animals Review Haemogregarines and Criteria for Identification Saleh Al-Quraishy 1, Fathy Abdel-Ghaffar 2 , Mohamed A. Dkhil 1,3 and Rewaida Abdel-Gaber 1,2,* 1 Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] (S.A.-Q.); [email protected] (M.A.D.) 2 Zoology Department, Faculty of Science, Cairo University, Cairo 12613, Egypt; [email protected] 3 Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo 11795, Egypt * Correspondence: [email protected] 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. Citation: Al-Quraishy, S.; Keywords: haemogregarines; gamogony; sporogony; schizongony; molecular analysis Abdel-Ghaffar, F.; Dkhil, M.A.; Abdel-Gaber, R. Haemogregarines and Criteria for Identification. Animals 2021, 11, 170. https:// 1. Introduction doi.org/10.3390/ani11010170 Phylum Apicomplexa was described by Levine [1] to include parasitic protozoa shar- ing unique ultrastructural features known as the “apical complex” (Figure1). Haemogre- Received: 27 November 2020 garines (Figure2) are ubiquitous adeleorine apicomplexan protists inhabiting the blood Accepted: 7 January 2021 cells of a variety of ectothermic and some endothermic vertebrates [2–4]. They have also an Published: 12 January 2021 obligatory heteroxenous life cycle (Figure3), where asexual multiplication occurs in the ver- tebrate host; while sexual reproduction occurs in the hematophagous invertebrate vector [5]. Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- This family contains four genera, according to Levine [6]: Haemogregarina Danilewsky [7], ms in published maps and institutio- Karyolysus Labbé [8], Hepatozoon Miller [9], and Cyrilia Lainson [10]. Barta [11] conducted a nal affiliations. 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 Haemogre- garinidae family should be divided into at least three families (as suggested by Mohammed Copyright: © 2021 by the authors. Li- and Mansour [12]), were family Haemogregarinidae, containing the genera Haemogrega- censee MDPI, Basel, Switzerland. rina and Cyrilia; family Karyolysidae Wenyon [13], of the genus Karyolysus; and family This article is an open access article Hepatozoidae Wenyon [13], of the genus Hepatozoon, since the four genera currently in distributed under the terms and con- the family do not constitute a monophyletic group. The picture is further complicated by ditions of the Creative Commons At- evidence from a study by Petit et al. [14] of a new Brazilian toad haemogregarine parasite tribution (CC BY) license (https:// Haemolivia stellata. creativecommons.org/licenses/by/ 4.0/). Animals 2021, 11, 170. https://doi.org/10.3390/ani11010170 https://www.mdpi.com/journal/animals Animals 2021, 11, x FOR PEER REVIEW 2 of 26 Animals 2021, 11, Animalsx FOR PEER2021, REVIEW11, 170 2 of 26 2 of 25 Figure 1. The generalFigure 1.structureThe general for the structure apical complex for the apical for Apicomplexa. complex for Apicomplexa. Figure 1. The general structure for the apical complex for Apicomplexa. Figure 2. Haemogregarines as a part of phylum Apicomplexa. Figure 2. HaemogregarinesFigure as 2. aHaemogregarines part of phylum Apicomplexa. as a part of phylum Apicomplexa. Animals 2021, 11, x FOR PEER REVIEW 3 of 26 Animals 2021, 11, 170 3 of 25 Figure 3. The life cycle Figureof the apicomplexan 3. The life cycle parasites. of the apicomplexan parasites. It undergoes sporogonicIt undergoes development sporogonic in development its tick host’s in gut its wall tick host’sand has gut a wallcomplex and has a complex life cycle that resembleslife cycle thatKaryolysus resembles speciesKaryolysus much morespecies than much Hepatozoon more than, HaemogregarinaHepatozoon, Haemogregarina, , and Cyrilia species.and HaemogregarinesCyrilia species. Haemogregarines can be morphologically can be classified morphologically based on classified the de- based on the velopmental detailsdevelopmental of sporogonic details phases of sporogonic of the parasite phases in of the the vector, parasite which in the provide vector, the which provide the main charactersmain for classification, characters for the classification, morphology the of morphologygametocytes ofin gametocytesthe red blood in cells, the red blood cells, and an evaluationand of an the evaluation stages of of developm the stagesent of development[15,16]. Although [15,16 useful,]. Although this methodol- useful, this methodology ogy is not sufficientis not for sufficient a taxonomic for a taxonomic diagnosis diagnosis[17,18] also [17 the,18 ]classical also the systematics classical systematics has has been been problematicproblematic because of because the variability of the variability to which morphological to which morphological details are detailssubjected are subjected [19]. [19]. Therefore,Therefore, the use of themolecular use of molecular methods methodsfrom blood from or bloodtissue orsamples tissue samples[20–22], with [20–22 ], with appro- appropriate molecularpriate molecular phylogeny phylogeny study, became study, an became essential an essentialadjunct to adjunct existing to morpho- existing morphological logical and biologicaland biological characters characters for use in for the use inference in the inferenceof evolutionary of evolutionary history relation- history relationships ships among haemoprotozoanamong haemoprotozoan parasites parasites[23–25]. [Molecular23–25]. Molecular data has databeen has carried been out carried out based based using PCRusing assays PCR targeting assays targeting the nuclea ther 18s nuclear ribosomal 18s ribosomal RNA gene, RNA which gene, have which been have been exten- extensively appliedsively to applied characterize to characterize hemopara hemoparasitessites DNA more DNA fully morein the fully absence in the of absencecom- of complete plete life cycleslife [26–32]. cycles [26–32]. In the present criticalIn the presentreview of critical the haem reviewogregarines of the haemogregarines complex, the proper complex, classifica- the proper classifica- tion, the criteriation, of generic the criteria and ofunique generic diagnosis, and unique and diagnosis,the cosmopolitan and the distribution cosmopolitan of distribution of haemogregarineshaemogregarines among the vertebrate among and the vertebrateinvertebrate and hosts invertebrate are examined hosts because are examined of because of their relevant characteristictheir relevant and characteristic taxonomic andrevisions. taxonomic revisions. 2. Materials and Methods 2. 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 Animals 2021, 11, 170 4 of 25 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 morpholog- ical 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: Table1 representing the characteristic features for the haemogregarines genera, Tables2–6 showing haemogre- garines species, the vertebrate host, site