Keys to the avian malaria parasites

Background Malaria parasites (genus Plasmodium) are widespread in birds. These pathogens cause pathology of blood and various organs, often resulting in severe avian malaria. Numerous recent studies have reported DNA sequences of avian malaria parasites, indicating rich genetic diversity and the possible existence of many undescribed species. However, the majority of reported Plasmodium lineages remain unidentified to species level, and molecular characterization is unavailable for the majority of described Plasmodium parasites. During the past 15 years, numerous new Plasmodium species have been described. However, keys for their identification are unavailable or incomplete. Identification of avian malaria parasites remains a difficult task even for experts, and this precludes development of avian malariology, particularly in wildlife. Here, keys for avian malaria parasites have been developed as a baseline for assisting academic and veterinary medicine researchers in identification of these pathogens. The main obstacles and future research priorities have been defined in the taxonomy of avian Plasmodium species. Methods The data were considered from published articles and type and voucher material, which was accessed in museums in Europe, the USA and Australia. Blood films containing various blood stages of the majority of described species were examined and used for the development of dichotomous keys for avian Plasmodium species. Results In all, 164 published articles were included in this review. Blood stages of avian Plasmodium parasites belonging to subgenera Haemamoeba, Giovannolaia, Novyella, Bennettinia and Huffia were analysed and compared. Illustrated keys for identification of subgenera and species of these parasites were developed. Lists of invalid and synonymous Plasmodium parasite names as well as names of doubtful identity were composed. Conclusion This study shows that 55 described species of avian Plasmodium can be readily identified using morphological features of their blood stages. These were incorporated in the keys. Numerous synonymous names of Plasmodium species and also the names belonging to the category species inquirenda exist, and they can be used as reserves for future taxonomy studies. Molecular markers are unavailable for 58% of described Plasmodium parasites, raising a task for the current avian malaria researchers to fill up this gap.

Life cycles of avian malaria parasites are similar in their basic features to those of human and other mammal Plasmodium species [1,2,8,13,14,20]. Malaria parasites are obligate heteroxenous protists, with merogony in cells of fixed tissues and also blood cells. Gametogony occurs in red blood cells, and sexual process and sporogony are completed in Culicidae mosquitoes. However, the life cycles of avian Plasmodium species differ from those of the parasites of mammals, particularly due to their relatively low host specificity and marked variation in patterns of development in avian hosts and vectors. For example, Plasmodium (Haemamoeba) relictum infects and completes its life cycle in birds belonging to over 300 species and 11 orders, and Plasmodium (Huffia) elongatum, Plasmodium (Novyella) vaughani and many other species also have a broad range of avian hosts [6,8,[21][22][23]. Erythrocytic merozoites of many avian malaria parasites can induce secondary tissue merogony in birds [24,25]. The exo-erythrocytic merogony occurs in cells of the reticuloendothelial and haemopoietic systems, but has not been reported in hepatocytes [2,4,8,23,26]. Pedunculated oocysts were discovered in Plasmodium (Bennettinia) juxtanucleare; these oocysts possess leglike outgrowths which attach the oocysts to the mosquito midgut wall [27]. These and some other features are not characteristics of malaria parasites of mammals, and this is reflected in genetic differences between these groups of parasites and their different position in molecular phylogenies [28][29][30][31][32][33].
Malaria, the disease caused by parasites of the genus Plasmodium, has traditionally been viewed as a disease of the blood and blood forming tissues of vertebrate hosts, with exo-erythrocytic stages of development causing little or no pathology [1,13,14,34]. While available evidence still supports this view for the primate and rodent malarial parasites, there is increasing evidence that the pathogenicity of tissue stages of avian species of Plasmodium has been significantly underestimated [25]. Even more, avian malaria is often a more severe disease than human malaria. There is recent experimental evidence of unexpected pathology associated with obstructive development of secondary exo-erythrocytic stages of Plasmodium in brain capillaries that can lead to ischaemia and rapid death in birds that have very low intensity parasitaemias during chronic stage of infection [24,25,35]. Importantly, the severity of disease caused by a given lineage of Plasmodium often varies markedly in different species of avian hosts, from absence of any clinical symptoms to high mortality [4,17,19,[36][37][38][39][40][41].
