Introduction
The life cycle of acanthocephalans involves two obligatory hosts: intermediate (a crustacean or insect) and definitive (a vertebrate). In many species, the life cycle can be complicated by an additional paratenic host, which is most frequently a vertebrate consumed by the definitive host. In the intermediate host, acanthocephalans invade the body cavity and, after going through one or more acanthella stages, develop into the cystacanth stage. This is infectious for the following definitive host, or paratenic host, if present. While residing in an intermediate host, acanthors, acanthellae, and cystacanths can be subject to cellular responses of the host organism, particularly ‘hemocyte encapsulation’ (Rotheram & Crompton, Reference Rotheram and Crompton1972; etc.), against which they must defend themselves. The components of such defence may be an envelope surrounding cystacanths and a thick layer of glycocalyx on the surface of their tegument (Lackie & Rotheram, Reference Lackie and Rotheram1972; Lackie & Lackie, Reference Lackie and Lackie1979; Zhao & Wang, Reference Zhao and Wang1992; Taraschewski, Reference Taraschewski2000; Nikishin & Skorobrekhova, Reference Nikishin and Skorobrekhova2015; Nikishin, Reference Nikishin2018; etc.). This structure has been referred to in the literature by several terms, including the neutral term ‘envelope’, as well as cyst (Petrochenko, 1956, 1958; Emde et al., Reference Emde, Rueckert, Kochmann, Knopf, Sures and Klimpel2014; Davydenko & Nikishin, Reference Davydenko and Nikishin2024; etc.) and capsule (Crompton, Reference Crompton1964, Reference Crompton1967; Dezfuli et al., Reference Dezfuli, Bosi and Rossi1992; Nikishin, Reference Nikishin1992; etc.); this inconsistency in terminology may result in confusion (Nikishin, Reference Nikishin2022). The use of different terms for defining the same structure arises from the lack of clear and experimentally confirmed criteria for its composition and formation process. The neutral term ‘envelope’ does not meet the criteria because it does not indicate the source of its origin. The more accurate term ‘capsule’ usually refers to an envelope formed around the parasite only by host cells, (e.g., macrophages, fibroblasts, etc.,), while the term ‘cyst’ in parasitology is commonly referred to as a structure formed by the parasite itself (Crompton, Reference Crompton1964, Reference Crompton1967; Walker & Wittrock, Reference Walker and Wittrock1992; Quist et al., Reference Quist, Bower and Hubert2007; Nikishin & Skorobrekhova, Reference Nikishin and Skorobrekhova2020; etc.). Differences between capsules and cysts can be exemplified by metacercariae of some trematodes. For instance, in metacercariae of Bolbogonotylus corkumi, ‘…metacercarial cyst consisted of an outer host capsule and an inner parasite cyst’ (Walker & Wittrock, Reference Walker and Wittrock1992). Another example is the trematode Stephanostomum sp., in which ‘…each metacercaria is surrounded by a cyst of parasite origin and by a host reaction of varying intensity’ (Olson & Pierce, Reference Olson and Pierce1991). It is also known that the encapsulation of encysted metacercariae of the genus Uvulifer causes black spot disease in fish: ‘infection occurs when a fish encounters cercariae that penetrate its integument and develop into encysted metacercariae, …. The characteristic black spot is the result of the fish forming a capsule of connective tissue containing melanophores around the encysted metacercaria’ (Quist et al., Reference Quist, Bower and Hubert2007).
Typical encapsulation by host cells is characteristic of acanthocephalans infecting paratenic hosts (Amin et al., Reference Amin, Heckmann, Inchausti and Vasquez1996; Choi et al., Reference Choi, Lee, Go, Park, Chai and Seo2010; etc.). The structure of typical cell capsules and the process of their formation have been explored using the example of Corynosoma strumosum encapsulated in paratenic hosts, which are fish of various species (Nikishin & Skorobrekhova, Reference Nikishin and Skorobrekhova2020). Cases of typical encapsulation of acanthellae in intermediate hosts have also been described in the literature, although analysis of these cases is beyond the scope of our present article. However, it is noteworthy that several authors have highlighted that some cases of such encapsulation lead to the death of the parasite (Skorobrekhova & Nikishin, Reference Skorobrekhova and Nikishin2019). These cases should not be confused with the primary reaction of a specific intermediate host to infection: hemocytes surrounding acanthors that have penetrated from the host’s intestine into its body cavity. Such cases were previously reviewed in detail in a monographic study by Taraschewski (Reference Taraschewski2000). We also note that this reaction stops within a few days, allowing acanthocephalans to continue their development.
