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Harl et al. Malar J (2019) 18:305 https://doi.org/10.1186/s12936-019-2940-6 Malaria Journal RESEARCH Open Access The nuclear 18S ribosomal DNAs of avian haemosporidian parasites Josef Harl1, Tanja Himmel1, Gediminas Valkiūnas2 and Herbert Weissenböck1* Abstract Background: Plasmodium species feature only four to eight nuclear ribosomal units on diferent chromosomes, which are assumed to evolve independently according to a birth-and-death model, in which new variants origi- nate by duplication and others are deleted throughout time. Moreover, distinct ribosomal units were shown to be expressed during diferent developmental stages in the vertebrate and mosquito hosts. Here, the 18S rDNA sequences of 32 species of avian haemosporidian parasites are reported and compared to those of simian and rodent Plasmodium species. Methods: Almost the entire 18S rDNAs of avian haemosporidians belonging to the genera Plasmodium (7), Haemo- proteus (9), and Leucocytozoon (16) were obtained by PCR, molecular cloning, and sequencing ten clones each. Phy- logenetic trees were calculated and sequence patterns were analysed and compared to those of simian and rodent malaria species. A section of the mitochondrial CytB was also sequenced. Results: Sequence patterns in most avian Plasmodium species were similar to those in the mammalian parasites with most species featuring two distinct 18S rDNA sequence clusters. Distinct 18S variants were also found in Haemopro- teus tartakovskyi and the three Leucocytozoon species, whereas the other species featured sets of similar haplotypes. The 18S rDNA GC-contents of the Leucocytozoon toddi complex and the subgenus Parahaemoproteus were extremely high with 49.3% and 44.9%, respectively. The 18S sequences of several species from all three genera showed chimeric features, thus indicating recombination. Conclusion: Gene duplication events leading to two diverged main sequence clusters happened independently in at least six out of seven avian Plasmodium species, thus supporting evolution according to a birth-and-death model like proposed for the ribosomal units of simian and rodent Plasmodium species. Patterns were similar in the 18S rDNAs of the Leucocytozoon toddi complex and Haemoproteus tartakovskyi. However, the 18S rDNAs of the other species seem to evolve in concerted fashion like in most eukaryotes, but the presence of chimeric variants indicates that the ribosomal units rather evolve in a semi-concerted manner. The new data may provide a basis for studies testing whether diferential expression of distinct 18S rDNA also occurs in avian Plasmodium species and related haemospo- ridian parasites. Keywords: 18S ribosomal RNA, Plasmodium, Haemoproteus, Leucocytozoon, Birth-and-death evolution, Concerted evolution *Correspondence: [email protected] 1 Department of Pathobiology, Institute of Pathology, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Harl et al. Malar J (2019) 18:305 Page 2 of 19 Background hundred copies. Te individual rDNA units do not evolve Ribosomal RNAs of eukaryotes independently, but according to a model of concerted Ribosomal RNAs (rRNAs) form the core part of the ribo- evolution, a process that leads to homogenization [3–5]. somes and are essential for protein synthesis in all liv- Concerted evolution involves mechanisms such as une- ing organisms. Due to functional constraints rRNAs are qual crossing over during recombination, gene duplica- among the most conserved nucleic acids in nature, and tion, and inter-chromosomal gene conversion, and is at the same time they constitute also the vast majority assumed to afect the evolution of members of most mul- of RNA molecules in cells, e.g., the rRNA content (dry tigene families [6]. However, there are exceptions to this weight) of Escherichia coli cells varies from 20% in the rule. Multigene families involved in the immune system early exponential growth phase to 2% in developed cells such as immunoglobulins and the major histocompatibil- [1]. Tese characteristics render rRNAs suitable targets ity complex (MHC) do not evolve in a concerted fashion for various molecular genetic approaches. Ribosomal [7], but they are assumed to follow a model of birth-and- RNAs and their genomic counterparts, the ribosomal death evolution [7, 8]. In this model, new copies originate DNAs (rDNAs), are the most common targets in hybridi- by gene duplication, whereas others become non-func- zation and screenings assays. tional and deleted over time. In respect to the evolution Cytoplasmic ribosomes of eukaryotes are composed of of their rDNAs, human, simian and rodent Plasmodium four rRNAs and over 50 proteins, which together form species are considered exceptional within the eukaryote the small ribosomal