Apicomplexa Levine 1970

Introduction

Apicomplexa contains a large assortment of single celled eukaryotic organisms, known to parasitise vertebrates (including humans) and invertebrate hosts. It is estimated that out of a probable 1.2 - 10 million apicomplexan species, only about 0.1% have been named and described to date (Adl et al., 2007). As a group, apicomplexans are complex organisms that exhibit a wide variety of morphological shapes depending on their genus and lifecycle stage. They are also regarded to be reasonably host specific, inhabiting a wide array of terrestrial and marine environments depending on their host niche environments.

This group of obligate parasites evolved from a free-living photosynthetic ancestor (=chromerids) and it is closely related to free living single-cell predators (=colpodellids).

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Hypothetical tree of Apicomplexa. Three principal parasitic groups are coloured and their life cycle indicated, as well as Cryptosporidium that likely emerged from within gregarines. Numbers on branches and thickness indicates diversity (i.e. named species). © 2011 Jan Slapeta

While a large portion of the apicomplexans are known parasites of virtually all vetrebrates including humans, they are equally diverse in both marine and terrestrial invertebrates. In fact, we are sure there is at least one unique apicomplexan parasite per any invertebrate or vertebrate. 

The most eminent of the apicomplexan parasites of humans are Plasmodium spp. from the Hematozoa clade, which cause malaria responsible for >1 million deaths annually (Manguin et al., 2010; Mackintosh et al., 2004). Another parasite, Toxoplasma gondii is present in aproximatelly 30% of humans worldwide as brain cysts causing no apparent disease; however, in immunonaive pregnat women it may cause serious disease afecting her foetus. Apicomplexan diseases of domestic animals are associated mainly with farmed animals and are renowned for the large economic costs incurred by the agricultural industry. Of these Eimeria spp. is known to be responsible for a $1.5 billion loss to the poultry broiler industry each year worldwide (Sharman et al., 2010). Similarly, Neospora caninum is linked to losses in the dairy as well as beef industry (Trees et al., 1999).

Parasitism in wildlife populations has been recorded as frequently as in domestic populations. However, in a wild setting, parasitism seems to act to reduce vigor and alter behaviour of the host rather than cause significant disease and losses (Hamilton and Zuk, 1982, Horak et al., 2004, Bouma et al., 2006, Morin-Adeline et al., 2011). How parasite burden and pathology in captive animals translate into the wild is of much interest, especially when large resources are being placed into captive breeding programs of endangered species.

Characteristics

The use of electron microscopes has led to the discovery of unique intracellular structures that led to the description of Apicomplexa as well as their sub-grouping (Levine, 1973).

Ultrastructural Characteristics of Apicomplexa

• Polar rings
• Conoid
• Micronemes
• Rhoptries
• Dense granules
• Pellicle (i.e. inner membrane complex)
• Micropores

The apical complex is the flag trait required for classification as Apicomplexa (Lee et al., 2000, Levine, 1973). It is a set of ultrastructurally and functionally homologous components found at the anterior end of certain stages, most notably the infective stages, displacing the nucleus and mitochondria towards the posterior end (Aikawa et al., 1978). The structural (cytoskeletal) components of the apical complex are known as the conoid, polar rings and sub-pellicular microtubulles. Three types of distinct secretory organelles are typically present: rhoptries, micronemes and dense granules. Rhoptries and micronemens are always associated with the anterior end, while dense granules are often in the posterior end as well. 

The feature that unifies Apicomplexa with other alveolates (Alveolata) is a distinct membrane complex that envelopes all infective stages. The outermost membrane part is called plasmalema. The inner part is composed of two membranes runnig parrallel to each other forming the flattened inner membrane complex (a.k.a. alveoli).  The pellicle is interrupted at the anterior end by the polar rings associated with the apical complex and a single or several micropores. Besides the above, they are comprised of endosymbiontic derived organelles (mitochondrion, apicoplast), universal eukaryotic organelles (nucleus, endoplasmic reticulum, Golgi apparatus, ribosomes), and specific compartments (i.e. acidocalcisomes), all enclosed by a complex membranous structure termed the pellicle.

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Schematic drawing of a typical Apicomplexa. © 2011 Jan Slapeta

Although all apicomplexans contain rhoptries, micronemes and dense granules, the number and general shape of these secretory organelles alter between different groups (Blackman and Bannister, 2001, Leander, 2007a). For instance, most coccidia and hematozoa are known to possess two rhoptries in their merozoites (Bannister and Mitchell, 1989), however a few others have been documented to contain more, an example being the triplet found in Babesia caballi (Kawai et al., 1999). Nonetheless, the proteins secreted from these secretory structures are associated with initiation and regulation of the host cell invasion process, controlled by the secretion of a vast array of proteins (Carruthers, 1999, Bannister and Mitchell, 1989, Proellocks et al., 2010). However, the conoid is not present in the hematozoan group (e.g. Plasmodium spp.) known by some authors as Aconoidasida.

