Open Vets. 2021; 2: 40–54

Communication

Fatik Baran Mandal, Ph.D. Hurdle in : A case of parasites and other Haemosporidia

https://10.1515/ovs-2020-0110 are: morphological, cytological, biochemical, ecological, Received Mar 29, 2021; accepted Jul 13, 2021 and ethological. Morpho taxonomy is called the traditional taxonomy. Each approach of taxonomy has Abstract: A crucial progress in taxonomy matches own advantages and drawbacks. However, there is a with the growth of various branches of biological conflict between the traditional taxonomy and molecular sciences. This validates the taxonomic positions of level systematics. The progress of molecular biology and many organisms. , and introducing of new tools and techniques help in finalizing , the members of the Haemosporidia, are the taxonomic position of the organism along with its the well recognized parasitic genera. Revisiting the evolutionary history. Taxonomy refers to the classification progress in taxonomy appears to be important to of groups of organisms. Systematics arranges various evaluate our studies. Haemosporidia being microscopic, taxa like phyla, subphyla, classes, orders, suborders etc. their taxonomy specially the morphotaxonomy has following a hierarchy of evolutionary interrelated groups. sometimes created confusion. Therefore, analysis of Taxonomy puts names for organisms and groups of progression of the taxonomy of the avian Haemosporidia related organisms. Systematics deals with the taxonomy demands special attention. Modern phylogenetic analyses and evolution of organisms to establish an evolutionary revealed a wealth of information, which is undoubtedly relationship between taxa meaningfully. Thus, both the useful for protozoology and other related sciences. taxonomy and systematic depend on each other. Techniques of molecular taxonomy are applied to draw Zoologists took the role of principal taxonomists the phylogeny covering members of Haemosporidia. The for in the early period. Definitely, they had study of the life cycles of both hosts and parasites are less opportunity for studying the taxonomic position of absorbing. Besides, analysis of their evolution through protozoans with the traditional tools and techniques. Also, molecular phylogenetics appears to be vital in studying authoritarianism by the zoologists perhaps played a major haemosporidians and to gain insight for basic and role in fixing the systematic position of the lower fungi, applied sciences. This article examines the potential of protozoa, and algae at the then time [1]. Now Protozoa are molecular phylogenetics in refining systematics of avian not the sole of protozoologists. Members of the Haemosporidia and to explain a holistic view of the group. Protozoa have important implications in the medical and veterinary parasitology as well as in microbiology. Gradual Keywords: Phylogenetics, Hemoproteus, Plasmodium, development of the classification scheme and progress of cytochrome b biochemistry, genetics and molecular biology improved our knowledge of protozoa and to fill up the gaps in our knowledge thereof. Many taxonomists still work with traditional techniques of protozoa. The classification of 1 Introduction protozoa was complicated due to lumping of protozoa, with algae and some other eukaryotic microorganisms Systematics, taxonomy, classification, synonyms, and together as Protista and by data collected from modern homonyms are becoming difficult for students, teachers, biological techniques. For a more detailed account in this and researchers. This applies to the field of zoology, context, the reader may consult the classical works [2-8]. microbiology, parasitology, medical parasitology, Similarly, improvements in microscopic methods and entomology and medical entomology. Difficult situations molecular biology revealed the morphological, cellular sometimes arise in dealing with taxa like Protozoa, and subcellular characteristics of protozoa in detail. Rotifera and Insecta. Various approaches to taxonomy

Open Access. © 2021 Fatik Baran Mandal, Ph.D., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 Public License. Hurdle in taxonomy: A case of malaria parasites and other Haemosporidia 41

The efforts of scientists throughout the globe through identification of specimens. The process of identification centuries have developed the present-day classification relies on information about the characters of the concerned of the protozoa. Interesting history of those efforts would organism. Specific taxonomic approach (morphological/ also attract readers. ecological/ behavioral/cytological) requires specific Here, I review the taxonomy of protozoa, especially information to classify organisms into groups. Taxon is the avian Haemosporidia to present the complexity of considered as a group of biological organisms (taxa, plural). the assemblages of diverse groups of within Taxa in a classification have rank, with the basic rank (basal the group. I shall also deal with the brief descriptions, rank) as . The higher ranks include , subgenus, general characteristics, life cycles, and systematics of the tribe, family, , suborder, class, subclass, phylum, and three genera of avian Haemosporidia. Other groups of . Scientific names are the primary but critical keys to animals involved in systematics of avian Haemosporidia unlock biosystematics information about living organisms. will also be taken into account, as and when required, Scientific names are unique. Typically, only one valid for the sake of clarity. Then I shall discuss the present scientific name indicates a specific organism throughout the trend of research on avian Haemosporidia as a case world. Disagreements in classification are due to differences study to illustrate how the hurdles in morphotaxonomy in paradigm and/or information. Theoretically, there should are gradually decreasing along with the development of be only one correct classification. However, in practice there biological tools and techniques. were multiple classifications in the past; some of which are in use today. For the correct classification, we must develop a nomenclatural system that supports two unique 2 Methods keys (valid name and original name). The valid name is the correct name and the best name to provide the full value of While consulting literature on animal classification, scientific name for a taxon. The original name refers to the opportunity presented itself to examine the progress in valid name in the classification in which it was proposed, taxonomy and to realize how sometimes, synonyms and which would be useful only to retrieve information across homonyms create confusion to get the valid information multiple classifications. of a genus, or a taxon. Besides, I had to consult the Classifications also include terms like synonymy literature on taxonomy of avian haemoparasites or blood which indicate two or more names for the same species. inhabiting parasites especially of the genus Haemproteus Homonymy shows various species having the same (Haemosporidia: Haemosporina) during 1986-1991 for my name. However, the rules of nomenclature can solve PhD work. At that time morpho-taxonomy was mostly such conflicts. For example, if homonymy becomes an followed. In the present study, I have analyzed the trends obstacle, the rule of uniqueness can address it.The rules of of progress of taxonomy and systematics of the avian typification can solve the problem of synonymy following Haemosporidia from my experience of consultation of the convention. The scientific name to be used is settled by classic literature, as well as the survey of recent literature giving priority to the oldest name. The codes of zoological in the field following standard procedure. nomenclature can solve the problems of homonymy Ethical approval: The conducted research is not related and synonymy, and can guide us for proper formation to either human or animals use. and documentation of scientific names. Linnaeus and Fabricius, following Aristotelian essentialism/typology, 3 Findings developed the first classification. Organisms are grouped together as they share the criteria of the group, which The findings are given in subheadings and in sequence as is the type. Later, to develop systematics and to get a below for convenience. holistic view, evolution was added to the taxonomy. The classifications then lead to phylogeny, which is the genealogical hypothesis of relationship. Later, 3.1 Systematics and taxonomy various ways of deciphering phylogeny and translating phylogenetic information into a hierarchical classification The science of classification of organisms following the valid (phylogenetics vs cladistic methods) have developed. A rules is called taxonomy. We get a picture of biodiversity, perusal of literature reveals that much has been written and the information required for entire biology from the about the relative advantages of phenetics, evolutionary taxonomy. Taxonomy mostly depends on the accurate systematics, and cladistics [2-8]. 42 Fatik Baran Mandal, Ph.D.