subunit (SSU) and the large ribo- domain because their rDNA units are assumed to evolve somal subunit (LSU). Te 18S rRNA is the rRNA com- according to a birth-and-death model under strong puri- ponent of the SSU, while 28S rRNA, 5.8S rRNA, and fying selection. Plasmodium species feature only four to 5S rRNA are the rRNAs of the LSU. Eukaryote nuclear eight rDNA units per haploid genome, which are located rDNA genes are arranged in units containing 18S rDNA, on diferent chromosomes and are assumed to accumu- 5.8S rDNA, and 28S rDNA, separated by the internal late mutations independently [e.g., 9] (Fig. 1b). Moreover, transcribed spacers ITS1 and ITS2. Te rDNA units of distinct units were shown to be expressed during difer- multicellular eukaryotes also include a non-transcribed ent developmental stages of the parasites in the verte- spacer (NTS) and an external transcribed spacer (ETS), brate and mosquito hosts [10]. which like ITS1 and ITS2 are not involved in the forma- tion of the ribosomes, but are assumed to play a role in The 18S rRNAs of Plasmodium rDNA transcription [2] (Fig. 1a). Te 5S rDNAs are usu- Te frst notable studies on 18S rDNAs of Plasmodium ally not directly associated with the other rDNA units but were published in the early 1980s when DNA sequenc- are located in diferent genomic regions. ing was still at the very beginning. Although complete Te rDNA units of most eukaryotes are arranged in sequences were not available at that time, mapping of clusters of so-called tandem repeats on one or several rDNAs by restriction enzyme analysis and Southern chromosomes, with each cluster containing up to several blot hybridization allowed determining the number Fig. 1 a Ribosomal units of multicellular eukaryotes and arrangement in ribosomal clusters (NTS: non-transcribed spacer, ETS: external transcribed spacer). b Arrangement of ribosomal units in Plasmodium species. The ribosomal units of Plasmodium spp. do not contain NTS and ETS regions and are not arranged in clusters with multiple units Harl et al. Malar J (2019) 18:305 Page 3 of 19 of distinct units. By using these methods, four nuclear lophurae using Southern blot analysis and restriction rDNA units were identifed in the rodent malaria para- analysis, supporting the presence of six copies, which site Plasmodium berghei in diferent genomic regions, may be divided into four classes [25]. and they were found not to be arranged in tandem Te temperature was shown to be a major factor arrays like in other eukaryotes [11]. Moreover, the four involved in transcription of distinct rDNA types. In labo- units could be assigned to two classes, putatively being ratory cultures of P. falciparum, both A-type genes were expressed during diferent developmental stages [11, preferentially expressed at higher temperatures and the 12]. Te so-called A-type rDNA units were found to S-type genes at lower temperatures. Tis pattern might be expressed in asexual blood-stages in the vertebrate relate to diferences in body temperatures between the host, whereas the expression patterns of the C-type vertebrate and mosquito hosts [18]. Since parasites enter- units were still unknown at that time [13]. Te naming ing the mosquito host face a severe drop in glucose con- of rDNA types is quite inconsistent because expres- centration compared to the vertebrate host, glucose is sion patterns of the C-type rDNAs were unknown at another potential regulator of gene transcription. Asex- frst. Later, McCutchan et al. [14] proposed consist- ual blood stages of P. falciparum subjected to glucose ently using the term S-type for the sporozoite specifc starvation decreased expression of A-type genes and stages instead of C-type. Tis recommendation is fol- increased expression of the S-type genes. Tis efect was lowed here by using the terms A-type or S-type in the shown to be in synergy with temperature changes related fgures and discussion. Te frst 18S rDNA sequences of to the host shift [26]. P. berghei were published several years later, allowing to For phylogenetic approaches 18S rDNAs expressed directly compare the variants for the frst time, and to in asexual stages were sequenced from a wider range of design oligonucleotide probes specifcally targeting the Plasmodium species, including Plasmodium fragile from A-type or the C-type [S-type] genes. Hybridization of macaques [27] and Plasmodium malariae from humans oligonucleotide probes to rRNA isolated from diferent [28], and Plasmodium lophurae [29] and Plasmodium life stages lead to the discovery that the C-type [S-type] gallinaceum [27] from poultry. Te A-type 18S rDNAs is expressed in the sporozoite stage in the mosquito of the primate malaria species Plasmodium knowlesi and vector [15].

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