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Protistology 3 (4), 257-264 (2004)
Protistology
Genetic diversity of insect trypanosomatids from subarctic and North-West Russia revialed by UP-PCR typing
Alexei Y Kostygov, Elena P. Slisarenko, Petr A. Merkulov and
Sergei A. Podlipaev
Zoological Institute RAS, St. Petersburg, Russia
Summary
A fast and reliable method of genotyping (UP-PCR) was applied to trypanosomatids isolated from insects collected in North and North-West Russia. These isolates were earlier shown to be situated in the crown of the trypanosomatid 18S rDNA phylogenetic tree. Relationships between parasites of two Heteroptera families — Nabidae and Saldidae — were investigated with a particular focus on isolates from supralittoral bugs Salda and Saldula. Several trypanosomatid species can apparently infect one insect species, as it was demonstrated for Leptomonas sp. PL and Wallaceina sp. Wsd isolated at the same place and day from different individuals of Salda littoralis. Very similar trypanosomatids, probably belonging to one species (Leptomonas sp. PL and Sld), were found in different insect genera, which points to a broad host specificity of these parasites. To our knowledge, the present paper is the first one aimed at revealing the presence and biodiversity of insect trypanosomatids from hosts inhabiting high latitude areas. The investigation has demonstrated that trypanosomatid fauna in subarctic regions is diverse.
Key words: Trypanosomatidae, Leptomonas, Wallaceina, Hemiptera, Nabidae, Saldidae, White Sea, UP-PCR typing
Introduction
Trypanosomatid protozoa include several mono-xenous (one host) genera, Blastocrithidia, Crithidia, Herpetomonas, Leptomonas, Rhynchoidomonas, and Wallaceina, which are usually referred to as insect trypanosomatids according to the host taxa most often
© 2004 by Russia, Protistology
parasitised. Out of ten known trypanosomatid genera, six consist of monoxenous insect parasites, insects serving as vectors for the other four (Wallace, 1966; Podlipaev, 1990). Diversity, phylogeny, host specificity and geographic distribution of insect trypanosomatids are very poorly known in contrast to thoroughly investigated insect-transmitted parasites of vertebrates
from the genera Trypanosoma and Leishmania. Thus, our knowledge of the entire family remains fragmentary.
Biodiversity of insect trypanosomatids is far from being well known. A recent 18S rRNA phylogenetic study showed that insect trypanosomatids included at least several unanticipated major lineages on the tree (Merzlyak et al., 2001b) and led to the suggestion that the group was extremely diverse.
Less than two hundred trypanosomatid species were described from insects (Podlipaev, 1990) but the search for flagellates ofinsects was restricted to 2000-2500 host species. As there are about one million insect species, only a tiny fraction of the predicted multitude of parasite species was sampled (Stevens, 2001). Most ofthe species described were studied only at the level of light microscopy, and their relationship with other species is obscure.
Insect trypanosomatids were searched only in several regions (Podlipaev, 2000). Such under-sampling provides very limited information on genetic diversity of insect trypanosomatids, that may be one of the most diverse groups within Kinetoplastida (Podlipaev, 2001). Trypanosomatids from insects inhabiting high latitude regions are rarely present in culture collections.
Host specificity of insect trypanosomatids is very broad and probability of nonspecific or/and occasional infection is high, especially in predators which may be invaded while feeding on infected prey (Bulat et al., 1999; Podlipaev, 2003). Supralittoral Hemiptera from the family Saldidae were chosen as hosts ecologically isolated from the others. Salda littoralis and Saldula pallipes are the only Hemiptera inhabiting the supra-littoral zone of the White Sea, where they live under a thick layer of stranded fucoids in a unique habitat, situated on the border of the tidal zone. Salda and Saldula were not found in other biotopes in the region under discussion, and no other Hemiptera were found in this particular habitat. A special investigation of such a specific biotope as seaweed washed ashore might be of interest not only for hydrobiology but also for parasitology, since parasite circulation has to be relatively restricted in this habitat.
