Trichonymphea, Parabasalia) from Neotropical Rhinotermitids
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The Journal of Published by the International Society of Eukaryotic Microbiology Protistologists J. Eukaryot. Microbiol., 58(6), pp. 487–496 r 2011 The Author(s) Journal of Eukaryotic Microbiology r 2011 International Society of Protistologists DOI: 10.1111/j.1550-7408.00575.x Morphology and Molecular Phylogeny of Pseudotrichonympha hertwigi and Pseudotrichonympha paulistana (Trichonymphea, Parabasalia) from Neotropical Rhinotermitids JUAN F. SALDARRIAGA,a,Ã GILLIAN H. GILE,a,1,Ã ERICK R. JAMES,a ALES HORA´ K,a,2 RUDOLF H. SCHEFFRAHNb and PATRICK J. KEELINGa aDepartment of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4, and bResearch & Education Center, University of Florida, 3205 College Avenue, Davie, Florida 33314, USA ABSTRACT. Pseudotrichonympha is a large hypermastigote parabasalian found in the hindgut of several species of rhinotermitid termites. The genus was discovered more than 100 years ago, and although over a dozen species have since been described, this represents only a small fraction of its likely diversity: the termite genera from which Pseudotrichonympha is known are all species rich, and in most cases their hindgut symbionts have not been examined. Even formally described species are mostly lacking in detailed microscopic data and/or sequence data. Using small subunit ribosomal RNA gene sequences and light and scanning electron microscopy we describe here the morphology and molecular phylogenetic position of two Pseudotrichonympha species: the type species for the genus, Pseudotricho- nympha hertwigi from Coptotermes testaceus (described previously in line drawing only), and Pseudotrichonympha paulistana from Heterotermes tenuis (described previously based on light microscopy only). Key Words. Diversity, DNA barcoding, intestinal symbionts, Isoptera, lower termites, Parabasalia, Rhinotermitidae, Teranymphidae, Trichonymphida. SEUDOTRICHONYMPHA is a large hypermastigote para- type species of Pseudotrichonympha is therefore most reasonably P basalian belonging to the Teranymphidae (Cepicka, Hampl, concluded to be a symbiont of C. testaceus. and Kulda 2010) and found in the hindgut of several species of Since the description of P. hertwigi many other species of rhinotermitid termites. In 1910, the German biologist Max Hart- Pseudotrichonympha have been described. Almost all are sym- mann investigated the protozoan symbionts of a then unnamed bionts in rhinotermitid termites of eight genera: Coptotermes, species of the termite genus Coptotermes (Isoptera, Rhinotermit- Heterotermes, Parrhinotermes, Prorhinotermes, Psammotermes, idae) collected in Manguinhos, Rio de Janeiro, Brazil. Having Termitogeton, Rhinotermes, and Schedorhinotermes (Kitade and been interested throughout his career in mechanisms of sexuality, Matsumoto 1998; Noda et al. 2005; Yamin 1979), although Pseu- Hartmann (1910) described three organisms inside that termite dotrichonympha has also been reported from the serritermitid that he interpreted as the sexual stages and juveniles of a single genera Serritermes and Glossotermes (Cancello and DeSouza species of protozoan, his Trichonympha hertwigi. The Italian 2004; Costa-Leonardo and Kitayama 1991), and there is also zoologists Giovanni Battista Grassi and Anna Foa` (1911) objected one report of Pseudotrichonympha in Rugitermes, a kalotermitid to Hartmann’s classification of those protozoans as members of termite (De Mello 1954a). Recent studies have made it clear that the genus Trichonympha. They also disagreed with his interpre- the diversity of Pseudotrichonympha is likely underdocumented, tation of the three morphotypes as sexual stages of a single species like that of many other genera of protists. Noda et al. (2007) re- of protist, and redescribed the ‘‘male’’ and ‘‘female’’ forms of vealed that the evolutionary history of at least some members of T. hertwigi as two new genera: Pseudotrichonympha and Holo- the genus Pseudotrichonympha, based on molecular phylogenies, mastigotoides, respectively, though they provided no further mor- reflects very closely not only that of their rhinotermitid termite phological observations. Confusingly, in the original paper the hosts, but also that of their bacterial endosymbionts, which account correspondence of Pseudotrichonympha and Holomastigotoides for more than two-thirds of the total bacterial population in the to male and female forms was misprinted and given in reverse, a termite’s gut. Each of the termite species they investigated was mistake that was corrected later (Grassi 1917). As for the ‘‘juve- foundtoharboronlyonePseudotrichonympha species, as defined nile’’ forms, Grassi and Foa` (1911) assigned them to a third new by 498% small