Haemogregarines and Criteria for Identification

Simple Summary Taxonomic classification of haemogregarines belonging to Apicomplexa can become difficult when the information about the life cycle stages is not available. Using a self-reporting, we record different haemogregarine species infecting various animal categories and exploring the most systematic features for each life cycle stage. The keystone in the classification of any species of haemogregarines is related to the sporogonic cycle more than other stages of schizogony and gamogony. Molecular approaches are excellent tools that enabled the identification of apicomplexan parasites by clarifying their evolutionary relationships. Abstract Apicomplexa is a phylum that includes all parasitic protozoa sharing unique ultrastructural features. Haemogregarines are sophisticated apicomplexan blood parasites with an obligatory heteroxenous life cycle and haplohomophasic alternation of generations. Haemogregarines are common blood parasites of fish, amphibians, lizards, snakes, turtles, tortoises, crocodilians, birds, and mammals. Haemogregarine ultrastructure has been so far examined only for stages from the vertebrate host. PCR-based assays and the sequencing of the 18S rRNA gene are helpful methods to further characterize this parasite group. The proper classification for the haemogregarine complex is available with the criteria of generic and unique diagnosis of these parasites.


Introduction
Phylum Apicomplexa was described by Levine [1] to include parasitic protozoa sharing unique ultrastructural features known as the "apical complex" (Figure 1). Haemogregarines ( Figure 2) are ubiquitous adeleorine apicomplexan protists inhabiting the blood cells of a variety of ectothermic and some endothermic vertebrates [2][3][4]. They have also an obligatory heteroxenous life cycle (Figure 3), where asexual multiplication occurs in the vertebrate host; while sexual reproduction occurs in the hematophagous invertebrate vector [5]. This family contains four genera, according to Levine [6]: Haemogregarina Danilewsky [7], Karyolysus Labbé [8], Hepatozoon Miller [9], and Cyrilia Lainson [10]. Barta [11] conducted a phylogenetic analysis of representative genera in phylum Apicomplexa using biological and morphological features to infer evolutionary relationships in this phylum among the widely recognized groups. The data showed that the biologically diverse Haemogregarinidae family should be divided into at least three families (as suggested by Mohammed and Mansour [12]), were family Haemogregarinidae, containing the genera Haemogregarina and Cyrilia; family Karyolysidae Wenyon [13], of the genus Karyolysus; and family Hepatozoidae Wenyon [13], of the genus Hepatozoon, since the four genera currently in the family do not constitute a monophyletic group. The picture is further complicated by evidence from a study by Petit et al. [14] of a new Brazilian toad haemogregarine parasite Haemolivia stellata.      It undergoes sporogonic development in its tick host's gut wall and has a complex life cycle that resembles Karyolysus species much more than Hepatozoon, Haemogregarina, and Cyrilia species. Haemogregarines can be morphologically classified based on the developmental details of sporogonic phases of the parasite in the vector, which provide the main characters for classification, the morphology of gametocytes in the red blood cells, and an evaluation of the stages of development [15,16]. Although useful, this methodology is not sufficient for a taxonomic diagnosis [17,18] also the classical systematics has been problematic because of the variability to which morphological details are subjected [19]. Therefore, the use of molecular methods from blood or tissue samples [20][21][22], with appropriate molecular phylogeny study, became an essential adjunct to existing morphological and biological characters for use in the inference of evolutionary history relationships among haemoprotozoan parasites [23][24][25]. Molecular data has been carried out based using PCR assays targeting the nuclear 18s ribosomal RNA gene, which have been extensively applied to characterize hemoparasites DNA more fully in the absence of complete life cycles [26][27][28][29][30][31][32].
In the present critical review of the haemogregarines complex, the proper classification, the criteria of generic and unique diagnosis, and the cosmopolitan distribution of haemogregarines among the vertebrate and invertebrate hosts are examined because of their relevant characteristic and taxonomic revisions. It undergoes sporogonic development in its tick host's gut wall and has a complex life cycle that resembles Karyolysus species much more than Hepatozoon, Haemogregarina, and Cyrilia species. Haemogregarines can be morphologically classified based on the developmental details of sporogonic phases of the parasite in the vector, which provide the main characters for classification, the morphology of gametocytes in the red blood cells, and an evaluation of the stages of development [15,16]. Although useful, this methodology is not sufficient for a taxonomic diagnosis [17,18] also the classical systematics has been problematic because of the variability to which morphological details are subjected [19]. Therefore, the use of molecular methods from blood or tissue samples [20][21][22], with appropriate molecular phylogeny study, became an essential adjunct to existing morphological and biological characters for use in the inference of evolutionary history relationships among haemoprotozoan parasites [23][24][25]. Molecular data has been carried out based using PCR assays targeting the nuclear 18s ribosomal RNA gene, which have been extensively applied to characterize hemoparasites DNA more fully in the absence of complete life cycles [26][27][28][29][30][31][32].