The International Code of Zoological Nomenclature [11]. Identification of Protozoa has employed microscopic (ICZN) in its 4th edition consists mainly of a series of methods by tradition. Now molecular methods identify 18 chapters comprising 90 articles. The ICZN contains many cryptic protozoan species. The cryptic species recommendations and examples to address the conflicts indicates two or more species that were grouped as a single or misunderstandings in taxonomy. Five different codes nominal species due to their apparent morphological of nomenclature are now in use, one each for bacteria, similarities. In recent times, molecular analyzes can viruses, , cultivated plants, and animals. In the distinguish different species within the cryptic species. early 1990s it was attempted to develop a single code of This clears the controversy in the taxonomic position nomenclature for the living world which was followed and elucidates the systematics of the protozoa. The use by a publication of draft BioCode. Use of such BioCode of electron microscopes presented a golden epoch of is significant in taxonomy and in broader aspects in the research in protozoology in the mid-20th century. Some study of systematics. Naming establishes a taxon as the parasitic protozoa are ectoparasites, while some are basic unit of biodiversity research. Giving a proper name endoparasites. There is blood inhabiting, skin and tissue to a species first can only provide the scope to consider inhabiting and visceral- protozoa. The protozoa had about the infraspecific entities which are found in various rules four times the number of taxonomic units above the family of nomenclature. The higher-level classification and the level than the classification scheme developed by Butschli Tree of Life depend on species. The Linnaean binomial (Honigberg Report of 1964) [12]. There are variations in nomenclature, often considered as outdated now, still nature, survival strategies, pathogenicity, and immune allows to maximize the number of named species. This responses among various groups of parasites. Parasites nomenclature places the species into the Tree of Life inhabit like Pisces, Amphibia, Reptilia, Aves, along with their genus, which is difficult with alternative and Mammalia. Researchers have classified the parasites non-Linnaean systems. However, with molecular level following their own realization, which sometimes fail advances and with implementation of the Nagoya to follow the principles of taxonomy and thus vary Protocol, new challenges are emerging. Taxonomy substantially [13-16]. Besides, the nature of parasites provides the framework to name, classify, and understand belongs to various taxa like Protozoa, Platyhelminthes, our natural environment. Besides, every branch of applied Nematoda, and Nematomorpha and even of Insecta varies science deal with organisms which require accurate extensively. naming and identification of the organism. Several policy The classification of eukaryotic protists has been in matters became obstacles in the progress of taxonomy. flux for over two centuries [2] following the 19th century For example, in the past few decades, the focus was on authors like Owen (kingdom Protozoa, 1858), Hogg revenue-generating science. The governments allocated (kingdom Primigenum, 1860), and Haeckel (kingdom more research budgets for applied research work, but much Protista, 1866). In the 1970s, a trend developed to divide less to basic science research. This affects the taxonomic the group protozoa into several subkingdoms. Since advancement, with permanent employment positions then, new discoveries of ultrastructure of protists have being eliminated, or replaced by other disciplines with driven to propose explicit phylogenies for protists. In a emphasis on applied research [9, 10]. conservative approach, Cavalier-Smith [2] opted to retain a single kingdom Protozoa including all except the primitive Archezoa and the four eukaryotic kingdoms 3.2 Systematics and taxonomy of Protozoa (Chromista, Plantae, Fungi, and Animalia). In his elegant review of 42 pages along with over 150 references, Cavalier Protozoa were accepted as a subset of the kingdom –Smith [2] stated to raise the taxonomic rank of kingdom Animalia in the middle of the 20th century. Protozoans Protozoa which was created by the Owen (originally are single-celled organisms in which a single cell protozoa as a class; Goldfuss, 1818). Cavalier –Smith [2] performs every function of an individual. So, to designate separated protists from animals and plants and divided the protozoans as acellular is more meaningful. The term protozoa into 18 phyla namely , Ciliophora, protozoon is equivalent to the German word Urthiere that Dinozoa, Entamoebia, Euglenozoa, Haplosporidia, means primitive animals. Leeuwenhoek observed protozoa Percolozoa, Heliozoa, Mycetozoa, Myxosporidia, Opalozoa, with microscopes made from simple lenses. Members of Paramyxia, Percolozoa, Radiozoa, Reticulosa, Rhizopoda, the Protozoa are either free living, or parasitic, ranging Choanozoa, and Mesozoa. Cavalier –Smith also defined from one micrometer to one, or two millimeters in length protozoa with diagnostic characters as follows: Hurdle in taxonomy: A case of malaria parasites and other Haemosporidia 43

