Background
There are more than 50 morphologically described species of
Plasmodium that infect birds (i.e., causing avian malaria) throughout the world, but there may be several thousand evolutionary independent lineages that act as separate species on their avian hosts [
1]. For over a decade molecular studies have been conducted on avian malaria. These were primarily based on cytochrome
b diversity (different cytochrome
b haplotypes are hereafter referred to as cyt
b lineages) and in a few cases combined with a handful of other nuclear and mtDNA genes [
1‐
3]. By combining published data on avian malaria parasites in communal databases, a vast lineage diversity, as well as remarkable host ranges and geographic spread of some parasite species has been observed [
4]. One of the well-known, morphologically defined parasites,
Plasmodium relictum, consists of several different lineages (e g, SGS1, GRW4, GRW11, LZFUS01), is found on all continents except Antarctica and is transmitted in both tropical and temperate regions [
5]. One of the lineages (GRW4) belonging to this taxonomically defined species has caused major population declines when invading endemic bird fauna [
6].
Plasmodium relictum also exhibits extraordinary levels of host generalism; the lineages SGS1 and GRW4 have been found in 50 bird species belonging to 15 different families and 53 species belonging to 17 families, respectively (MalAvi data base 2013-06-04 [
4]). But how has this parasite spread around the globe and into so many bird species? In order to comprehend these processes there is a need for variable genetic markers as well as an understanding of the genetic variation of genes involved in the invasion stage of the host cells.
The vaccine candidate gene, merozoite surface protein 1 (
msp1) is one of the more variable genes found in the human malaria parasite
Plasmodium falciparum[
7]. Within
P. falciparum the msp1 gene encodes a 190 kDa protein that is cleaved into four polypeptides of different length (p83, p42, p38 and p30) during erythrocytic merogony (i.e., schizogony) [
8]. These four peptides are bound together on the surface of the merozoite during adhesion to the host’s erythrocyte and are anchored to the parasite’s membrane via glycosylphosphatidylinositol (GPI) located in the p42 fragment [
9]. During erythrocyte invasion, the p42 peptide is cleaved into two new polypeptides, p33 and p19, resulting in the loss of the merozoite’s peptide coat. Once inside the erythrocyte, p19 is all that remains of
msp1[
10]. Several studies have found that antibodies to the p19 peptide frequently occur in populations with high malaria prevalence and can be associated with immunity to the parasite [
11,
12]. Further, due to the high variability of the gene,
msp1 has frequently been used to infer population structures and understand the epidemiology of primate malaria parasites [
7,
13‐
15]. Apart from the different polypeptide fragments, the
msp1 gene has been divided into 17 different blocks based on variability of the amino acid composition. Of these 17 blocks, six are conserved, four are semi-conserved, six are variable and one block consists of repetitive elements [
16].
Obtaining data on nuclear genes involved in the host invasion process has been notoriously difficult in avian malaria parasites due to the fact that the hosts (i. e., birds) have nucleated erythrocytes and the genome sizes between the host and parasite differ 52-fold. However, during erythrocytic merogony, (i.e., when the parasites undergo asexual reproduction in the blood stream and invade numerous new red blood cells (RBCs)), genetic activity in the parasites is high [
17,
18], thus making it possible to utilize mRNA sequencing in order to acquire sequence data of nuclear genes of the parasites [
19].
By using expressed sequence data from a partially sequenced transcriptome of birds infected with the SGS1 lineage of
P. relictum[
19] it was possible to: 1) identify and develop Sanger sequencing protocols for
msp1 in
P. relictum and, 2) investigate the degree to which the
msp1 gene in avian malaria parasites shares the characteristics of the
msp1 gene found in
P. falciparum, which makes it a good candidate gene for future population studies and investigations focusing on the RBC invasion mechanisms across diverse malaria parasite species.
Discussion
Malaria parasites (i.e., Plasmodium spp.) are found to infect a huge diversity of hosts, including birds, mammals and reptiles. By understanding how genes taking part in the invasion of host cells and tissues have evolved across this vast host range might help us to identify genes and processes that are evolutionarily constrained or are of importance for limiting a parasite to one or several host species. One such gene that is involved in the invasion process is msp1. The msp1 gene encodes for one of the most abundant proteins on the surface of the merozoite (the stage of the parasite that invades the RBC of the host), and is thought to be involved in the initial adhesion to the RBC. This gene is, therefore, highly important for the understanding of invasion biology of malaria parasites as well as for the understanding of how genes involved in the invasion of the host have evolved across different host ranges.
This study identified and investigated the
msp1 gene originating from two different cyt
b lineages of the avian malaria parasite
P. relictum (SGS1 and GRW4). The phylogeny of the
msp1 peptides placed the avian malaria parasites into a strongly supported monophyletic cluster. Two
P. relictum lineages showed a genetic divergence below that found between different
P. falciparum isolates MAD20 and K1. The genetic distance between
P. relictum/
P. gallinaceum exceeded the genetic distance seen between
P. falciparum (K1)/
P. reichenowi (Figure
5).