Materials and Methods
In the present critical review of the haemogregarines complex, the proper classification, the criteria of generic and unique diagnosis, and the cosmopolitan distribution of haemogregarines among the vertebrate and invertebrate hosts are examined because of their relevant characteristic and taxonomic revisions.

Materials and Methods
This review included all related published scientific articles from January 1901 to December 2020. This article was conducted by searching the electronic databases NCBI, ScienceDirect, Saudi digital library, and GenBank database, to check scientific articles and M.Sc./Ph.D. Thesis related to the research topic of this review. Studies published in the English language were only included and otherwise are excluded.
Relevant studies were reviewed through numerous steps. In the first step, target published articles were identified by using general related terms related to the morphological features, such as "Haemogregarines" and "Apicomplex". The second step involved screening the resulting articles by using highly specific keywords of the generic features for stages in the life cycle of haemogregarines species, including "Merogony", "Gamogony", "Sporogony", "Infective stages", "Motile stage", "Infection sites", and "sporozoites". The last step of the review focused on selected studies involving the use of molecular analysis for accurate taxonomic identification by using highly specific keywords, including "PCR", "Genetic markers", "Variable regions", "18S rRNA", and "Phylogenetic analysis".
The obtained data were presented in tables and figures and were: Table 1 representing the characteristic features for the haemogregarines genera, Tables 2-6 showing haemogregarines species, the vertebrate host, site of the merogonic stage, the invertebrate vectors, site of gamogony and sporogonic stages, geographical locality for hosts, and the authors for publishing data, Table 7 with the primer sets used for the amplification and sequencing for the appropriate gene of 18S rRNA for haemogregarines, and Table 8 representing all the sequenced and deposited haemogregarines in the GenBank database until now.