Predominantly unicellular, plasmodial or colonial Fundraising to procure tools and establish a good phagotrophic eukaryotes, wall-less in the trophic taxonomic laboratory is still problematic in numerous state. Primitively possessing mitochondria and countries, and also in individual research institutes peroxisomes (unlike Archezoa); when mitochondria and or universities. However, necessary skills enhance the peroxisomes are both secondarily absent (Parabasalia, marketability of the new generation of taxonomists in Entamoebia, Lyromonadea, and anaerobic the job market [18]. Many publications [19-22] show the only), hydrogenosomes and/or Golgi dictyosomes are hurdle in taxonomy which is being discussed here in the present instead. Ciliary hairs are never rigid and tubular section 3.4 and 3.5. (unlike most chromists); haptonema absent (excludes Many novel criteria emerged from the use of electron nonphotosynthetic [94] haptophytes). Chloroplasts, when microscopy. Those criteria helped in developing kingdom present (some euglenoids and only), systems of classification, avoiding the dichotomy contain neither starch nor phycobilisomes (unlike in of animal-or-vegetable. In 1860 John Hogg[23] first Plantae), have stacked thylakoids, and usually have three, proposed the kingdom Protoctista to separate them from rather than two, envelope membranes. Chloroplasts are animals and plants. Ernst Haeckel [24]. in the year 1866 located in the cytosol, never within a smooth periplastid also proposed the third kingdom Protista. Haeckel, a membrane inside either the lumen of the rough biological giant of the second half of the 19th century, endoplasmic reticulum or a fourth smooth membrane created the Protista, to contain the microscopic and (unlike Chromista); ejectisomes never of the double-scroll unicellular organisms. He believed that those organisms cryptist type (this excludes the cryptist Goniomonas); the placed in the Protista should be from the then existing few multicellular species have minimal cell differentiation kingdoms containing the macroscopic and multicellular and altogether lack collagenous connective tissue plants and animals. This evolutionarily based systematic sandwiched between two dissimilar epithelia (unlike concept was again refined in 1878. Haeckel’s concept was Animalia). controversial from its inception to now. However, this Source: Cavalier-Smith [2]. is valuable to focus on the phylogenetic component of taxonomy and on the microscopic eukaryotic organisms All 41 species and 6 genera belonging to phylum called the protists. Attempts were taken in the 20th Apicomplexa were named between 1826 and 1850. In the century to improve the systematics of all protists. One of year 1987, those numbers stood respectively at 4516 and the major challenges to the workers is to determine ways 339 including 444 species of in 9 genera of including information from phylogenetic cladograms [17]. All members of Apicomplexa are parasitic, having into ranked hierarchical schemes of classification [22,25]. apical complexes to penetrate the host cell which signifies Corliss’s work [1,4-6] help us immensely in unravelling the name Apicomplexa. They belong to either the class the early history of protozoa. Haeckel’s [24] idea of origin , or class Sporozoea, and exhibit alternation of protist, animal, and independently from various of generation with cycles of sporogony (sexual) and precellular ancestors led Woese and Fox [26] to propose schizogony (asexual cycle) besides gametogony. The a ‘progenote’ ancestor. Cavalier-Smith [3] considered gametocytes and sporozoites are the invasive or infecting Apicomplexa to rank as a subphylum and Sporozoa as an stages in the life cycle of the members of Apicomplexa. infraphylum. Haemosporidians belong to the suborder Haemospororina Revision of the classification of protozoa called the in the phylum Apicomplexa of the subkingdom protozoa. ‘Levine Report’ of 1980 [17] acknowledged seven phyla in The avian Haemosporina comprises three genera, the Subkingdom Protozoa with 229 taxa above the family namely Haemoproteus (), Leucocytozoon level. The commonly used guide to the Protozoa appeared (Leucocytozoidae) and Plasmodium (). with its classification scheme based on the Levine Plasmodium is a typical malaria parasite. Perhaps most Report [17]. This scheme classified protozoa into seven taxonomic papers published before and to a major extent phyla, namely Sarcomastigophora, Labyrinthomorpha, now contain morphological descriptions of a taxon. Apicomplexa, Microspora, Ascetospora, Myxozoa and New species are often designated without information Ciliophora. However, Zhang [27] has placed Myxozoa as on behavior, natural history, key for identification, a separate phylum of the Kingdom Animalia, although and phylogenetic hypotheses. Taxonomy changes Myxozoa was a group of protozoa before. Molecular rapidly due to molecular biology, sometimes combining phylogenetic analyses support the close relationship multiple, both old and novel tools. Currently, individual between Myxozoa and Cnidaria [22,28] showing its taxonomists apply modern tools for taxonomic research. benefits. 44 Fatik Baran Mandal, Ph.D.