Because of broad vertebrate host specificity, the same Plasmodium species can infect distantly related birds. In other words, vertebrate host identity cannot be used as a taxonomic feature during identification of avian malaria parasites [1,12,42]. This raises questions about parasite species identification if the same pathogen is found in unusual avian hosts. Molecular characterization is helpful in diagnosis of malaria infections, and has been developed for detection of some avian Plasmodium species [21,40]. Molecular markers are essential in diagnosis and identification of exo-erythrocytic and vector stages, which cannot be identified using morphological features [11,43,44]. However, molecular diagnostics using general primers (the main diagnostic tool currently used in wildlife malariology) is often insensitive in distinguishing of avian Plasmodium spp. co-infections, which are common and even predominate in many bird populations [45][46][47][48]. Specific molecular markers for the majority of avian Plasmodium species have not been developed, and currently are difficult to develop due to significant genetic diversity of malaria parasites, which remain undescribed in wildlife. Morphological identification using microscopic examination of blood films remains important in malaria diagnostics in the wild, and is particularly valuable if it is applied in parallel with polymerase chain reaction (PCR)-based diagnostic tools [5,30,49,50].
During the past 15 years, numerous avian Plasmodium parasites were named and described using morphological features of their blood stages [49,[51][52][53][54][55][56][57][58][59]. However, molecular markers for parasite detection were developed in a handful of these descriptions. The keys that are available for identification of avian Plasmodium species [8], should be reworked in the light of the newly available information.
The main aim of this review is to develop easy-to-use keys for identification of avian malaria parasites using morphological features of their blood stages as a baseline for assisting academic and veterinary medicine researchers in identification of these pathogens. Lists of synonymous names of Plasmodium species as well as invalid species names were updated and compiled. The Plasmodium parasite names of unknown taxonomic position (incertae sedis) and the species of doubtful identity requiring further investigation (species inquirenda) were specified as well. The information about useful molecular markers, which can be used for described Plasmodium species detection and comparison was also summarized. This review might be helpful for wildlife malaria and veterinary medicine researchers aiming identification of avian malaria infections.

Methods
Full-length papers with descriptions of new Plasmodium species published in peer-reviewed journals were considered. In all, 164 articles were reviewed, and 152 papers containing most representative information about taxonomy of these parasites were incorporated in the References.
Type and voucher preparation as well as images of blood stages of avian Plasmodium parasites were obtained from the collections of Natural History Museum (London, UK), International Reference Centre for Avian Haematozoa (Queensland Museum, Quensland, Australia), the US National Parasite Collection (National Museum of Natural History, Washington DC, USA), Muséum National d'Histoire Naturelle (Paris, France), Grupo de Estudio Relación Parásito Hospedero, Universidad Nacional de Colombia (Bogotá, Colombia) and Nature Research Centre (Vilnius, Lithuania). All accessed preparations were studied. An Olympus BX61 light microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP70 digital camera and imaging software AnalySIS FIVE (Olympus Soft Imaging Solution GmbH, Münster, Germany) was used to examine preparations and prepare illustrations.
A method of dichotomous key was applied for identification of Plasmodium species. This tool consists of steps divided it two alternative parts, which allow to determine the identity of a specimen due to a series of choices that lead the user to the correct name of a given specimen. The most difficult choices, which do not exclude ambiguity, were accompanied with references to the corresponding pictures, which illustrate meaning of the text information. This simplifies the comparison of diagnostic features used in the keys. All parasite names in the keys are accompanied with references to the original parasite descriptions and (or) reviews containing description and (or) illustrations of corresponding species.