In Toxoplasma gondii and presumably other apicomplexans, the conoid is made of tubulin fibres arranged in a unique manner and it has the potential to expand and reduce in a Ca2+ dependent manner to produce some form of movement (Hu et al., 2002). Therefore, in addition to invasion of host cells the apical complex may have a vital role to play in parasite orientation, support and movement prior to and after invasion. In gregarines, the conoid structure is often not observed and is replaced instead with a homologous mouth piece known as a mucron that attaches to host cell extracellularly for feeding (Dyson et al., 1994, Leander, 2007a). It has been suggested that these organisms are dependent on the attachment of the mucron to the host cell for structural support (Leander, 2007b).

Apicoplast

The apicoplast is a remnant non-photosynthetic plastid characteristic of apicomplexans consisting of a 35kb circular genome (Wilson et al., 1996). Discovery and recognition of this organelle occurred as recently as fifteen years ago in the genus Plasmodium with the aid of in situ hybridisation techniques to a 16S plastid rRNA gene (McFadden et al., 1996). Since then much emphasis has been placed on research of this organelle due to its potential to act as a drug target for the treatment of diseases caused by apicomplexan parasites (Williamson et al., 2002). It is hypothesised that all photosynthesising organelles within higher organisms originated from a primary endosynbiont process that initiated after a eukaryote engulfed and enslaved a photosynthesising cyanobacterium, resulting in a photosynthesising eukaryote (Green, 2011). During the course of time, another (aka secondary) endosymbiont event is thought to have occurred whereby the photosynthesising eukaryote was engulfed by a heterotrophic organism to give rise to the multiple membrane plastid (Cavalier-Smith, 1999). It was later confirmed that this sequence of events seemed likely as the origin of the apicoplast in apicomplexan parasites, and with the discovery of Chromera velia in 2008, the origin of the apicoplast has been traced back to red algal origins despite being significantly reduced in nature (Fast et al., 2001, Janouskovec et al., 2010, Waller and McFadden, 2005).

Plastid rRNA gene analyses performed on multiple apicomplexans have confirmed that all apicomplexans seem to posses the plastid-like structure with the exception of Cryptosporidium spp. and Gregarina niphandrodes - a eugregarine species (Lang-Unnasch et al., 1998, Zhu et al., 2000, Toso and Omoto, 2007). The conservation of this relic structure within the majority of apicomplexans implies that apicoplasts still carry out an important function required for parasite survival (Lang-Unnasch et al., 1998). The apicoplast has been identified as critical for parasite survival as it is responsible for the anabolic FASII and DOXP pathways, representing fatty acid synthesis and isoprenoid synthesis respectively, both of which have been deemed critical to parasite survival (Brooks et al., 2010).

Invasion of Host Cells

Invasion of host cells by apicomplexan parasites is highly conserved between related protozoa, with the possible exception of Theileria (Shaw, 2003).  Attachment to the host cell is initiated via interaction of its surface proteins to the host cell's plasma membrane, an example being the SAG glycoprotein in Toxoplasma gondii (Grimwood and Smith, 1996). The apical region of the infecting parasite (zoite) then connects to the host cell forming a depression in the cell plasma membrane, taking the shape of the zoite while creating a condensed, electron-dense area at the point of attachment (Aikawa et al., 1978). Rhoptry ducts extend from the apical complex and through the junction formed between the two cells (Aikawa et al., 1978). This step is followed by the release of vast array proteins from the microneme and rhoptry of the apical complex which have the capacity to induce formation of the protective parasitophorous vacuole (PV) that surrounds the parasite once inside the host cell (Bannister and Mitchell, 1989). The actual invasion of the parasite is mediated by the formation of a moving junction around the infecting parasite which is so named because it moves along the length of the parasite resulting in the engulfment of the parasite within the host cell (Besteiro et al., 2009).

General Life Cycle

The basic life cycle of the apicomplexan parasite is typically complicated. Generally, there are four transformations to complete the cycle: the zygote, a sporozoite, a merozoite, and a gametic stages. The typical lifecycle of apicomplexa is well established as they all undergo a series of asexual and sexual reproduction involving either one or two hosts whereby a diploid zygote is formed by the union of gametes.  It then undergoes a meiotic division to form infective haploid sporozoites. Within the body, sporozoites are thought to target specific cells by the use of a range of molecular armaments which include surface adhesions such as parasite surface proteins and binding antigens to enter host cells (Baum et al., 2008). The sporozoites then transform to form merozoites within the host cells, followed by several additional generations of asexual division to produce more merozoites and eventually sexual gametes are produced leading to fertilization and the production of a diploid zygote followed by formation of infective diploid sporozoites (Levine, 1973; Lee et al., 2000).

