all of the sources were infected with the symbiont. In (sp) within a bursa copulatrix. addition to T. molitor, TMS was seen in the nerve chords of Zophobas atratus, another tenebrionid beetle that was obtained from commercial producers of both it and T. molitor. Phylogenetic analysis. The SSU rDNA was 1,292 bp (Gen- Unlike T. molitor infections, the spores in Z. atratus were not Bank JN699060). The trees inferred from parsimony, maxi- detected in testes, suggesting that transmission was per os. mum likelihood, and Bayesian analyses were highly similar, Tissue specificity and morphology. The symbiont was found especially with regard to the phylogenetic position of T. moli- in fat body, testes, and nerve chord (Fig. 1). In the ventral tor symbiont and its relationship to other taxa. Therefore, nerve chord, the spores were concentrated in clusters in or only the bootstrap consensus parsimony phylogenetic tree is adjacent to ganglia. The number of spores/cluster varied from shown (Fig. 5); the Bayesian and maximum likelihood trees less than ten to greater than 50. The testes were especially are available upon request from the authors. In the parsimony heavily infected, with clusters in the epithelium and between analysis, 951 characters were constant, 250 characters were testes lobes with occasional penetration of the lobes. At mat- variable, but parsimony uninformative, and 368 characters ing, spermatophores carried spores into the bursa copulatrix were parsimony informative. The parsimony analysis resulted (Fig. 2). Whether the transferred spores infected the eggs in a single tree of 1,343 steps. Although several monophyletic could not be determined, but no infection was seen in sections lineages were recovered by the parsimony analysis, some of of eggs either within the ovaries or after oviposition. Uninu- the deeper branches of the ingroup taxa were unresolved (see cleate and binucleate prespore stages developed in clusters Fig. 5), a result that is not surprising considering the relatively within vesicles and contained mitochondria with apparently small number of parsimony informative characters. flat cristae and thick-walled vesicles that contained a dense According to all three analyses, the TMS and C. mesnili are body (Fig. 3). Similar thick-walled vesicles were common in monophyletic with a high degree of statistical support (i.e. spores. The spores had thick, dense laminated walls resem- 98% of bootstrap replicates and Bayesian posterior probabil- bling those of the ichthyosporean, Caullerya mesnili (Lohr et ity of 1). The taxa Pirum gemmata and Abeoforma whisleri al. 2010) (Fig. 4) and measured 10.41 ± 0.84 x 6.6 ± 0.41 lm. were a sister clade to the T. molitor and C. mesnili clade, again Attempts to culture the organism in vitro were unsuccessful, with a high degree of statistical support. All of these species including in mycological media, such as potato dextrose broth fall within the order with the other species in and agar and in insect tissue culture media with or without Fig. 5 excluding the Dermocystidia, Rhynosporidium spp., and T. molitor nerve chords. spp. 248 J. EUKARYOT. MICROBIOL., 59, NO. 3, MAY–JUNE 2012