Results
Birds are often infected with different blood parasites belonging to same and different genera in the wild, and various combinations of different parasite co-infections often occur in same individual hosts. Haemosporidians (order Haemosporida) develop intracellularly, and they should be distinguished from other eukaryotic intracellular infections before identification of the parasite species identity. Haemosporidians can be readily distinguished from all other intracellular protists (species of Babesia, Isospora, Lankesterella, Haemogrerina, Hepatozoon, Toxoplasma) due to one particularly readily distinguishable feature. Mainly, gametocytes of all haemosporidians are characterized by sexually dimorphic features, which are readily distinguishable under the light microscope. Haemosporidian macrogametocytes possess compact nuclei and bluish-stained cytoplasm, and the microgametocyte nuclei are diffuse and the cytoplasm stains paler than in macrogametocytes (compare Fig. 1a, h with b, i). Some variation occurs in the size of nuclei and in the staining of the cytoplasm in different haemosporidian species. While, this also depends on staining protocols, macroand microgametocytes can be readily distinguished in each haemosporidian species. This is not the case in other intracellular protists, whose gamonts and other intracellular blood stages do not show sexually dimorphic features and all look similar under the light microscope ( Fig. 1j-l).
Based on current taxonomy, four families of haemosporidians can be recognized. These are Plasmodiidae, Haemoproteidae, Leucocytozoidae and Garniidae [1,4,8,30,60,61]. Malaria parasites are classified in the family Plasmodiidae, which contains one genus Plasmodium. When haemosporidians are found in blood films, Plasmodium parasites should be distinguished from species of related haemosporidians belonging to the families Garniidae, Haemoproteidae and Leucocytozoidae. The main distinctive features of parasites belonging to these families are summarized in Table 1.
Blood stages of species of Plasmodium are particularly similar to those of relatively rare haemosporidian parasites of the genera Fallisia and Garnia of the family Garniidae [8,[60][61][62]. Parasites of these three genera produce gametocytes and meronts (=schizonts) in blood cells (Fig. 1a-f ). However, species of Plasmodium do not digest haemoglobin completely and accumulate residual pigment granules (hemozoin), which are refractory and readily visible in blood stages under light microscope ( Fig. 1a-c). This is not true of species belonging to the genera Fallisia and Garnia or other garniids, which digest haemoglobin completely when they inhabit red blood cells and do not possess pigment granules in their blood stages ( Fig. 1d-f ).
When malaria parasites of the Plasmodium genus are reported in blood films, the next step is to distinguish subgenera of this genus. The main characteristics of different subgenera are summarized in Table 2.
When the subgenus of a malaria parasite has been identified, the next step is the species identification using the keys to species (Tables 3, 4, 5, 6).

Discussion
There are three main groups of obstacles, which a researcher usually faces during morphological identification of malaria parasites using microscopic examination of blood samples collected in the field. First, the quality of microscopic preparations is essential for correct parasite identification, but often is insufficient due to thick blood films or artefacts of their drying, fixation, staining or storage. This precludes visualization of some important features for species identification. It is essential to master these simple methods of traditional parasitology before sample collection, and this can be readily achieved Main morphological features of blood stages, which are used for identification of families of haemosporidian (Haemosporida) parasites (a-i). Mature gametocytes (a, b, g-i) and meronts (c-f) of Plasmodium (a-c), Garnia (d, e), Fallisia (f), Haemoproteus (g) and Leucocytozoon (h, i) parasites belonging to the families Plasmodiidae (a-c), Garniidae (d-f), Haemoproteidae (g) and Leucocytozoidae (h, i). Note presence of malarial pigment in