In the regions investigated the most infected bugs are Nabis flavomarginatus (Nabicula flavomarginata) (fam. Nabidae) and Gerris spp. (fam. Gerridae) (Podlipaev, 1985; Podlipaev et al., 1991). Nabidae bugs inhabit forests and meadows, Gerridae bugs, freshwater reservoirs in woods. Salda littoralis and Saldula pallipes were not found in the Leningrad region. Saldidae bugs inhabiting the White Sea shore migrate to the water border even during the highest tide, whereas Nabidae or Gerridae were never recorded in this habitat. Therefore, the contact between the representatives of the Saldidae, the Gerridae and the Nabidae in the region of investigation is very improbable and we could
predict that some uncommon parasites might be found in such isolated hosts as Salda and Saldula.
The first trypanosomatid from Salda littoralis, Leptomonas rigidus, was described a decade ago. Its very peculiar morphological and ultrastructural characters discriminated this species from the trypanosomatids of Nabidae and Gerridae bugs (Podlipaev et al., 1991); moreover, L. rigidus had a unique shizodeme of kinetoplast DNA of small minicircle size and maxicircle restriction pattern (Kolesnikov et al., 1990).
However, Leptomonas rigidus proved to be difficult to cultivate and the culture was lost in several years. Having started complex investigations of subarctic insect trypanosomatids, we have isolated some new cultures from Saldidae. The aim of the current paper is to study diversity of Nabidae and Saldidae parasites in the Leningrad region and on the White Sea shore near the polar circle using Universally Primed PCR (UP-PCR) typing.
Material and methods
Methods of culture isolation, purification and cultivation were described earlier (Podlipaev, 1985; Podlipaev and Frolov, 1987). Laboratory cultures were isolated from insects collected in North-West and North Russia (Table 1). All newly isolated cultures grow well, e.g., the culture of Leptomonas sp. PL11 achieves its maximum (106 cells/ml) in 9-10 days (Fig. 1).
Universally Primed PCR (UP-PCR) is a PCR fingerprinting method (Bulat et al., 1992), close to the well-known RAPD technique (Williams et al., 1990), that enables amplification of DNA from any organism without previous knowledge of DNA sequences and generation of multibanding profiles (fingerprints) by agarose gel electrophoresis. The main advantages of UP-PCR are in the use of high annealing temperatures, fast ramping and relatively long primers, resulting in increased reproducibility, poor reproducibility being an inherent problem of the RAPD technique (e.g. Karp et al., 1997). In our previous study primers were chosen and UP-PCR was performed on 16 isolates of different insect and plant trypanosomatids (Bulat et al., 1999). Both papers use the same three isolates, which allows us to show reproducibility of isolates grouping. The results obtained with UP-PCR (Bulat et al., 1999) are in good correspondence with 18S rDNA-based phylogeny (Merzlyak et al., 2001b), which confirms the choice of UP-PCR as a reliable and fast method to estimate the genetic diversity of insect trypanosomatids.
For DNA isolation cells were concentrated by means of centrifugation 10 min at 5000g, and total DNA was extracted by standard phenol-chloroform
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Fig. 1. Leptomonas sp. PL11 growth curve.
method (Sambrook et al., 1989). About 100 ng of this DNA was used for PCR amplification.