not been cultured in vitro. The original members of the group are pathogens of fish (Ragan et al. 1996), and most others are pathogens of aquatic organisms (Mendoza et al. 2002). Only the Eccrinales, which were formerly thought to be zygomycete fungi belonging to the class Trichomycetes, have been reported in association with insects, attaching to the hindgut cuticles of their aquatic hosts (Benny and O’Donnell 2000; Cafaro 2005). The TMS is the first representative of the class to be found in a terrestrial invertebrate. Of the Ichthyosporea for which morphological features are known, only C. mesnili resembles the TMS. Caullerya mesnili is a common parasite of Daphnia spp. and was recently trans- ferred to the Ichthyosporea based on its nuclear SSU rDNA sequence (Lohr et al. 2010). It had been placed in the Haplos- poridia (Chatton 1907) where the TMS was thought to belong (Poinar and Thomas 1984). Caullerya mesnili too falls into the order Ichthyophonida, which was erected by Cavalier-Smith (1998). Mendoza et al. (2002) characterized Ichthyophonida by the presence of a motile amoeboid stage, but none has been seen in either C. mesnili or TMS, nor is one present in some other species (Marshall et al. 2008). Unlike TMS, C. mesnili infects only gut tissue and is clearly pathogenic. Like TMS, C. mesnili has an arthropod host and has similarly ovoid spores with thick, dense laminate walls (Fig. 4). The TMS, C. mesnili, and , a parasite of crayfish (Vogt and Rug 1999), are the only ichthyosporeans known to infect ar- thropods internally beyond the gut lumen. Two recently described Ichthyosporea, Abeoforma whisleri and Pirum gem- mata (Marshall and Berbee 2011), are members of the same clade as the TMS and C. mesnili according to our analysis of SSU rDNA sequences and fall within the Ichthyophonida line- age of Cavalier-Smith (1998). These two species are quite mor- phologically distinct from the TMS and C. mesnili in having more rounded spores with thinner walls and many lucent vac- uoles in spores and vegetative stages. They also differ from TMS and C. mesnili in having been isolated from cultures that were started from the gut contents of marine Lophotrochozoa and in being culturable (Marshall and Berbee 2011). Mitochondrial cristae, which have been used as taxonomic characters, are flat in the TMS, A. whisleri, and P. gemmata, whereas those of C. mesnili have not been described and are not clear in published micrographs (e.g. Fig. 11 in Lohr et al. 2010). In any case, cristae shape should be carefully evaluated together with other morphological and physiological charac- teristics to avoid misinterpretations (Mendoza et al. 2002; Zettler et al. 2001). The morphological similarity between C. mesnili and TMS was reflected in the phylogenetic analysis of the SSU rRNA gene. In all three methods of phylogenetic inference used (i.e. maximum parsimony, maximum likelihood, and Bayesian), Fig. 3-4. Electron micrographs of the ichthyosporean symbiont the TMS and C. mesnili were sister taxa with strong statistical of Tenebrio molitor. 3. Immature stages with one or two nuclei (n) support. Furthermore, A. whisleri and P. gemmata were the clustered in a vesicle within the host’s testis. Insert shows thick-walled sister clade to the TMS and C. mesnili clade, a relationship vesicle (tv) and tubular mitochondrion with flat crista (mc). 4. Mature that was strongly supported in all three phylogenetic analyses. spore showing thick laminate wall (sw) and vesicle-filled cytoplasm. Overall, the phylogenetic analyses of TMS, C. mesnili, A. whis- leri, and P. gemmata are indicative of a common descent, A 1,581 bp LSU rDNA (GenBank JN699061) was not use- whereas morphology supports the phylogenetic analysis in ful for identifying the TMS as an ichthyosporean because no finding a closer relationship of TMS to C. mesnili than to other member of the group has an LSU rDNA sequence in A. whisleri, and P. gemmata. GenBank. In spite of infection of vital organs, the TMS does not pro- duce any apparent pathology, but this can only be confirmed by comparison of infected beetles with uninfected beetles. To DISCUSSION date, we have not been successful in obtaining a symbiont-free The class Ichthyosporea, comprising at least 30 genera of culture in spite of repeated efforts at surface disinfection of microbes, is a basal clade of protists near the fungi-animal eggs. It may be that eggs have internal infections that we have divergence. None is known to be free-living, and most have not detected. LORD ET AL.—ICHTHYOSPOREAN SYMBIONTS OF TENEBRIO MOLITOR 249

Rhinosporidium seeberi 99.8 AF158369

93.2 Rhinosporidium cygnus AF399715

87.6 Dermocystidium salmonis U21337

93.7 Dermocystidium percae AF533949

Sphaerothecum destruens AY267345

Abeoforma whisleri 100 GU810145

Pirum gemmata 99.1 GU810144

Caullerya mesnili 98 GU123072 100 T. molitor symbiont JN699060

Astreptonema gammari AY336709

56 Palavascia patagonica AY682845

99.9 Enteropogon sexuale AY336705

Enterobryus oxidi 88.3 AY336710 89 Astreptonema sp. Ichthyophonida AY336706

Creolimax fragrantissima EU124914 99.8 89.6 Y16260

Pseudoperkinsus tapetis AY192386

Psorospermium haeckelii U33180

Ichthyophonus hoferi U43712

70 Paramoebidium sp. 96.1 AY336708

86.1 parasiticum Y1915518

Eccrinidus flexile AY336698

Diaphanoeca grandis DQ059033 Fig. 5. Phylogeny inferred for the ichthyosporean symbiont of Tenebrio molitor and related taxa with GenBank accession numbers. The tree is a bootstrap consensus based on maximum parsimony. The choanoflagellate Diaphanoeca grandis was the outgroup. Numbers above the branches are bootstrap support values in percentage. 250 J. EUKARYOT. MICROBIOL., 59, NO. 3, MAY–JUNE 2012