3.3 Systematics and taxonomy of The vector, canariensis Macquart Haemosporidia (Diptera: ) was also described as Microlynchia pusilla, Ornithomyia avicularia, and Lynchia Haemoproteus, Plasmodium, and Leucocytozoon along hirsuta (Hippoboscidae) [46] (a case of synonymy). with other genera, form the group Haemosporida. Similarly, misidentification of Members of Haemosporidia parasitize amphibians Kruse as H. maccallumi from the avian host Zenaidura through mammals. There are pitfalls in the identification macroura and as H. palumbis from the host (Columba of hosts and their haemosporidian parasites. Members of palumbus) present cases of synonymy. In a similar way, the family Haemoporoteidae Doflein, 1916 are the most Haemoproteus nettionis was described inadvertently as common parasites of . Danilevski [29] first noted H. anatis and H. anseris [47]. Thus, the concept of host pigmented parasites in the RBC of pigeons. Later in specificity stands no longer valid now. In case of vector 1890, Kruse [30] erected the genus Haemoproteus for the specificity in human malaria, Anopheles is the sole vector. pigmented parasite, which was similar to Plasmodium. However, mosquitoes belonging to genera like Culex, Adie [31], O’Roke [32], Coatney and Roudabush [33], Haiba Anopheles, Mansonia, Aedes, Culiseta, and Aedeomyia [34], White and Bennett [35] studied the taxonomy of transmit species of avian Plasmodium [48]. Again, the rule avian haemoproteids. The International Reference Centre of vector specificity does not explain such transmission for avian haematozoa deals with various aspects of avian in birds. Hematozoa was thought to contain Haemosporea haemoparasitology. In fact, taxonomic errors were due and Piroplasmea before. Levine et al. [17] considered to ‘one host-one parasite’ belief. Besides, morphological haematozoa to contain Haemoproteus, Trypanosoma, variations of the same parasite in various hosts also Leucocytozoon, Plasmodium, , and Lankesterella. created errors. Molecular studies show the evolutionary The term haematozoa is now less in use following origin and prevalence of Plasmodium species in birds of classification of Protozoa [17]. The members of the group various orders across different countries [36-39]. The level Haematozoa lacks sporocysts, conoids, and nine-triplet of host order in case of Haemoproteus and Leucocytozoon centrioles which are typical of eukaryotes. is limited. Host based parasite taxonomy was also a source Controversy exists regarding the number of families of taxonomic confusion, which required re- classification in Haemosporina. Some claim that it should contain the of avian fauna. family Plasmodiidae only, while others claim three families. The use of molecular markers for avian In the latter case, the three families are Haemoproteidae, haemosporidians began in 2000 [40]. Valkiunas [41] (genus ), and Plasmodiidae. According considers the Haemoproteidae as the most primitive group, to another opinion, the family Leucocytozoidae is distantly and Leucocytozoidae as the most recent. The excellent related to the above-mentioned groups. It is proposed review on Valkiuna’s study by Catherine Cosgrove [42] to include Plasmodium, Haemoproteus, is mention-worthy here. Taxonomy of the Leucocytozoon and Plasmodium in a monophyletic clade as the malaria is being revised using molecular techniques to achieve parasites. Leucocytozoon is also considered as a malaria accuracy. This is supposed to change the parasite- vector- parasite in another approach [49]. This proposal has associations. However, Garnham and Duggan [43] created debate among the protozoologists and researchers included at least 17 genera and subgenera inhabiting of other related fields. various reptile, bird and mammal species in the order Haemosporidia. These genera and subgenera are Akiba, Fallisia, , , , , 3.4 Haemosprodia Rayella, , , , , Carinamoeba, Haemocystidium, Ophidiella, Sauramoeba, Members of avian Haemosporidia represent an excellent Saurocystozoon and Hepatocystis. However, Levine et al. model system for studying vector borne protozoan [17] in their revised classification of the Protozoa have diseases like malaria. The morphology and life cycles not used the term Haemosporidia. A total of 145 avian of Plasmodium, Haemoproteus, and Leucocytozoon haemoproteid species along with 35 spp. as nomen nudum are complex. The family Garniidae comprises the and one as numen dubium were listed in 1988 while this lesser-known genus Fallisia. Mixed infections with number was 196 in 1982 [44]. Multi host parasites may Plasmodium and Haemoproteus are common. Similarities explain this [45] instead of strict host specificity. This and differences in the life histories of Plasmodium, again is a case of taxonomic hurdle. Hepatocystis, Haemoproteus, and Leucocytozoon can be Hurdle in taxonomy: A case of malaria parasites and other Haemosporidia 45 generally summarized. Laveran [50] for the first-time have one and some have two types of gametocytes [43]. observed Plasmodium in 1881 in humans. Danilevski [51] Leucocytozoon marchouxi is pathogenic for doves and realized its parasitic nature. Since then, many studies [57] while L. danilewskyi for some species. focused on the biology Haemosporidia. Laboratory studies Schizonts of Leucocytozoon occur in muscle, cause damage of centered on the . and necrosis [58]. Extent of tissue damage causes mortality. This parasite stands as one of the first models for human Leucocytozoon causes a disease called leucocytozoonosis. malaria. Avian haemosporidians are important for study The disease is also called gnat fever, malaria, and for their association with birds’ health. Introduction of duck malaria which can be fatal in poultry. Number of bird malaria into Hawaii in the 19th century affected the described species of Leucocytozoon is over 100 [59,60]. endemic birds. Outbreaks of the haemosporidian infection However, vectors are known only for a few species of cause serious economic losses in poultry. Five subgenera Leucocytozoon. anatinum and S. rugglesi transmits of genus Plasmodium are Giovannolaia, , L. simondi. L. smithis and L. simondi are economically Novyella, Haemamoeba, and Huffia. Plasmodium causes important. Simulium meridionale and S. slossonae transmit the disease malaria. Female Anopheles transmits L. smithi that inhabits turkeys. Molecular work has added Plasmodium. MacCallum [52] discovered the asexual more diversity of Leucocytozoon in its vector, the black stages of Plasmodium in RBC of birds. Ronald Ross [53] [61,62]. Birds serve as a host for asexual development described the sporogony of in for Leucoctozoon (schizogony and gametogony; reviewed culicine mosquitoes. Shortt and Garnham [54] discovered by Adler et al. [63]. Birds with chronic infections that Plasmodium completes schizogony in the show weak