species of Plasmodiidae (a-c) and Haemoproteidae (g) and its absence in species of Garniidae (d-f) and Leucocytozoidae (h, i). Macrogametocytes (a, g, h) and microgametocytes (b, i) are readily distinguishable due to presence of sexually dimorphic features. Common avian intracellular non-haemosporidian parasites (j-l) are shown for comparison with haemosporidians. These are Isospora (synonym Atoxoplasma) (j), Hepatozoon (k) and Babesia (l). Long simple arrows-nuclei of parasites. Simple arrowhead-pigment granules. Triangle arrowheads-developing merozoites. Long simple wide arrow-nucleolus. Simple wide arrowheads-host cell nuclei. Short simple wide arrow-cytoplasm of host cell. Scale bar = 10 µm. Explanations are given in the text in each laboratory using available protocols [1,8,63,64]. Second, Plasmodium species parasitaemia is often light in natural infections in the wild. In other words, malaria parasites might be reported in blood films, but not all stages, which are needed for parasite species identification, are present. This might limit the use of the keys. Sampling of large number of birds (20-30 individuals) belonging to the same species at a study site is often helpful to detect relatively high parasitaemia of the same pathogen and to access the full range of blood stages allowing parasite species identification. Third, co-infections of Plasmodium species might occur, and requires some experience to distinguish between different pathogens [45,48,56]. These obstacles strengthen the need for the development of molecular characterization in avian malaria diagnostics, which is still only available for 44% of described parasite species, whose validity is obvious ( Table 7). This is particularly timely for itemizing Plasmodium species phylogenies, which currently are based mainly on mitochondrial cytb gene sequences in avian malariology [5,7,23,29,33].
Molecular markers are sensitive for distinguishing different parasite species and their lineages, and they are essential for identification of cryptic Plasmodium species [35]. Molecular characterization is best developed for Novyella parasites (molecular markers are available for 59% of described species of this subgenus), and is weakest for Giovannolaia parasites (only two species or 12.5% of this subgenus have been characterized molecularly). Lack of molecular markers for many described malaria pathogens [51,53,54,56,57,59,65] precludes biodiversity research on Plasmodium species and recognition of new malaria pathogens, for whose detection, detailed comparison with already described and genetically characterized parasites is needed. The development of molecular markers for diagnosis of disease agents is an important task of current avian malariology (Table 7).
This study shows that 55 described species of avian malaria parasites can be readily distinguished (Tables 3,  4, 5, 6, 7). Among them, 12, 16, 22, 4 and 1 species belong to subgenera Haemamoeba, Giovannolaia, Novyella, Huffia and Bennettinia, respectively. The great majority of described avian Plasmodium species were reported only in birds that live in tropical and subtropical countries or in Holarctic migrants wintering in the same regions, indicating that transmission of these pathogens occurs mainly in countries with warm climates. Those malaria parasites, which have adapted for transmission globally and have become cosmopolitan, are exceptions. Among these, Plasmodium relictum, Plasmodium elongatum, Plasmodium circumflexum, Plasmodium matutinum and Plasmodium vaughani should be mentioned first of all [6,8,21,23,[66][67][68][69][70]. These are invasive infections, which are often virulent in non-adapted hosts, and they are worth particular attention in bird health.
Limited available experimental information indicates that some Novyella species (P. ashfordi, P. rouxi) may cause severe and even lethal malaria in some birds due to blood pathology [1,8,74,75], but the complete mechanism of their pathogenicity remains unresolved, mainly due to lack of information about exo-erythrocytic development [72]. Investigation of life cycles and virulence of infections caused by Novyella species is an important task in current avian malaria research.