Lohr, J. N., Laforsch, C., Kroner, H. & Wolinska, J. 2010. A Daph- ACKNOWLEDGMENTS nia parasite (Caullerya mesnili) constitutes a new member of the We thank Dr. Carlos Lange for suggestions to improve the Ichthyosporea, a group of protists near the animal-fungi diver- – manuscript, Drs. Jeff Wilson and Dan Boyle for assistance gence. J. Eukaryot. Microbiol., 57:328 336. Marshall, W. L., Celio, G., McLaughlin, D. J. & Berbee, M. L. 2008. with electron microscopy, and Tim Vocke of Nature’s Way Multiple isolations of a culturable, motile ichthyosporean (Meso- for providing insects. A special thanks to Andrea Schrage for mycetozoa, Opisthokonta), n. gen., n. sp., cheerfully performing over a thousand testes dissections. Men- from marine invertebrate digestive tracts. , 159:15–433. tion of trade names or commercial products in this publication Marshall, W. L. & Berbee, M. L. 2011. Facing unknowns: living cul- is solely for the purpose of providing specific information and tures (Pirum gemmata gen. nov., sp. nov., and Abeoforma whisleri, does not imply recommendation or endorsement by the U.S. gen. nov., sp. nov.) from invertebrate digestive tracts represent an Department of Agriculture. USDA is an equal opportunity undescribed clade within the unicellular lineage Ichthy- provider and employer. osporea (). Protist, 162:33–57. Mendoza, L., Taylor, J. W. & Ajello, L. 2002. The class Meso- mycetozoea: a heterogeneous group of microorganisms at the ani- LITERATURE CITED mal-fungal boundary. Annu. Rev. Microbiol., 56:315–344. Poinar, G. O. & Thomas, G. M. 1984. Laboratory Guide to Insect Adl, S. M., Simpson, A. G. B., Farmer, M. A., Andersen, R. A., Pathogens and Parasites. Plenum, New York, 392 p. Anderson, O. R., Barta, J. R., Bowser, S. S., Brugerolle, G., Fen- Ragan, M. A., Goggins, C. L., Cawthorn, R. J., Cerenius, L., Jamien- some, R. A., Fredericq, S., James, T. Y., Karpov, S., Kugrens, P., son, A. V. C., Plourde, S. M., Rand, T. G., So¨derhall, K. & Gutell, Krug, J., Lane, C. E., Lewis, L. A., Lodge, J., Lynn, D. H., Mann, R. R. 1996. A novel clade of protistan parasites near the animal- D. G., Mccourt, R. M., Mendoza, L., Moestrup, O., Mozley- fungal divergence. Proc. Natl. Acad. Sci. USA, 93:1190–11912. Standridge, S. E., Nerad, T. A., Shearer, C. A., Smirnov, A.V., Sprague, V. 1979. Classification of the Haplosporidia. Ma. Fish. Rev., Spiegel, F. W. & Taylor, M. F. J. R. 2005. The new higher level 41:40–44. classification of with emphasis on the of pro- Spurr, A. R. 1969. A low-viscosity epoxy resin embedding medium tists. J. Eukaryot. Microbiol., 52:399–451. for electron microscopy. J. Ultrastruc. Res., 26:31–41. Benny, G. L. & O’Donnell, K. 2000. Amoebidium parasiticum is a pro- Steenkamp, E. T., Wright, J. & Baldauf, S. L. 2006. The protistan tozoan, not a trichomycete. Mycologia, 92:1133–1137. origins of animals and fungi. Mol. Biol. Evol., 23:93–106. Cafaro, M. 2005. Eccrinales (Trichomycetes) are not fungi, but a Swofford, D. L. 2002. PAUP*. Phylogenetic Analysis Using Parsi- clade of protists at the early divergence of animals and fungi. Mol. mony (*and Other Methods). Version 4. Sinauer Associates, Sun- Phylogenet. Evol., 35:21–24. derland, Massachusetts. Cavalier-Smith, T. 1998. Neomonada and the origin of animals and Vogt, G. & Rug, M. 1999. Life stages and tentative life cycle of fungi. In: Coombs, G., Vickerman, K., Sleigh, M. & Warren, A. Psorospermium haeckeli, a species of a novel DRIPs clade from the (ed.), Evolutionary Relationships among Protozoa. Chapman & animal-fungal dichotomy. J. Exp. Zool., 283:31–42. Hall, London. p. 375–407. White, T. J., Bruns, T., Lee, S. & Taylor, J. W. 1990. Amplification Chatton, E. 1907. Caullerya mesnili n. g. n. sp. Haplosporidie parasite and direct sequencing of fungal ribosomal RNA genes for phyloge- des Daphnies. C. R. Seances Soc. Biol., 62:529–531. netics. In: Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. Gadzama, N. M. & Happ, G. M. 1974. The structure and evacuation J. (ed.), PCR Protocols: A Guide to Methods and Applications. of the spermatophore of Tenebrio molitor L. (Coleoptera: Tenebri- Academic Press, New York. p. 315–322. onidae). Tissue Cell, 6:95–108. Zettler, L. A. A., Nerad, T. A., O’Kelly, C. J. & Sogin, M. L. 2001. Hillis, D. M. & Dixon, M. T. 1991. Ribosomal DNA: molecular The nucleariid amoeba: more protists at the animal-fungal bound- evolution and phylogenetic inference. Quart. Rev. Biol., 66:411– ary. J. Eukaryot. Microbiol., 48:293–297. 453. Huelsenbeck, J. P. & Ronquist, F. 2001. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics, 17:754–755. Received: 09/28/11, 02/09/12; accepted: 02/16/12