The fine details within this life cycle vary within different apicomplexan groups. For example in coccidian parasites, access to atmospheric oxygen levels induces sporulation of the zygote to form haploid sporozoites, so oocysts are released into the environment non-infectious (Levine, 1973). In contrast, oocyst formation and sporulation in haematozoan parasites occurs within the gut cells of an invertebrate host and the released sporozoites are injected into a new host upon invertebrate feeding (Krotoski, 1985). The presence of an additional dormant phase in the Plasmodium vivax life cycle is also well established and is associated with the relapse of diseases seen during infections. Few apicomplexans have been documented to having this phase, but it is suggested that it may be a common occurrence in many related apicomplexans which have been overlooked (Markus, 2010). Furthermore, differences between species arise in the localization of each stage. For example, in the Plasmodium spp. it is well understood that merozoites target erythrocytes while sporozotes target hepatocytes (Baum et al., 2008). This is contrary to the merozoites in the genera Eimeria and Isospora which are located in the intestinal epithelial cells of the villi (Fernando, 1974, Morin-Adeline et al., 2011).

The life cycles of different groups vary in terms of the number of hosts involved and the type of cellular invasion. While coccidians and gregarines are known to parasitise a single host and an environmental stage to complete their lifecycle, haematozoans have to make use to two hosts with no stages occurring in the environment (Cox, 2010, Lee et al., 2000, Levine, 1973, Moller and Arriero, 2008, Prensier et al., 2008). Moreover, while gregarines infect only invertebrate hosts and coccidia infect only vertebrate hosts, haematozoans make use of both invertibrate and vertebrate hosts (Cox, 2010, Lee et al., 2000, Prensier et al., 2008).

Discussion of Phylogenetic Relationships

The apicompexans, dinoflagellates and the ciliates are classified as Alveolata, along with the newly discovered Chromera velia, an organism that shares many similarities with the putative ancestor of Apicomplexa (Lee et al., 2000, Moore et al., 2008).  Analysis of the small subunit rRNA has confirmed that apicomplexans are more closely related to dinoflagellates then to ciliates (Van de Peer and De Wachter, 1997, Fast et al., 2002). However more recently, investigations of plastid genes has uncovered a more intimate relationship with the phylum Chromerida (Moore et al., 2008). Furthermore, 18 plastid genes were found in common between the alveolate lineage and red algae, confirming the red algal roots of apicomplexa (Janouskovec et al., 2010).

Phylum: Apicomplexa Levine, 1970 (syn. Sporozoa Leucart, 1879)

Traditionally the grouping called Apicomplexa comprises obligate parasites. Nevertheless, the phylum Apicomplexa Levine, 1970 was conceived to include free living marine flagellates of the genus Perkinsus and the traditional Sporozoa Leucart, 1879 emended to include gregarines and coccidia only, therefore omitting hematozoa (Levine, 1970). Perkinsus spp. are no longer considered Apicomplexa, therefore Sporozoa and Apicomplexa are equally good descriptors for the group. Apicomplexa has become frequent in recent literature but it is not used exclusively. We consider both terms synonyms, but use Apicomplexa throughout the text.

One of the distinct characters of Apicomplexa is their parasitic life style. Therefore, free-living predatory flagellates classified as Colpodellida Cavalier-Smith, 1993 are not included within Apicomplexa. Colpodellida Cavalier-Smith, 1993 and Chromerida Moore et al., 2008 are considered independent groups.

Apicomplexa comprise five principal groups commonly known as 'gregarines', 'haemogregarines', 'coccidia', 'malarial parasites' and 'piroplasms'. Besides these dominant five groups, there is a myriad of small transitional groups or species, for example the cryptosporidia forming an independent group.

Formally Apicomplexa is split into three principal groups; the Coccidia (=coccidia , haemogregarines), Gregarina (=gregarines) and Hematozoa (=malarial parasites, piroplasms), all of which are obligate parasites. It has been postulated that before any morphological characteristic is utilised as the basis of taxonomical classification, its association with that particular group of organism has to be confirmed with clade analysis (Clopton, 2009, Morrison, 2009). The lack of such support for morphological traits, and additional errors associated with inappropriate representation of taxa and misuse of genetic analysis tools in the classification of organisms within the three different groups has complicated this field over the years (Morrison, 2009). Recently, much emphasis has been placed on identification of clade-supported traits in an attempt to improve classification criteria (Clopton, 2009, Jirku et al., 2008, Imam, 2009, Adl et al., 2007).

Recently, Apicomplexa became the home for one of the longest-standing protist enigmata - Nephromyces (Saffo et. al., 2010). Nephromyces are endosymbiotic marine protists, whose classification was uncertain ever since they were described in the 19th century. These protists inhabit the lumen of a ductless, urate- and calcium oxalate-rich organ of uncertain function (a.k.a. renal sac), in molgulid ascidian tunicates (Urochordata: Chordata). These protists are ubiquitous nonhereditary symbionts, transmitted horizontally to new hosts. Using rDNA and some apicomplexa-like ultrastructure, Nephromyces form a monophyletic group most closely related to Cardiosporidium cionae, a parasite found in pericardial bodies of an ascidian, Ciona intestinalis (Ciancio et al., 2008; Saffo et al., 2010). Together these protist form a group within Hematozoa (Aconoasida). These examples indicate how little we know about the diversity and habitats that Apicomplexa were able to invade and establish themselves in. No doubt more surprises are still out there.

Other Names for Apicomplexa Levine 1970

• Sporozoa Leucart 1879

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