immune response and low reproduction. before starting the blood cycle in the year1948. In 1982, Leucocytozoonosis had serious effects on the poultry in Krotoski and others [55] described the dormant stages North America [63]). The U.S. Agricultural Research Service of the sporozoites called hypnozoites in the liver [56]. estimated an average loss of nearly $750,000 per year in the Plasmodium causes avian malaria with sublethal effects US during 1942 to 1951 due to leucocytozoonosis in turkeys. on host fitness. The most detrimental long-term impact of In the 1970s, the last major outbreaks of leucocytozoonosis avian malaria is the reduction of the host population due in domestic turkeys happened. Turkeys now are raised to effects on the host’s reproductive system. primarily in poultry houses, reducing the incidence of Members of the family Hippoboscidae and disease because the vectors generally do not venture inside (Diptera: Ceratopogonidae) are shelters [64]. Leucocytozoon enlarges the liver and the vectors of the subgenera Parahaemoproteus and and causes anemia [63]. Haemoproteus of genus Haemoproteus, respectively. Host specificity of a parasite ranges from specialists Parahaemoproteus does not infect doves and pigeons infecting one, or a few related host species to generalists (Columbiformes). Number of species is the highest in the which infects many host species. Host specificity has genus Haemoproteus among avian Haemosporidia. Some no relationship to the abundance, or evolutionarily species of Haemoporoteus pose threats to their avian distinctiveness of the host(s). Haemoproteus is the most hosts. Haemoproteus produces subclinical infections with common genus encountered in bird species, followed enlargement of kidneys, liver, spleen, and gizzards and also by Plasmodium and Leucocytozoon. Most species of invades the . Anorexia, anemia, and depression have Haemoproteus and Leucocytozoon are relatively host been reported in some birds infected with Haemoproteus. specific and found in closely related species within the The subgenus Haemoproteus infects non-passerine bird same host family. Species of avian Plasmodium have much species from the Columbiformes, Pelecaniformes, and broader host specificity and may occur in several avian Charadriiformes. Parahaemoproteus infects birds across families [43,65,66]. all the avian phylogeny [38]. Leucocytozoon and Akiba are the two subgenera of the family Leucocytozoidae. Only one species, Akiba, is known for avian Leucocytozoidae. Life 3.5 Life cycles of members of avian cycle of Leucocytozoon is similar to that of Haemoproteus. Haemosporidia Gametocytes of various species of Leucocytozoon develop either in RBC, or lymphocytes. Gametocytes lack Members of Haemosporidia completes their sexual phase haemozoin granules like Plasmodium and Haemproteus of life cycle in the vector and they use their host but distort the host cell. Morphologic forms of gametocytes for completion of their asexual phase of life cycle. Both are either round or elongated (fusiform). Some species the phases of life cycles are described below: 46 Fatik Baran Mandal, Ph.D.

3.5.1 Sporogony gametes unite to produce zygote in the vector. Zygote develops through stages such as ookinete and oocyst The sexual cycle takes place in dipteran vectors and results in the infective stage, which is called the in all three genera. Hematophagous dipterans sporozoite. In Hepatocystis, the ookinete penetrates the (Insecta: Diptera) of the families Ceratopogonidae, stomach wall of and moves into its hemocoel Hippoboscidae, Culicidae, and Simuliidae serve as the to develop into oocyst. vectors. Both mosquitoes and biting midges of the order Two types of oocyst are described in Haemoproteus. Diptera are the vectors for Plasmodium and Hepatocystis, H. columbae in the hippoboscid develop into large respectively. Hippoboscid flies and biting midges serve oocysts with many sporozoites. Oocysts of H. columbae that as the vectors of Haemoproteus spp. Simuliids and mature in Culicoides are slightly larger than the zygotes biting midges transmit Leucocytozoon. Life histories from which they arise. Two types of sporozoites develop of Plasmodium, Haemoproteus and Leucocytozoon bear differently from two types of oocysts. In Leucocytozoon, distinctiveness to place them into three families namely the oocysts are relatively small; some of which develop Leucocytozoidae, Haemoproteidae, and Plasmodiidae intracellularly, some intercellularly, and some below the respectively. The sporogony (sexual cycle) comprises basement membrane. Sporozoites in Leucocytozoon have stages like gametes, zygote, ookinete, oocyst, as well one round and one pointed end similar to sporozoites as the end product, the sporozoites and they occur of Haemoproteus in hippoboscid vectors. Sporozoites of in vector fly. Life histories of the said three genera Haemoproteus in Culicoides possess pointed ends similar show a limited specificity for the dipteran vectors. to that of Plasmodium and Hepatocystis. Mosquitoes (e.g. Anopheles, Culex bitaeniorynchus, Culex vishnui, Aedes butleri, Mansonia uniformis, Aedes pseudomediofasciatus) transmit avian malaria. 3.5.2 Schizogony Hippoboscid flies transmit Haemoproteus columbae of pigeons. Culicoides also transmit some species of Schizogony is the asexual lifecycle in which sporozoites, Haemoproteus. the end product of sporogony, transform into schizonts Several ornithophilic simuliids transmit and upon maturity schizont bursts to liberate merozoites. Leucocytozoon. A female ingests the gametocytes Two and possibly three types of schizonts are found in of Leucocytozoon during taking its bloodmeal from the Leucocytozoon. Hepatic schizonts occur in cells of the infected bird. The parasite then undergoes both asexual liver. Large megalo-schizonts of L. simondi occur in and sexual development. It completes its life cycle over the liver, kidney, pancreas, brain, , gizzard wall, 3–4 days on the fly. During the next blood meal, the fly intestinal wall, and in other locations. Most of the transmits the infected form of the parasite, the sporozoites merozoites released from the schizont invade blood to the host. In Leucocytozoon sporozoites are injected into cells to become gametocytes; some also produce another the host blood by the vectors, the black fly, during their generation of schizont. Thus, a cycle of schizogony blood meal. Members of the genus Leucocytozoon infect continues to happen for ultimate production of the end a wider range of host cells. However, when they infect the product (merozoite) of schizogony to continue the life RBC, Leucocytozoon digests hemoglobin without forming cycle of the parasite. In Plasmodium, schizogony occurs the hemozoin granules. In the case of Leucocytozoon and in the blood, liver and other tissues. Schizogony