UP-PCR was performed in 21 ^l volume contain-ing 2 mM dNTP, 6mM MgCl2 500ng primer, 1U of Taq polymerase (SRI Genetika, Moscow, Russia) and an appropriate PCR buffer. Four primers were used for this investigation: L45: 5'-GTAAAA CGACGGCCAGT-3', L15/ AS19: 5'-GAGGGTGGCGGC TAG-3', AA2M2: 5'-GAG CGA CCCAGAGCGG-3', AS15: 5'-
Table 1. Description of insect trypanosomatid strains
Species/Isolate name
Insect Host
Place and time of isolation
Leptomonas sp. P
Leptomonas nabiculae D2 Podlipaev, 1985
Leptomonas peterhoffi 101 Podlipaev, 1985
Leptomonas sp. CL8
Leptomonas sp. F2
Leptomonas sp. F5
Leptomonas sp. F6
Leptomonas sp. F7
Leptomonas sp. F8
Leptomonas sp. PL11
Leptomonas sp. Sld
Wallaceina sp. Wg Wallaceina sp. Wsd
Panorpa communis Mecoptera: Panorpidae
Nabicula flavomarginata (Nabis flavomarginatus) Hemiptera: Nabidae
Nabicula flavomarginata (Nabis flavomarginatus) Hemiptera: Nabidae
Nabicula flavomarginata (Nabis flavomarginatus) Hemiptera: Nabidae
Nabicula flavomarginata (Nabis flavomarginatus) Hemiptera: Nabidae
Nabicula flavomarginata (Nabis flavomarginatus) Hemiptera: Nabidae
Nabicula flavomarginata (Nabis flavomarginatus) Hemiptera: Nabidae
Nabicula flavomarginata (Nabis flavomarginatus) Hemiptera: Nabidae
Nabicula flavomarginata (Nabis flavomarginatus) Hemiptera: Nabidae Salda littoralis Hemiptera: Saldidae
Saldula pallipes Hemiptera: Saldidae
Gerris rufoscutellatus Hemiptera: Gerridae Salda littoralis Hemiptera: Saldidae
Leningrad region,
North-West Russia, 1988 by S.
Podlipaev
Leningrad region,
North-West Russia, 1983 by S.
Podlipaev
Leningrad region,
North-West Russia, 1982 by S.
Podlipaev
Leningrad region,
North-West Russia, 1984 by A. Frolov
Cape Kartesh, Tshupa bay,
White Sea, North Russia,
1986 by S.Podlipaev
Cape Kartesh, Tshupa bay,
White Sea, North Russia,
1986 by S.Podlipaev
Cape Kartesh, Tshupa bay,
White Sea, North Russia,
1986 by S. Podlipaev
Cape Kartesh, Tshupa bay,
White Sea, North Russia,
1986 by S. Podlipaev
Cape Kartesh, Tshupa bay,
White Sea, North Russia,
1986 by S. Podlipaev
Cape Kartesh, Tshupa bay,
White Sea, North Russia,
2001 by E. Slisarenko & P. Merkulov
Cape Kartesh, Tshupa bay,
White Sea, North Russia,
2000 by S. Podlipaev
Leningrad region, North-West
Russia, 2000 by M. Malysheva
Cape Kartesh, Tshupa bay,
White Sea, North Russia,
2001, by E. Slisarenko & P. Merkulov
Fig. 2. UP-PCR banding profiles for trypanosomatids examined, generated with L45 primer. Lines from left to right: M - Molecular Weight Markers (Lambda DNA/EcoRI+HindIII); Leptomonas sp. PL11; Leptomonas sp. Sld; Wallaceina sp. Wsd; Wallaceina sp. Wg; Leptomonas peterhoffi 101; Leptomonas sp. P; Leptomonas sp. D2; Leptomonas sp. F2; Leptomonas sp. F5; Leptomonas sp. F7; Leptomonas sp. F8; Leptomonas sp. CL8. Isolate designations are given in Table. 1.
Fig. 3. Live cells of Leptomonas sp. PL11, phase contrast.
GGCTA AGCGGTCGTTAC-3'. The reaction was carried out in the Genamp 2700 thermal cycler (Applied Biosystems). The temperature profile was as follows: first 95°C for 3 min, then 40 cycles 95°C for 1min, 55°C for 1 min, 72°C for 1min 15 sec, and finally 72°C for 5 min. Amplification products were tested on 10 cm long 1.5% agarose gel stained with ethidium bromide (Fig. 2).
The UP-PCR profiles were analyzed by means of Gel-Pro Analyzer 3.1 software package (Media Cybernetics). The data obtained from individual gels were combined and input in Treecon v. 1.3b program (Y. Van de Peer, Copyright 1994-2001, University of Konstanz, Germany) to construct the tree of genetic distances. The tree was created using Nei and Li distance estimation and clustering with UPGMA method.
Results and discussion
Parasite prevalence in Salda littoralis (in total, about 90 insects were dissected) achieved almost 50% and did not significantly vary in different years (47,6% in 2001; 40% in 2002; 43% in 2003). Saldula pallipes collected from the same locality showed much lower parasite prevalence: about 1%.