Results and Discussion
In this review, the different stages of the apicomplexan life cycle were used to identify haemogregarines. However, in most cases, their assignment to one or another genus cannot be considered more than provisional. Accordingly, about 82 haemogregarines in 155 research articles were identified previously. Osimani [33] stated that the differences between the haemogregarines relied more on the host's identity than the parasite's characteristics. Mohammed and Mansour [12] reported that haemogregarines gamonts morphology does not provide generic identification with a reliable key. However, Telford et al. [34], and Herbert et al. [35] stated that the determination of generic haemogregarines should not be based exclusively on the gamonts' form, the type of parasitized host cells, and their effect on the host and site merogony in host cells. While the most characteristic feature for the basic identification via the sporogonic stage.
The reviewed species belonged to the four genera within Hemogregarinidae (Table 1). Following the parsimony analysis in the phylogenetic study of the representative genera in phylum Apicomplexa performed by Siddall and Desser [36] primarily based on ultrastructural observations, it was concluded that the variations between the different haemogregarines genera are mainly reflected by the sporogony features. Besides, Dvořáková et al. [37] added that the host specificity, together with the haemogregarine's careful morphological and biological analysis, is a sound criterion for accurate identification. These species are common in different animals as fish (Table 2), amphibians (Table 3), reptiles (Tables 4-7), birds (Table 8), and mammals (Table 9). Table 1. Characters of different groups of haemogregarines used in the parsimony analysis carried out by Barta [19] and Siddall and Desser [36].          Hepatozoon luiperdjie Panthera pardus Leukocytes --Limpopo Province, South Africa Van As et al. [125] Hepatozoon canis Dogs Intra-erythrocytic --Manila, Philippines Baticados et al. [126] In the schizogony (merogony) stage, haemogregarines are characterized by their considerable ability to invade and develop within different organs and cell types inside the vertebrate host (Tables 2-9). Bray [127] proposed that haemogregarines with schizonts in the liver should be placed in the genus Hepatozoon. In contrast, those species that precede schizogony in other organs should belong to another genus as Haemogregarina or Karyolysus. However, only in the lung of the river turtle, Trionyx gangeticus infected with Haemogregarina gangetica, was described by Misra [87]. In addition to the usual location of merogonic development in the liver, lung, and spleen, Ball et al. [71] have found certain merogonic stages in the highly infected snakes' brain and heart. Siddall and Desser [84] described merogonic stages in the lacunar endothelial cells of the circulatory system of the leech and its proboscis, besides the liver, lung, and spleen in the turtle. Yanai et al. [128] also described nodular lesions containing schizonts and merozoites of Hepatozoon sp. of the heart's martens, perisplenic, and perirenal adipose tissues, the diaphragm, mesentery, and tongue. Úngari et al. [102] reported that the genus Haemogregarina underwent schizogony in the circulating blood cells as in turtles and fish, and the genus Hepatozoon underwent schizogony in the liver. Additionally, there are two morphologically different meronts were the micro-and macromeronts. The presence of these two forms of meronts was mentioned to be a fundamental feature of the whole haemogregarine [74,129,130].

