Protistology 13 (4), 206–214 (2019) Protistology Crithidia dobrovolskii sp. n. (Kinetoplastida: Try- panosomatidae) from parasitoid fly Lypha dubia (Diptera: Tachinidae): morphology and phylogenetic position Anna I. Ganyukova, Marina N. Malysheva, Petr A. Smirnov and Alexander O. Frolov Zoological Institute, Universitetskaya nab. 1, 199034 St. Petersburg, Russia | Submitted November 17, 2019 | Accepted December 11, 2019 | Summary The article provides characteristics of a new parasite, Crithidia dobrovolskii sp.n., which was isolated from the tachinid fly captured in the Leningrad Region of Russia. The presented description of Crithidia dobrovolskii sp.n. is based upon light microscopic, ultrastructural, and molecular phylogenetic data. Molecular phylogenetic analyses of SSU rRNA gene and GAPDH gene sequences have demonstrated that the new species is most closely related to Crithidia fasciculata. Key words: Crithidia, Trypanosomatidae, phylogeny, SSU rRNA, GAPDH, ultra- structure Introduction et al., 2013; Maslov et al., 2013), as well as the fact that it is monoxenous insect parasites that are now Flagellates belonging to the Trypanosomatidae considered ancestral forms of all representatives of family are widespread parasites of animals, plants and the family (Frolov, 2016). One of the most signi- protists. Dixenous (i.e. “two-host”) parasites from ficant findings in the history of the family study the genera Trypanosoma and Leishmania, the most was the discovery and description of the new genus well-known representatives of the group that are Paratrypanosoma. Monoxenous flagellates P. con- pathogens of humans and animals, have significant fusum, found in the gut of culicid mosquitoes, economic and medical importance. Until recently, are located at the base of the phylogenetic tree of monoxenous (i.e. “one-host”) trypanosomatids, trypanosomatids, occupying a position between parasitizing mainly in representatives of two insect free-living kinetoplastids and other species of the orders Diptera and Hemiptera, were overshadowed family Trypanosomatidae (Flegontov et al., 2013). by dixenous species of the family. However, in recent The results of numerous studies upon insect- years, interest in monoxenous trypanosomatids parasitizing trypanosomatids obtained in the last of insects has increased significantly. This fact is decade entailed revision of many views on diver- explained by the discovered high biodiversity of the sity, origin and entire system of the family Trypa- group, a wide range of hosts (Podlipaev, 2001; Týč nosomatidae (Podlipaev, 2001; Teixeira et al., 2011; doi:10.21685/1680-0826-2019-13-4-4 © 2019 The Author(s) Protistology © 2019 Protozoological Society Affiliated with RAS Protistology · 207 Flegontov et al., 2013; Kostygov et al., 2014; To visualize DNA containing trypanosomatid Votýpka et al., 2014). New species have been structures, DAPI staining with fluorescent dye described and continue to be described (Yurchenko was used according to the methods described et al., 2014), as well as new taxa of a higher rank previously (Ganyukova et al., 2017). The slides (genus and subfamily), for example, the genus were studied using a Leica DM 2500 microscope. Jaenimonas (Hamilton et al., 2015) and the genus Microphotographs were obtained using a 14 Mps Kentonomas (Votýpka et al., 2014), the subfamilies USB camera UCM0S14000KPA (TOUPCAM). Phytomonadinae (Yurchenko et al., 2015) and Flagellate cell sizes were measured using the Toup Strigomonadinae (Teixeira et al., 2011). View software (version 3.7, 2013). Statistical data Despite the growing interest in monoxenous processing was carried out in the LibreOffice Calc parasites of flies, as representatives of one of the program. The statistical significance of differences in least studied groups of trypanosomatids, their the average values of morphological characteristics fauna remains studied in a fragmented manner. was evaluated using Student’s t criterion (n=25) At the same time, the geography of the research (significance level of P<0.01). is extremely heterogeneous and tends to study the parasite fauna in the tropics and subtropics zones. TRANSMISSION AND SCANNING ELECTRON MICROSCOPY The present work was carried out in the context of investigation of the diversity of trypanosomatids For electron microscope studies, the flagellate parasitizing dipteran insects in the north of Eurasia. culture, previously purified from concomitant orga- The article provides description of a new species nisms, was precipitated by centrifugation (3000 Crithidia dobrovolskii sp.n. from the intestines of the rpm), the supernatant was discarded, and the sedi- fly Lypha dubia Fall. (Diptera: Tachinidae). ment was fixed with 1.5 % glutaraldehyde in 0.1 M cacodylate buffer (1 hour), after which the cells washed in a 0.1 M solution of cacodylate buffer, Material and methods followed by postfixation with a 2 % solution of OsO4 in cacodylate buffer for 30 minutes. Then the FIELD WORK, INSECT’S DISSECTION AND CULTIVATION