starts Haemoproteus, exflagellation, the process of male gamete after the entry of sporozoites in the host. In case of other formation, occurs rapidly in comparison to Plasmodium two genera, it occurs only in the tissues. The schizogony gallinaceum. (asexual cycle) with the end product of merozoites, Culicoides adersi transmits Hepatocystis kochi in as well as the gametogony, occurs in avian hosts, or mammals. Hepatocystis kochi constitutes an important vertebrate hosts. Gametogony results in the production position in phylogenetic hypotheses for the relationship of male and female gametocytes. The exo-erythrocytic for several genera of Haemosporidia inhabiting cycle takes place in endothelial cells of capillaries, mammals, lizards and birds [49]. Thus, Plasmodium lymphocytes, spleen, and in liver parenchymal cells. In and Hepatocystis develop in mosquitoes and simulids, Haemoproteus, a prepatent period lasts for two to three respectively. The parasite in the form of sporozoite weeks. In Leucocytozoon simondi, it lasts for less than enters the vector fly during sucking their host blood. six days. The prepatent period is the time gap between The male and female gametocytes on entering the vector the entry of the infective stage in the host and clinical fly produce male and female gametes, respectively. The manifestation of the disease. Hurdle in taxonomy: A case of malaria parasites and other Haemosporidia 47

3.6 Molecular phylogenetics in resistance in Plasmodium to control malaria infection. Haemosporidia Molecular phylogeny enables us to control such diseases. Molecular approach uses the genetic complement, Microscopy was the method to identify avian DNA –hybridization, DNA bar-coding to name a few. haemosporidia with morphological data before 2000. Haemosporidia is a highly diverse group with a wide This was and is the traditional taxonomy in case of host range. Thus, phylogenetic and ecological studies microscopic protozoa. Several morphological differences propelled the use of molecular tools [70]. mtDNA is the help to identify various parasite genera in a blood smear/ smallest in the haemosporidian among all eukaryotes. tissue smear. Vector competency, life history traits, This DNA has cytochrome c oxidase subunit1 (cox1), and morphological data together formed the earliest cytochrome b(cytb) and cytochrome c oxidase subunit phylogenies of Haemosporidia before the 1990s. Renewed 3(cox3). mtDNA genes identify species including cryptic interest in avian haemosporidian emerged from the species to draw phylogenetic relationships. It also explores development of reliable molecular methods. These tools the biogeographical and historical patterns [62,70,71]. ease the screening of haemosporidian infection. They are Realizing the importance of mtDNA, Pacheco et al. [70] also important for reexamining the global epidemiology of reviewed the rate of mtDNA evolution. They used a data infection with haemosporidian. Molecular data has helped set comprising 114mt DNA genomes from Haemoproteus, to alter and clarify this original phylogeny. Polymerase Plasmodium, Hepatocystis, and Leucocytozoon. Pacheco chain reaction (PCR) became a prominent molecular and others in the year 2017 estimated the phylogeny tool to screen the haemosporidians. For Plasmodium and divergence times for the distribution of major and Haemoproteus, and Leucocytozoon, the PCR screens haemosporidians of birds. The study shows that mtDNA targeting the cytb gene stand reliable often. Mitochondrial genome is a suitable biomarker for phylogenetic analysis. cytochrome b (cytb) sequences of H. sacharovi have In the mtDNA phylogeny, Plasmodium of lizards and birds been identified. Phylogenetic relationships of subgenus appeared as monophyletic. However, the group contains Haemoproteus show that H. sacharovi and H. turtur branch five clades, four of which are well-supported based on from the clade along with the Parahaemoproteus. The their biomarker-based similarities. In a similar case, study provided barcodes for H. sacharovi, and predicted a only six species of Plasmodium were examined for their revised classification of avian haemoproteid species. One relatedness from lizard hosts. As the number of species can get a clear picture of the systematics of the concerned examined were few, it was inconclusive that the said taxa from the study as well. species of Plasmodium could form a monophyletic group. The current subgeneric classification of avian It is interesting to note that 101 species and subspecies of haemoproteids is generally effective. However, the Plasmodium inhabit various species of lizards. Pacheco position of some species requires revision [67]. This et al. [70] also showed that whole mtDNA was vital again shows that systematics is still enigmatic, at least for phylogenetic analysis. Recently, Nilsson et al. [71] in some cases. The identification of avian malaria reported multiple cryptic species of Haemoproteus majoris parasites was revolutionized by many researchers [37,68]. from its avian host. In more recent times, true diversity of Bensch et al. [37] designed a primer set to amplify a haemosporidians is believed to be over 260 species [72]. 478-nucleotide part of the cytb gene from Haemoproteus Variation in the bar-coding region of the cytb gene has and Plasmodium of birds. Designing more primers since resulted in over 3600 lineages. Besides, phylogenetic then amplified many nucleotide fragments. Some primers analysis of avian haemosoridian was detailed by many amplify Leucocytozoon fragments homologous to those of workers [for example 73-79]. The cytb gene is useful to Haemoproteus and Plasmodium. This makes it possible to identify parasite lineage and to know the evolutionary compare the same fragment in all three genera [69]. history of avian haemosporidian. Multigene phylogenetic Traditional taxonomy described 500-odd species of analysis is useful and most reliable in this context [73,74]. avian haemosporidia, of which less than half were valid Phylogenetic evidence emerged from the molecular [41]. Molecular approach is promising to construct the data has questioned traditional taxonomic schemes. As a evolutionary history of an organism through molecular result, major changes in the taxonomic practices are also phylogeny. Parasites existed before the emergence arising [80]. The acquisition of reliable evidence is the key of mammals or birds. Wild animals are the source of for a stable scheme in taxonomy. Phylogenetic evidence many human diseases called zoonotic diseases like is crucial to get a picture of phylogeny using modern human malaria. We must trace out the origin, spread, taxonomy [80]. Multigene phylogenies are appearing insecticide resistance in vector mosquitoes and drug for many genera, including Plasmodium [81,82]. The 48 Fatik Baran Mandal, Ph.D.