Romanowsky-Giemsa stained cells from new cultures displayed blurred prochoanomastigote morphotype for Leptomonas sp. PL and Sld, as well as for Leptomonas sp. F2, F5, F7 and F8 (typical morphotypes are shown on Figs 3, 4). Leptomonas sp. F6 showed very similar cell shape (Merzlyak et al., 2001b). Wallaceina sp. Wsd (Fig. 4 D) and Wallaceina sp. Wg (Fig. 4 I) demonstrated endomasti-gotes typical of Wallaceina genus (Podlipaev et al., 1990). Leptomonas nabiculae, Leptomonas peterhoffi, Leptomonas sp. P and Leptomonas sp. CL8 were promastigote-like. On the denrogram obtained (Fig. 5) endomastigote-bearing parasites did not form groups of any taxo-nomic significance but inter-calated among promastigote-like flagellates.
From the dendrogram obtained we can see that Leptomonas nabiculae and Leptomonas sp. D2 are united in the same cluster, which was reliably demonstrated in our previous work (Bulat et al., 1999). Therefore, we can designate the genetic distances close to 0.3 as the level of cluster reliability.
On the phylogenetic tree based on 18SrDNA with isolates from insects (Merzlyak et al., 2001b) most parasites from North and North-West Russia constitute the tree crown with unresolved relationships between parasites. Since they were isolated in northern Russia they belong to postglacial fauna (Podlipaev, 2001) that invaded the territory under discussion not earlier than 15 thousand years ago (Nikonov and Shlykov, 2002). In previously constructed phylogenies the trypanoso-matids' divergence time was estimated as hundred million of years (Fernandes at al., 1993). Therefore, postglacial fauna can attract special attention since it gives a recent checkpoint for molecular phylogenies.
Isolates from the Saldidae differ strongly from those from the Nabidae (Table 1, Fig. 5). Promastigote-like Leptomonas sp. Sld and PL are very close to each other, both differing from endomastigote-bearing parasite of Salda - Wallaceina sp. Wsd.
Isolates F2 — F8 — parasites of Nabis flavomarginatus (Nabicula flavomarginata) on the White Sea shore — form one cluster, with different genetic distances
Fig. 4. Romanowsky-Giemsa- stained cells. A — Leptomonas sp. PL11 from Salda littoralis; B — Leptomonas sp. PL11 from laboratory culture; C — Leptomonas sp. Sld, culture; D- Wallaceina sp. Wsd, culture; E - Leptomonas sp. F2, culture; F- Leptomonas sp. F5, culture; G - Leptomonas sp. F7, culture; H — Leptomonas sp. F8, culture; I — Wallaceina sp. Wg, culture.
between isolates. All these isolates probably belong to one or two closely related species. At least F5 and F6 isolates correspond to the crown group that had very short branches in the 18S rRNA-based trees (Merzlyak et al., 2001b), very similar ND8 mRNA editing patterns (Merzlyak et al., 2001a) and 5S rRNA sequences (Podlipaev et al., in press).
Wallaceina sp. Wg from Gerris in Leningrad region is clustered with "F" group but has less genetic similarity. Leptomonas peterhoffi and Leptomonas sp. CL8 isolated from Nabis flavomarginatus (Nabicula flavomarginata) in the Leningrad region constitute the
next branch. This association of isolates ("F" group, Wallaceina sp. Wg, Leptomonas peterhoffi and Leptomonas sp. CL8) very likely belongs or is very close to the Wallaceina genus, which corresponds to the data obtained earlier. On the 18S rDNA-based phylogenetic tree Leptomonas sp. F6 and Leptomonas peterhoffi are grouped close to Wallaceina and both are located in the crown (Merzlyak et al., 2001b). In addition, L. peterhoffi occupied a similar position on UP-PCR dendrogram (Bulat et al., 1999). It is worth mentioning that proceeding from the data obtained earlier (Merzlyak et al., 2001a; Bulat et al., 1999) the two known Wallaceina
species, and the isolates associated with this genus, appear to be in the tree crown only. Here we report the first case when Wallaceina-like trypanosomatids (isolate Wsd) are obviously separated from the crown group. This fact is supported by 5S rDNA based phylogeny (Podlipaev et al., in press).