Similar patterns of conserved and variable blocks were found across the gene when comparing the block variability observed between
P. relictum (SGS1)/
P. gallinaceum with the variability between the two different
P. falciparum isolates (Figure
2). In addition, when comparing the
msp1 haplotypes of
P. relictum (SGS1 and GRW4), the same blocks that have been defined as being variable in
P. falciparum[
16] were also the most variable in this comparison. These similarities might indicate that the properties and function of the avian malaria
msp1 gene is similar to the mammalian malaria
msp1. However, when comparing the degree of variation in the different blocks between the two different
msp1 isolates of
P. falciparum (MAD20/K1) some of the conserved blocks (block 3, 5 and 12, Figure
2) were as conserved as between the two
P. relictum lineages (representing the same morphological defined species), while some of the variable blocks (block 4, 6, 8, 10, and 16, Figure
2) showed variability that exceeded that found between two distinctly different avian malaria parasite species (
P. relictum/
P. gallinaceum). This variability exceeded that of
P. relictum/
P. gallinaceum although the overall genetic distance between the
P. relictum/
P. gallinaceum msp1 gene is bigger than that between
P. falciparum (K1)/
P. reichenowi (Figure
5). The differences in the degree of within species variability at the variable blocks might indicate different selection pressures acting upon the different regions in
P. relictum compared to
P. falciparum. However, in order to make comparisons between the two species and to know how and in what direction selection has acted upon the avian malaria gene, there is a need for larger population studies of the
msp1 gene. Larger population samples are needed in order to pinpoint sections of stabilising versus diversifying selection as well as being able to compare the degree of allelic polymorphism between the two.
Molecular characterization of avian malaria strains has, to date, mainly utilized cyt
b sequences [
2,
4,
24], together with a handful of nuclear genes, of which none show nuclear variation below the defined cyt
b lineages [
1,
3]. In order to understand the epidemiology of avian malaria parasites that have large geographical transmission areas [
4,
25] as well as broad host ranges [
26], there is a need for more variable nuclear markers. Although
msp1 block 2, which is often used for population studies in human malaria species, seems to be absent in avian malaria, other parts of the gene might well serve as useful molecular markers for inferring population structures below the level of cyt
b lineages, in avian malaria. One such marker might be
msp1 gene block 14. Block 14 has diversified 5.3 and 10.2 times faster between
P. relictum lineages SGS1/GRW4 and SGS1/
P. gallinaceum, respectively, compared to the divergence rate of the cyt
b gene. It is important to note that the block diversification rate observed in Block 14 in the current study may change when including more isolates and lineages of
P. relictum. Still, it might serve as a good starting point for future studies wishing to examine the epidemiology and population structure of
P. relictum.
During merozoite formation the
msp1 peptide is anchored to the parasite membrane via glycosylphosphatidylinositol (GPI) located at C-terminus of the protein. The GPI anchors are the last remaining part of the peptide (p19) during erythrocyte invasion. For
P. relictum (SGS1), it was possible to identify the full 3′ end of the peptide; within this region the GPI anchor point was fully conserved at the amino acid level between
P. falciparum,
P. gallinaceum and
P. relictum (SGS1) with the characteristic “FCSSS” motif (Figure
3). The “FCSSS” motif has been found to be extremely conserved across almost all investigated species of malariae parasites [
27] and this was also the case when including more lineages of avian malariae species. In the p19 section of the avian malaria parasite peptides, it was also possible to identify the two epidermal growth factor-like domains (EGF), based on the six conserved characteristic cysteine residues [
28] (Figure
3). In
P. falciparum, the cysteine residues form disulphide bonds, which create a conserved structure similar to that of the epidermal growth factor-like domains. These conserved epitopes are acted upon by the host’s immune system through antibodies that provide the host with protective immunity against malaria. To some extent, antibodies against the p19 epitopes can give the host cross-immunity to other malaria strains. In the bird-malaria parasite system it is common that bird species can be infected with a broad diversity of different malaria species and lineages [
29‐
31]. Within this system,
msp1-p19 would be a promising target for investigating cross-immunity between different species and changes of this cross-immunity depending on the phylogenetic distance of the parasite. Further,
P. relictum has caused large population declines and mortality in endemic host communities, when introduced into formerly parasite-free areas (e.g., Hawaii) [
32‐
34]. Here,
msp1-p19 could be used as a candidate gene to investigate whether individual birds that survive malaria infection carry antibodies against this peptide, in order to examine whether diverse host species target the same structures and pathways in the parasite when fighting the disease.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
OH carried out the molecular work on the msp1 gene, designed the study, drafted the manuscript. MK developed and carried out the molecular work on the nuclear genes excluding msp1. VP and GV isolated Plasmodium parasite strains, designed and conducted bird infection experiments from which samples for molecular work were used. OH, MK, GC, VP, and SB all participated in interpreting the data and made significant contribution to drafting the manuscript. All authors read and approved the final manuscript.