Fig. 1 Reproduction of HAECxcr’s phylo- genetic tree of life (from HxEcKEL 1866. Plate I). Source: Corliss [5]. Hurdle in taxonomy: A case of malaria parasites and other Haemosporidia 49 availability of genome sequence for selected apicomplexan similar cryptic species, although appears to be a single parasites has revealed the genomic landscape of virulence species, may consists two or more species, which can be gene families [83]. Detailed information on virulence determined by the molecular method. genes is useful to control infectious diseases. Interested Phylogenetic parameters like molecular clock and readers may avail further information from the recent extinction rates for Haemosporidia are still lacking. This is publication [84]. Phylogenetic studies analyzed the due to the lack of fossil DNA. The rate of mtDNA divergence sequences from Hepatocystis (mammal), Haemoproteus between species is about 2% per million years. Studies (bird and lizard), and Plasmodium (mammal, bird, and suggest a slower clock for Haemosporidia than their lizard). Perkins and Schall [85] analyzed the cytb gene and vertebrate hosts. It is unclear how much slower this clock formed the tree that rooted with annulata. Two would be. Generation time is shorter for Haemosporidia included Leucocytozoon sequences were basal in the tree than vertebrates. Therefore, a slower haemosporidian as a close sister out-group to the in-group. Martinsen et al. clock is surprising. A divergence rate of 1.3% per million [49] used Leucocytozoon both as the out-group and to root years for Haemosporidia is lower than that of their avian the tree. Outlaw and Ricklefs [82] analyzed the same data hosts. This rate is higher than co-speciation estimates using an alternate rooting method. (Ricklefs and Outlaw [89]. If 1.3% divergence rate is Comprehensive phylogenies show that correct, certain haemosporidians could have diversified Parahaemoproteus and Haemoproteus are distinct. to infect many hosts in the last 20 million years [89]. They were intermediate between Plasmodium and Hellgren et al. [90] analyzed fragments of the mtcytb Leucocytozoon. Parahaemoproteus is more related to gene from six morphospecies of avian Haemoproteus. Plasmodium in birds. Plasmodium is not monophyletic due The average divergence between the morphospecies to Hepatocystis. In all three comprehensive phylogenies, was 5.5%. This suggests that separate morphospecies Plasmodium formed two clades based on vertebrate hosts resulted in sequences with a genetic difference >5%. Two (one in mammal and the other in lizard/bird). Evidence has morphospecies differed by only 0.7%. Krizanauskiene been mounting for a revision of Haemospordia taxonomy. et al. [91] found that a divergence of 2% was enough for Controversy arose to redesignate haemosporidians of species to show external distinctness. Closely related P. birds and reptiles. Most malaria trees contain an out- falciparum and P. reichenowi diverge by 2.3% across their group from the genus Leucocytozoon, which is related mtDNA. Insight into haemosporidian phylogeny can trace to, but not the malaria genera. The reason is that the the origin of Plasmodium in primates. Characterizing gametocytes of Leucocytozoon lack hemozoin. Meronts patterns of host–parasite associations in wild populations of the Leucocytozoon are not present in the blood of the would produce models. These models can predict the vertebrate host. For this, along with a high divergence rate, probability of infection with haemosporidian to hosts. Leucocytozoon was believed to be ancestral to Hepatocystis, Haemoproteus, and Plasmodium. Leucocytozoon is the ancestral genus and therefore its role as an out-group is 3.7 Phylogenetic Relationships among controversial. Leucocytozoon may be the most derived members of Haemosporidia genus and is a sister group to Haemoproteus. Plasmodium is ancestral to both (species infecting mammals being Diversity of Haemosporidian among raptorial birds was most ancestral). This is the opposite tree configuration estimated by DNA sequencing. mtcytb revealed raptor- from what was before found. Outlaw and Ricklefs [82] specific parasite clades in Parahaemoproteus, although viewed merogony in blood and hemozoin production as not in Plasmodium. A divergent clade of raptor parasites primitive. These traits evolved once in Haemosporidia was found as a sister clade to Leucocytozoon [92]. The and were then lost in some genera. Plasmodium and evolutionary relationships among malaria parasites Haemoproteus both keep the hemozoin trait. Only the is still an annoying phylogenetic problem. This is due Plasmodium keeps the merogony trait. Leucocytozoon, as to bar in sampling of taxon, character and nucleotide a most derived genus, has lost both traits. Plasmodium base composition biases characteristics of this clade. keeps the merogony trait, which was the basis for creating The phylogenetic analysis of Plasmodium from a diverse this genus, as merogony in blood was perhaps a recent vertebrate host were studied. Results showed that acquisition to Haemosporidia. Interestingly, levels of evolutionary history of Plasmodium through a complex species richness and morphologically cryptic species have series of transitions. Plasmodium is polyphyletic with their been under-estimated for tropical biodiversity [86-88] own life-history traits. The traits evolved in a dynamic due to rare use of DNA-based methods. Morphologically manner across the phylogeny. DNA sequencing estimated 50 Fatik Baran Mandal, Ph.D. the diversity of haemosporidian among raptorial birds. of species is likely to be higher than can be recognized Further studies in future covering various avian families by microscopy. Thus, both the parasites and their would contribute on the accurate diversity of bird hosts require study in