Table 2 Key to subgenera of Plasmodium parasites of birds
Main taxonomic characters of subgenera of avian malaria parasites [8] a Exo-erythrocytic merogony takes place in cells of the haemopoietic system. Erythrocytic trophozoites and growing meronts ( Fig. 2l-p) contain plentiful cytoplasm. Erythrocytic meronts develop in various immature red blood cells (Plasmodium huffi probably is an exception, but this needs confirmation). Fully grown erythrocytic meronts and gametocytes are variable both in form and size; elongate, roundish and irregularly shaped parasites might occur. Pedunculated oocysts are absent b Exo-erythrocytic merogony takes place in cells of the reticuloendothelial system. Erythrocytic trophozoites (Fig. 3d) and growing meronts (Fig. 2a, b) contain plentiful cytoplasm. The size of fully grown erythrocytic meronts exceeds that of the nuclei of infected erythrocytes (Fig. 1c). Fully grown gametocytes are roundish, oval or of irregular form, and their size exceeds that of the nuclei of infected erythrocytes ( Fig. 4t-x). Pedunculated oocysts are absent c Exo-erythrocytic merogony takes place in cells of the reticuloendothelial system. Erythrocytic trophozoites and growing meronts contain scanty cytoplasm (Fig. 3g). Growing erythrocytic meronts are nucleophilic. The size of fully grown erythrocytic meronts does not exceed that of the nuclei of infected erythrocytes (Fig. 3g …………………………………………… Novyella e Many species of Plasmodium inhabit numerous species of birds and use mosquitoes of different genera for transmission [1,8,9,11]. Within this spectrum of hosts and vectors, the same parasite species might exhibit diverse morphological forms and strain varieties. Because of these morphological variants, it has been conventional in old avian malaria research (approximately between 1927 and 1995) that any new Plasmodium species description should only be accepted if supported by a comprehensive package of taxonomic features, which not only included the full range of blood stages, but also data on the vertebrate host specificity, periodicity of erythrocytic merogony, tissue merogony, vectors and patterns of sporogonic development. It is not surprising that recent molecular studies supported the validity of the old Plasmodium species descriptions, which were detailed and precise ( Table 7). Application of molecular diagnostic tools in studies of avian haemosporidian parasites [29,69,76,77] opened new opportunities to distinguish haemosporidian parasites based on their unique DNA sequences. This stimulated biodiversity research of wildlife Plasmodium parasites, particularly because the molecular characterization, which was done in parallel with morphological description of blood stages, made each parasite species detection readily repeatable at all stages of life cycle (Table 7).
A list of synonymous names of avian Plasmodium species and the justification of the nomenclature status of these names are given in Table 8. The majority of these parasite descriptions are insufficiently complete and were not accompanied with molecular characterization. Due to the huge genetic diversity of avian malaria pathogens and numerous genetic lineages reported in birds, some of these names might be validated in the future, and they represent a reserve for future taxonomic work. However, available descriptions of these parasites do not provide sufficient information to readily distinguish them from parasites, whose validity is well established (Tables 3, 4, 5, 6). For clearness of scientific texts, it is preferable to avoid use of the synonymous names before additional data on their validity are available. Reports of parasite lineages and GenBank accessions of their DNA sequences in publications would be helpful to specify Plasmodium species identity in the future.
A list of the Plasmodium species names of unknown taxonomic position (incertae sedis) and also the names of species of doubtful identity, which require further investigation (species inquirenda), is given in Table 9. All these parasite descriptions are insufficiently complete and were not accompanied with molecular characterization. Taxonomic status of the majority of these names was justified in [8]. Twenty names of Plasmodium parasites were added to this list and their taxonomic status was explained ( Table 9). The majority of these parasite descriptions are based on preparations with co-infections of several Plasmodium parasites belonging to same and (or) different genera. This raises a question if all blood stages reported in the original descriptions truly belong to corresponding species.
Additionally, in many of such parasite descriptions, gametocytes were not described, but this stage is essential for the identification of some Plasmodium species (Tables 3, 4 ,5,6,Figs. 4,5). It is important to note that the descriptions of many Plasmodium parasites, which were incorporated in Table 9 and published during past 15 years, contain some information about their blood stages. Additionally, the type material was designated in many descriptions, but usually is insufficient for practical use and distinguishing parasites at the species level, particularly because (1) the type preparations contain coinfections and (2) single cells (meronts) were designated as holotypes. Single cells usually do not reflect entire morphological diversity of malaria parasites, so deposition of parahapantotype material is preferable in wildlife haemosporidian research [35,49,58,78]. Validation of some names listed in Table 9 is possible in the future, but it requires additional research, preferably based on new samples from the same avian hosts and type localities.