The taxonomy of trypanosomatids is very unreliable, especially in case of insect flagellates (Podlipaev, 2000, 2001). Almost century-old diagnoses of kinetoplastid genera are increasingly unsuitable, resulting in a situation, when it is easier to describe a new species than to assign it to a genus (Dollet, 2001). At least four insect parasites genera are polyphyletic (Hollar et al., 1998; Maslov et al., 2001; Merzlyak et al., 2001b; Huges and Piontkivska, 2003). We used two Wallaceina isolates in the present work and only one of them — Wallaceina sp. Wg — was shown to be close to the group related to the both species of Wallaceina described previously (W. brevicula and W. inconstans). To our knowledge, it is now described as Wallaceina vicina (Malysheva and Frolov, in press). As for the isolate named Wsd, it obviously should not be assigned to the genus Wallaceina (although it has typical endomastigotes) to avoid making one more genus polyphyletic.
Leptomonas sp. PL and Wallaceina sp. Wsd have been isolated from different individuals of S. littoralis at the same locality and day. Therefore we presume that the "one host - one parasite" paradigm is not applicable to insect trypanosomatids, since the two parasites are not closely related (Fig. 5). At the same time, leptomonads PL and Sld isolated from Salda littoralis and Saldula pallipes, respectively, seem to belong to one species. The two hosts belong to different genera of the family Saldidae and the cultures were obtained within a one-year-interval. This is an argument for low specificity of the parasite. One of the possible explanations is that Salda, as a predator, got infected while feeding on Saldula. Saldidae do not form colonies or feeding groups, therefore aggregation or common feeding may not play a role in transmission of these trypanosomatids, contrary, e.g., to the Drosophila spp. (Ebbert et al., 2001).
Leptomonas sp. P and Leptomonas nabiculae are very close to each other. Nevertheless, they were isolated from hosts belonging to different insect orders (Table 1). This fact, combined with the position of Wallaceina sp. Wg from Gerris among isolates from Nabicula, is another evidence of a very broad host specificity of monogenetic trypanosomatids (Podlipaev, 2003).
All trypanosomatids, isolated from Nabisflavomar-ginatus (Nabicula flavomarginata) at the White Sea, are clustered in one group with subgroups of isolates closely resembling each other. Cultures isolated in the Leningrad region from different host insects are situated in the central part of the dendrogram.
The isolates under discussion hardly group by host taxa, more likely the grouping is arranged according to territories and parasite taxa: all Wallaceina-related flagellates cluster together with the closest parasites isolated from the White Sea shore, other isolates including a Wallaceina-like one are out of the Wallaceina-group with a cluster of the White Sea isolates from Saldidae bugs.
Cluster analysis of the isolates obtained in NorthWest and North Russia has shown that almost each isolate represents definite typing unit, with different scale of relationships between them. Some isolates very likely belong to one species — Leptomonas sp. F2 — F8; Leptomonas sp. PL11 and Sld, and, probably, Leptomonas sp. P and D2; the rest of isolates are expected to belong to separate species and Wallaceina sp. Wsd might be a member of a new genus.
To our knowledge, the present paper is the first one aimed at revealing the presence and biodiversity of insect trypanosomatids from the hosts inhabiting high latitude areas. This study proves that trypanosomatid fauna in subarctic region is diverse.
Acknowledgements
This work was carried out in the Center "Taxon" of the Zoological Institute and was partly supported by the Federal Program "Integration of Science and Education, I0035/1342", and the Programs of RAS "Biodiversity Conservation in Russia" and "Dynamics of plant, animal and human genofonds".
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-Leptomonas sp. F2
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Address for correspondence: Alexei Y. Kostygov. Zoological Institute, Russian Academy of Sciences, 199034, St. Petersburg, Russia. E-mail: Alex_Kostygov@vs13059.spb.edu
Editorial responsibility: Alexander Frolov