subunit (SSU) ribosomal RNA (rRNA) gene se- genus, Spirotrichonympha, but did not select the form seen by quence identity among clones, and the phylogeny of Pseudotricho- Hartmann as its type species. The Coptotermes host of Pseudot- nympha species was fully congruent with the phylogeny of the host richonympha hertwigi was named Coptotermes hartmanni by termites. Of the 15 Pseudotrichonympha ‘‘species’’ investigated by Holmgren (1911), but he failed to provide a formal description Noda et al. (2007), only three have been formally named. and as a consequence this name is now considered invalid (Snyder Here we use light microscopy (LM), molecular methods, and 1949). Subsequent detailed surveys of neotropical termite diver- the first use of scanning electron microscopy (SEM) to investigate sity have shown that all native species of Coptotermes in Brazil the Pseudotrichonympha symbionts of two species of rhino- correspond to Coptotermes testaceus (RHS, unpubl. data), so the termitid termites collected in Colombia. One of the organisms that we investigate is the type species of the genus, P. hertwigi from C. testaceus, while the other is Pseudotrichonympha pau- Corresponding Author: P.J. Keeling, Department of Botany, Univer- listana described from Heterotermes tenuis (De Mello 1954b). sity of British Columbia, 3529-6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4—Telephone number: 11 604 822 4906; FAX number: 11 604 822 6089; e-mail: [email protected] MATERIALS AND METHODS 1 Present Address: Department of Biochemistry and Molecular Biol- Host termite collections. Termites were collected in May and ogy, Dalhousie University, 5850 College Street, Room 9-G1, Halifax, Nova Scotia, Canada B3H 1X5. June of 2009 in the framework of a faunistic study of the isopter- 2Present Address: Biology Centre, Institute of Parasitology, Czech ans of the Caribbean Basin. Rhinotermitid termites of the genera Academy of Sciences, and Faculty of Science, University of South Heterotermes and Coptotermes were collected above the town Bohemia, 37005 Ceske´ Budejovice (Budweis), Czech Republic. of Minca in the Sierra Nevada de Santa Marta, Magdalena, ÃContributed equally. Colombia (11.12561N, 74.119721W). Collected specimens were 487 488 J. EUKARYOT. MICROBIOL., 58, NO. 6, NOVEMBER–DECEMBER 2011 subsequently identified by morphology and DNA barcoding (mi- In order to confirm the identity of the host termites, partial mt tochondrial SSU [mt SSU] rRNA gene). SSU rRNA gene sequences were amplified using the primers 50- Light and SEM. Whole termite guts were dissected, sub- TTA CGC TGT TAT CCC TAA-30 and 50-CGC CTG TTT ATC merged in Trager medium U (Trager 1934), and macerated on AAA AAC AT-30 (Kambhampati and Smith 1995; Simon et al. glass slides. Light microscopy was performed on living cells using 1994) from genomic DNA extracted using the Epicentre Master- a Zeiss Axioplan 2 compound microscope (Zeiss, Oberkochen, pure Complete DNA and RNA purification kit. Polymerase chain Germany) with Plan Apochromat objectives and differential in- reaction products were sequenced directly on both strands. New terference contrast (DIC) optics. Image capture was carried out sequences determined in this study were deposited in GenBank using a Q-imaging Micro Imager II digital camera or using a under accessions HQ683705–HQ683708. Canon XL-M1S (Canon, Tokyo, Japan) for HD video, from which Molecular phylogenetic analyses. The identities of C. test- stills were captured and live video archived. aceus and H. tenuis were determined by BLAST similarity and For SEM, whole gut contents were diluted approximately 1:1 confirmed by phylogenetic analysis of their mt SSU rRNA gene with medium U, and prefixed with OsO4 vapor for 30 min. The sequences, because this gene has been the most commonly used prefixed material was transferred to small containers with a floor barcode for lower termites (e.g. see Scheffrahn et al. 2004; that consisted of a 10-mm polycarbonate membrane filter (Corning Szalanski et al. 2004). All available Coptotermes and Hetero- Separations Division, Acton, MA) and fixed for 30 min with a termes mt SSU rRNA gene sequences and select Reticulitermes mixture of 8% (v/v) glutaraldehyde and 4% (w/v) OsO4, giving a sequences with high BLAST similarity were downloaded from final concentration of 2.5% glutaraldehyde and 1% OsO4. Sam- GenBank, aligned with our new sequences using MAFFT (Katoh ples were postfixed with a few drops of 4% OsO4 for 30 min and et al. 2002), and refined by eye. To reduce the number of taxa washed 3 times in medium U to remove the fixative. Samples were included, sequences were removed from the alignment if they dehydrated using a graded ethanol series and critical point dried in were both identical to and from the same collection location as a Tousimis