Invalid Plasmodium parasite names (nomen nudum) are listed in Table 9. These names were not accompanied with descriptions so have no status in nomenclature. The names of this category can be used as a reserve for new parasite descriptions in the future, but it is preferable not to use them to avoid taxonomic confusion [78].

(2)
Cytoplasm of gametocytes is not highly vacuolated; if vacuoles are present in macrogametocytes, they are few and of small size (< 1 µm in diameter) (Fig. 4p-s)

Conclusion
Based on available morphological data and DNA sequence information, 55 species of avian Plasmodium parasites can be readily distinguished. Species of subgenus Novyella predominate among them. Dichotomous keys for identification of these parasites were compiled allowing identification of these pathogens using morphological features of their blood stages. The majority of described avian Plasmodium species are mainly transmitted in countries with warm climates. The obstacles for their global spread remain insufficiently understood, mainly because of limited information on life cycles and vectors of the majority of described parasites of tropical birds. The lists of synonymous names as well as names of the categories species inquirenda and incertae sedis should be considered in future taxonomic work of avian malaria parasite at species level. The majority of described Plasmodium parasites have not been characterized using molecular markers, which b Plasmodium beaucournui, P. bigueti, P. coggeshalli, P. dherteae, P. ghadiriani, P. golvani, P. jeanriouxi, P. lenoblei, P. papernai, P. reniai, P. snounoui, P. tranieri were named and described, and P. alaudae was re-described from individual birds co-infected with parasites belonging to subgenera Haemamoeba, Giovannolaia and Novyella [56,57,65,149]. The authors of the original descriptions have grouped the blood stages visible in blood films and attributed them to different species provisionally, which is particularly obvious in case of parasites with elongate gametocytes. This makes species description and validation of parasite names questionable. Only single cells (erythrocytic meronts) were selected as holotypes in these parasite descriptions. However, due to morphological variation of blood stages of Plasmodium and presence of parasites at different stages of growth in each blood film, such methodology of designation of the type material can work only in case of exceptionally distinctive cell characters, which is not the case in all these parasite descriptions, particularly belonging to subgenus Haemamoeba. Molecular characterization of all these parasites is unavailable. It is clear from the original descriptions, that many individual birds were infected by representatives of several subgenera. However, the reported blood stages were selected and attributed to certain species without providing convincing explanations, making identifications difficult or even impossible based on available information. Co-infections of Plasmodium parasites belonging to different subgenera are common in wildlife, and the described cases of co-infections with several malaria parasites are not unpredictable [45]. However, description of new species from such co-infections hardly possible if the unique morphological characters of blood stages are absent, which is the case with P. beaucournui, P. bigueti, P. coggeshalli, P. dherteae, P. ghadiriani, P. golvani, P. jeanriouxi, P. lenoblei, P. papernai, P. reniai, P. snounoui, P. tranieri and also in re-description of P. alaudae. These parasites are considered as species inquirenda. Recent molecular studies provided molecular markers for distinguishing blood stages of Plasmodium species (Table 7). Examination of blood films from experimental infections shows variations in morphological characters of same parasite lineages in different avian hosts, calling for careful application of minor differences in blood stage morphology in avian malaria parasite taxonomy, particularly during co-infections c Based on available information [53], P. buteonis cannot be distinguished from P. circumflexum and other similar parasites of Giovannolaia (Plasmodium gabaldoni, Plasmodium homocircumflexum). The main feature, which has been noted to distinguish P. buteonis from P. circumflexum in the original description [53], is the presence up to 36 nuclei in mature erythrocytic meronts of the former. Plasmodium circumflexum produce less number of nuclei in mature meronts. However, the description of P. buteonis is based on high parasitemia (6.6%), with numerous multiple infections of the