material was dehydrated in alcohols of increasing OF TRYPANOSOMATIDS concentration and acetone and embedded in a mix- ture of Araldite and Epon. Sections were obtained The collection of insects was carried out in June on a Leica UC-6 ultramicrotome, stained with an 2018 on a field near the village Vysokoklyuchevskoy aqueous solution of uranyl acetate and lead citrate of the Leningrad Region, Russia (59°47’N, 30°08’E). and examined under a Morgagni 268-D microscope The insects were euthanized in chloroform and (FEI Company). dissected in a drop of physiological saline after For scanning electron microscopy, the fixed that the intestines were isolated. Fragments of the and dehydrated material deposited on poly-L- intestine and its contents were studied for infection. lysine-coated coverslips was processed in an HCP-2 Intestinal fragments infected with trypanosomatids critical point dryer (Hitachi Ltd., Tokyo, Japan) were placed in tubes with Brain Heart Infusion and after coating with a 20 nm platinum layer in culture medium (BHI, Difco) supplemented with an IB-5 coating device Ion (Giko Co. Ltd., Tokyo, hemin (25 µg/ml), benzylpenicillin antibiotics at Japan) was examined under a Tescan Mira3 LMU a rate of 500 units/ml and streptomycin 500 µg / microscope with accelerating voltage of 25.00 kV. ml. Subsequently, the culture of flagellates was purified from related organisms in M-shaped tubes DNA ISOLATION, AMPLIFICATION AND SEQUENCING (Podlipaev and Frolov, 1987). Genomic DNA was isolated from cell culture LIGHT MICROSCOPY and alcohol-fixed insect tissues using the PureLink Genomic DNA Kit (Invitrogen). The host COI The cells morphology was studied using vital gene fragment was amplified using LCOI and slides and dry smears. Dry smears made from HCOI primers according to the previously described fragments of infected intestine or a drop of culture protocols (Cywinska et al., 2010). Fragments of the were fixed for 10 minutes in 96% ethanol, after which 18S and GAPDH genes were amplified using speci- Giemsa staining was performed for 25 minutes. fic primers S762-763 and M200-M201, respectively 208 · Anna I. Ganyukova, Marina N. Malysheva, Petr A. Smirnov and Alexander O. Frolov (Kostygov et al., 2014). The amplification protocol Table 1. Morphometry of Crithidia dobrovolskii and the composition of the reaction mixture are in the laboratory culture. described earlier (Kostygov and Frolov, 2007). The resulting PCR fragments were isolated from the Parameters Promastigotes Paramastigotes 7,74 ±0,86 7,58 ±0,92 reaction mix and purified using a Cleanup Standard Cell length (μm) kit (Eurogen) and sequenced on a 3500 xL Applied (6,39-9,18) (6,25-9,03) 2,97 ±0,37 2,91 ±0,31 Cell width (μm) Biosystems automated sequencer using Thermo (2,47-3,72) (2,39-3,63) Sequenase Cy5 Dye Terminator Kit. 8,12 ±1,12 7,95 ±1,05 Free fl agellum length (μm) (6,33-10,19) (6,28-10,10) 2,61 ±0,32 2,55 ±0,37 Nucleus length (μm) PHYLOGENETIC ANALYSES (2,12-2,78) (2,21-2,74) 2,36 ±0,13 2,26 ±0,23 Nucleus width (μm) The alignment of SSU rRNA gene sequences of (2,12-2,56) (2,13-2,43) trypanosomatids, representing known phylogroups Anterior end to nucleus 2,45 ±0,49 2,73 ±0,40 distance (μm) (1,75-4,02) (1,90-3,08) of these flagellates was prepared as described before Anterior end to kinetoplast 1,49 ±0,45 2,81 ±0,52 using the software package MEGA (Molecular distance (μm) (0,68-2,13) (1,95-3,19) Evolutionary Genetic Analysis Version 5.05, Tamu- ra et al., 2011, www.megasoftware.net) (Schwarz et al., 2014). The final file included 35 sequences and L. acus (Fig. 1). Summing it up, the sequence of the 18S rRNA gene of trypanosomatids. The analysis demonstrated that the studied trypanoso- number of positions in the final file with indices was matid C. dobrovolskii should represent a separate 2174. Phylogenetic analysis of the sequences was species. performed by the Bayesian algorithm in MrBayes The SSU rRNA and GAPDH C. doborvolskii are 3.2.7 program using the GTR+G+I model with the deposited to the GenBank system under accession following parameters: 2 runs, 4 Markov chains, 3 numbers MN809265 and MN807630 respectively. million generations, sampling every 100 generations, Flagellates are presented in culture by two main removal of the first 8 thousand trees when building morphotypes: promastigotes and paramastigotes consensus. (Fig. 2A). In promastigote cells kinetoplast is situ- ated in the anterior third of the cell; the nucleus is displaced to its middle. The average length of such Results cells is 7.74±0.86 µm, the flagellum is well defined, and its length reaches an average of 8.12±1.12 We have discovered C. dobrovolskii infection µm. The nucleus of the paramastigote is situated in a fly rectum identified as Lypha dubia (Diptera: in the middle of the cell or can be displaced to the Tachinidae) on the basis of the COI gene sequence. posterior end. However, the kinetoplast is situated The axenic culture of flagellates that is maintained at the level of the nucleus to its posterior edge.