terms of evolution. The diversity inhabiting Haemosporidia. of members of Apicomplexa with recent advances in To reconstruct the phylogenetic relationships among molecular phylogeny shows the drawbacks of the morpho- the Plasmodium researcher used SSUrRNA gene sequences taxonomy, and stresses the absence of lineages and taxa from 13 Plasmodium species inhabiting mammals, birds, in recent phylogenies [97]. and lizards. The phylogenetic tree thus constructed The importance of life history and epidemiological comprises two major clades. The first with bird and reptile characteristics of the genera Haemoproteus and Plasmodium parasites while the second with the rest of the species. are crucial to know their evolutionary relationships Bird parasites, P. gallinaceum and P. lophurae, do not with their vertebrate hosts and vectors [98]. Martinsen cluster with human, chimpanzee, or rodent parasites, but et al. [49] proposed to include the monophyletic clade cluster with lizard parasites [93]. comprising Hepatocystis, Plasmodium, Haemoproteus, Outlaw and Ricklefs [94] proposed a phylogeny where Parahaemoproteus under the malaria parasites. Braga Plasmodium and Hepatocystis of mammals were sister to et al. [99] thought only Plasmodium as the avian malaria all haemosporidians from reptiles and birds as proposed parasite. Identification of microscopic animals is carried in another phylogeny. Avian Plasmodium found at the base out following some morphological keys in morphological of avian Haemosporidia, followed by Parahaemoproteus. approach. As there are various developing stages of Haemoproteus was sister to the genus Leucocytozoon. the parasite and morphology of a particular stage may This example shows that to construct phylogeny in change depending on various conditions, it sometimes separate groups or taxa is difficult without considering leads to incorrect identification. This does not happen broad evolutionary processes. These processes are host with a molecular approach. Thus, this approach helps to switching, ecology of host, transmission opportunity eliminate controversy in the taxonomic position of the of the parasite from one host to the other. The first organism. study comparing molecular phylogenies with morpho- taxonomy got support from genetic and phylogenetic species concepts as well. For constructing a phylogenetic 4 Discussions and conclusion tree different criterion like molecular clock, nonreversible Three species concept defines a species based on model of DNA substitution and outgroup are in use. morphological, genetic, or phylogenetic characters. Huelsenbeck et al. [95] found outgroup and molecular The morphological species concept distinguishes clock as the best criteria for detecting the root of the tree. parasite species through similarities or dissimilarities The nonreversible model works in presence of highly in morphology using microscopy. The genetic species non reversible substitution process. Leucocytozoon concept is a molecular method that identifies parasites was found at the base of haemosporidians along with based on similarities or divergence of DNA, RNA, or amino avian Plasmodium and Haemoproteus as sister taxa acid sequences. The phylogenetic species concept requires (monophyletic) in their phylogeny [49]. that defined species are monophyletic. Martinsen et al. [49] Use of the ssrRNA gene in phylogenetic analysis reveals identified fourteen of fifteen Haemoproteus, Plasmodium, that avian Plasmodium subgenera do not correspond and Leucocytozoon parasite species using morphology. to their morpho-taxonomy. A two-gene mitochondrial Genetic and phylogenetic analyses supported such phylogeny (cytb and col) of avian Plasmodium got support identification. The exception being H. belopolskyi, which for three subgenera namely Huffia, Haemamoeba and belong to two separate phylogenetic clades. Three species Bennettinia, out of the five morphology-based subgenera concept validates five subgenera of Plasmodium that infect [96]. Perhaps the reptiles hosted several ‘ancient’ genera birds at molecular level. Analysis of the coI and mtcytb of Haemosporidia. Haemosporina of modern reptiles have genes validated the monophyly for Haemamoeba, Huffia, evolved from such an organism. As birds evolved from and Bennettinia. While most Novyella sequences formed a the saurischian dinosaurs, parasitologists do not rule clade, the two Novyella samples were outside the clade, out a parallel evolutionary process with the extant avian forming the most basal branches of the tree. The subgenus Fallisia, Haemoproteus, Leucocytozoon, and Plasmodium. Giovannolaia did not form a monophyletic group. Mammalian haemosporidians perhaps evolved from an The present article deals with the conflict between earlier reptilian stock than birds. High genetic diversity morphotaxonomy and molecular taxonomy to draw the in members of Haemosporidia shows that the number Hurdle in taxonomy: A case of malaria parasites and other Haemosporidia 51 attention of the scientists to end the conflict for furtherance Conflict of Interest of the protozoology and related subjects with an example Author states no conflict of interest. of the avian Haemosporidia. From the evolutionary Data Availability Statement relationship, mammalian Plasmodium species is the sister Data sharing is not applicable to this article as no datasets clade to reptilian and avian malaria parasites. It has been were generated or analyzed during the current study. concluded that “Hepatocystis appears first sharing a common ancestor closer to primate malaria parasites than the avian parasites. Haemoproteus columbae appears References to be closely related to two avian malaria parasites. Study of systematics of Plasmodium requires the study [1] Corliss JO. 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