same erythrocytes, so it is difficult to rule out that 2 mature meronts were present in same cell in case of so great number of merozoites. Additionally, parasite morphology often changes during high parasitemia, so such samples should be carefully used in taxonomical descriptions. Plasmodium buteonis might be a valid name, but more research is needed to prove its validity. Molecular characterization of this parasite is absent, but is essential to solve the question about its validity d Plasmodium ninoxi was described from owl Ninox scutulata in co-infection with Haemoproteus sp. [160]. Only one erythrocyte with 2 binuclear growing meronts was detected; no other data about merogony in the blood were provided. Plasmodium ninoxi gametocytes were reported to be rounded. Based on available information, it seems that infected blood was exposed to air, which stimulated rounding-up of haemoproteid gametocytes [8], which were attributed to P. ninoxi. DNA sequence was provided (AY099035.1), and it belongs to Plasmodium sp. Plasmodium ninoxi description is incomplete. Re-description is needed, and it is possible due to available sequence information. The most similar cytb sequence belong to P. gallinaceum, P. relictum and P. circumflexum e Descriptions of P. pachysomum, P. pfefferi, P. sergentorum, P. stellatum [54] are incomplete. Information about morphology of gametocytes is absent. Molecular characterization is unavailable. Species identification is questionable based on the available information f Paperna et al. [54] published re-description of P. rouxi from non-type avian host (Alauda arvensis, Alaudidae instead of Passer hispaniolensis, Passeridae whose is the type host). The re-description is based on samples, which were collected beyond of the type locality (France, instead of Algeria which is the type locality). This contradicts the Article 75.3.6 of the International Code of Zoological Nomenclature [78]. Additionally, according to [54], the erythrocytic meronts of the parasite from A. arvensis do not possess refractive globules and gametocytes possess few tiny pigment granules (Figs. 8, 9 in [54]). These are not characters of P. rouxi, which was described by Sergent et al. [111]. Sergent's original material from Algiers labelled "2198, 26.4.28, Institut Pasteur d'Algérie" is available in the Natural History Museum, London. Examination of this blood film showed that numerous erythrocytic meronts of this parasite possess refractive globules (Fig. 3f, j) and gametocytes possess few large ( Fig. 4o) pigment granules. The latter character is an important feature of P. rouxi. Based on available information, the parasite described in [54] as 'P. rouxi' cannot be attributed to P. rouxi and is considered as a species inquirenda. The parasite described by Paperna et al. [54] is characterized by presence of (1) the relatively prominent cytoplasm in growing meronts and (2) tiny size of pigment granules in gametocytes, so might belong to a new Plasmodium species. Additional investigation is needed to answer this question. In the same study, Paperna et al. [54] described a new species Plasmodium mohammedi, which was reported, Passer domesticus (the common host of P. rouxi in Mediterranian region [68]). Blood stages of P. mohammedi are indistinguishable from P. rouxi [111], particularly due to the presence of refractive globules in erythrocytic meronts and large pigment granules in gametocytes (see Figs. 18-21 in [54]). Plasmodium mohammedi is a synonym of P. rouxi. Molecular identification of P. rouxi (lineage pPADOM16) was developed [68]. Application of the barcoding indicates that the details of disposition of nuclei in erythrocytic meronts during different infections, particularly in different avian hosts, is variable in P.rouxi, but binuclear "bow-tie" form parasites often are present (Fig. 3f ) and can be used for this parasite species identification. Additionally, presence of few large pigment granules in mature gametocytes also is a characteristic feature, and it recommended to use for distinguishing P. rouxi infection (Fig. 4o) from other Novyella parasites producing tetranuclear erythrocytic meronts Macrogametocytes (a-g, k-u, w-y) and microgametocytes (h-j, v). Note long outgrowth (f), terminal position of pigment granules (e) and nucleus (g), granular (l, m) and vacuolated (n) appearance of the cytoplasm, slender (p-r) and circumnuclear (s) shapes of gametocytes, clumps of pigment granules located near the parasite margin (t, w), distinct smooth outline of nucleus (y). Symbols as in Figs. 1, 2, 3. Explanations are given in the text