Comparable Features
Gametocytes are usually the only stages of the parasite detected by scientists. Their morphology, unfortunately, does not provide a reliable clue to the generic differentiation. Together with other relevant data, their morphological characteristics offer a reliable basis for specific identification [35,67]. The haemogregarines gametocytes appeared as sausageshaped and generally lie singly within erythrocytes (Tables 2-9), but sometimes free in extracellular space, which is consistent with Telford et al. [34], Sloboda et al. [79] as the presence of free extracellular gametocytes. They are also observed in the leucocytes of fish (Table 2), birds (Table 8), and mammals ( Table 9).
The shape, size, and structure of infected blood-corpuscles often undergo considerable changes. Hypertrophy may result directly from the gametocyte's added intraerythrocytic volume or represent an erythrocyte adaptation to the gametocyte's presence [53,82,131,132]. An entirely different cell response occurred when the gametocytes of Hemogregarina sp. invaded erythrocytes of Rana berlandieri. The erythrocytes undergo hypertrophy, and the plasmalemma of the infected erythrocyte demonstrated numerous microvilli-like outgrowings. Hussein [133] also described the hypertrophy of Karyolysus-infected erythrocytes. Most haemogregarine gametocytes do not invade the host cell's nucleus but instead move it to the opposite side or the other host cell's other pole. This is contrary to the effect of the genus Karyolysus on the infected erythrocytes. Karyolysus has a karyolytic impact on the host cell's nucleus and is therefore identified Karyolysus Reichenow [134].
Little work had been done to identify the actual arthropod vectors of haemogregarines, as the transmission by inoculation of blood was rarely successful. In general, the invertebrate vectors of haemogregarines were the most challenging problem facing this group's research progress [49]. The haemogregarines displayed a wide distribution of vertebrate host infections, and a large number of invertebrate vectors (Tables 2-9). In all haemogregarines, fertilization is of Adelea type; both micro-and macrogamonts lie in syzygy within the same parasitophorous vacuole. Syzygy can stimulate the production of the associated gamonts in haemogregarines, since only the parasites found in pairs were mostly differentiated, which is consistent with Davies and Smit [42]. Regarding the number of microgametes produced by each microgamont, the members of the suborder Adeleidea were characterized by the production of only a few (four or less) microgametes [135]. Simultaneously, the formation of multiple microgametes has been identified in most haemogregarines species [52]. However, there are some suggestions that multiple microgamete formation does not occur in the entire genus Hepatozoon [111]. Regarding the number of flagella in microgametes in haemogregarines, contradictions were recorded. While monoflagellated microgametes have been described for haemogregarines species [74], biflagellated microga-metes were also recorded for other haemogregarines [52]. On the other hand, Michel [85] reported non-flagellated microgametes in Hepatozoon mauritanicum.
Fertilization follows, leading to the formation of a zygote that becomes an oocyst. The oocyst is surrounded by a flexible membrane rather than a wall, and it produces sporozoites that may undergo further merogony. Sporogony is elucidated for just a few known haemogregarines species, the vast majority of which is supposed to investigate this aspect of their life-cycle, as reported by Forlano et al. [113]. There is also another potential criterion for distinguishing between Hepatozoon and Haemogregarina based on the presence or absence of oocysts containing sporocysts in the invertebrate vector, which is consistent with Levine [6]. When the developing mite reaches the nymphal stage, the sporozoites attain their maturity. The sporozoites eventually get the nymph's stomach and pass out with their faeces, which are considered infection sources of the vertebrate host (lizard). The morphological characteristics of the gamonts and meronts found in the blood cells sometimes provide inadequate information for differential diagnoses [37], meaning that assigning species of haemogregarines to one of these genera must be based on the characteristics of its sporogony in the invertebrate vectors [6,64]. However, data on invertebrate vectors and sporogony are missing for the majority of species [23].
Until now, the current taxonomy of haemogregarines is facing a great challenge due to the high variation in gamont morphology, low host specificity, unknown invertebrate hosts in many cases, and fewer details of sporogony. Therefore, molecular approaches are now available to distinguish populations of morphologically identical but genetically different parasites, including DNA and polymerase chain reaction (PCR) based approaches [22,[136][137][138][139][140][141]. Some studies based on PCR-based assays as the reference diagnostic test for epidemiological studies, which given their greater sensitivity, particularly for testing different hosts with intermittent levels of parasitemia via a low infection rate by gamonts, as Otranto et al. [114], Haklová-Kočíková et al. [18], Jòzsef et al. [24], Ramos et al. [116], and Mitkova et al. [120]. Notably, all the molecular evidence comes from the complete and partial sequences of the small subunit (SSU) ribosomal DNA (rDNA) 18S gene is a sufficient phylogenetic marker to approximate ordinal level relationships and those within orders [68,98,119,[142][143][144][145]. Previous molecular studies of Harris et al. [22] and Barta et al. [19] demonstrated that the haemogregarine species are clustered in sister clades with interspecies linked more with the host geographic distribution, rather than host species. There are universal primer sets that were able to molecularly characterize haemogregarines, as mentioned in Table 10. However, many species with sequences deposited in the GenBank database are not identified correctly at the generic level. Table 11 expressed only haemogregarines identified at the species level and others identified at the generic level are excluded. Table 10. Primer sets used in the phylogenetic analysis of haemogregarines by 18S rRNA gene.

Primer Set
Primer Sequence Reference

Conclusions
Few haemogregarine characteristics provide a reliable basis for the related parasite to recognized genera. Details of the sporogonic cycle seem to be the only reliable criterion as they are the "Key-stone" in the classification system. Morphological characteristics of the gametocytes do not help in this respect. Features of the schizogonic stages, when these are known, are not much better as criteria of generic value. Molecular phylogenetic studies using the appropriate genetic markers are helpful tools for the accurate taxonomic identification for haemogregarines. Further studies are recommended to include other nuclear and mitochondrial genes to provide more information about the genetic variability among haemogregarines.