In this paper, we consider the composition and origin of the structure for which the term ‘cyst’ has been used, thus emphasizing not only its location around acanthella/cystacanth, but also its formation by the parasite itself. As an additional argument for this term, we note its wide use in various fields of parasitology. Besides the above-mentioned cases of trematode metacercariae, it is relevant to also mention cysticercoids of cestodes (metacestodes) from the suborder Hymenolepidata, which are enclosed in one or two cysts (Chervy, Reference Chervy2002; etc.). Thus, if the outer, non-cellular cyst is present, it may be externally similar to the cyst of acanthellae and/or cystacanths (Nikishin, Reference Nikishin2011). Finally, the term ‘cystacanth’ itself is used by many authors (Taraschewski, Reference Taraschewski2000; Emde et al., 2014; etc.) to refer to an organism surrounded by a cyst.
Earlier, we attempted to resolve the above-mentioned terminological confusion in the literature between the cellular capsule formed by the host around the parasite and the non-cellular cyst formed by the acanthocephalan itself. We also emphasized the fallacy of using a single term, e.g., ‘capsule’, to refer to both structures (Nikishin, Reference Nikishin1999, Reference Nikishin2022). Furthermore, an assumption was made that the cyst may be an envelope obligatorily surrounding any formed cystacanths of the classes Palaeacanthocephala and Archiacanthocephala, whereas cystacanths of the class Eoacanthocephala, e.g., Neoechinorhynchus rutili, lack such an envelope (Dezfuli, Reference Dezfuli1996). The latter fact has recently been confirmed by examination of the acanthella of N. beringianus (Kusenko & Nikishin, Reference Kusenko and Nikishin2024). In the present article, we have summarized information about the structure and mechanisms of cyst formation in cystacanths of acanthocephalans that are members of the class Palaeacanthocephala.
Cyst formation involving microvilli
The almost complete process of cyst formation was first studied experimentally using Filicollis anatis that develop in isopods, Asellus tschaunensis (Nikishin, Reference Nikishin1992). Quite predictably, the source of the material forming most of the cyst turned out to be the tegument and its derivatives, i.e., microvilli. It should be emphasized that short microvilli on the tegument surface in this species emerged at the early acanthella stage and, although these grew in length with the development of acanthella, their length rarely reached 0.3–0.8 μm in mature acanthellae.
Certain components of the cyst’s lamellar layer were first observed among hemocytes around the early acanthella on day 20 of the experiment (Figure 1a). Subsequently, the number of these components increased to 10–20; these became more densely arranged, with the components of the outer half of the layer most densely organized. By day 50 of the experiment, the total thickness of the layer could be greater than 1 μm. The origin of the lamellar layer remains unclear. Considering that the first components appear among hemocytes during the early acanthella stage and later become more densely arranged in the outer half of the layer, we cannot rule out the probability that the host organism forms the lamellar layer or that it has a parasitic origin. At the late acanthella or cystacanth stage, the lamellar layer may be modified or even completely vanish, therefore it may not always be observed (Nikishin, Reference Nikishin1992). The cyst vesicular layer in this species is formed of fragments of microvilli or apical vesicular expansions separated from microvilli on the tegument surface, which is the classic micro-apocrine secretion (Figure 1b, c). Throughout the acanthella formation period, the material of the amorphous layer accumulates in ampoule-shaped extensions of invaginates of the tegument’s outer plasma membrane (Figure 1c); at the late acanthella stage (day 60 of the experiment), this is released outside the tegument, where it is deposited on microvilli (Figure 1d). This process is similar, to some extent, to the merocrine secretion. Subsequently, this material and the microvilli are detached from the tegument and form the inner layer of the cyst (Figure 1e) (Nikishin, Reference Nikishin1992, Reference Nikishin2004). Thus, fragments of microvilli become part of both the vesicular and amorphous layers of the cyst. After the microvilli are detached from the tegument and the cyst is formed, a thick layer of glycocalyx is deposited on the surface of the acanthocephalan that has already reached the cystacanth stage.
Cyst formation around acanthella of Filicollis anatis in an experimentally infected isopod, Asellus tschaunensis (electron microscopy; according to Nikishin, Reference Nikishin1992, Reference Nikishin2004). (a) – Acanthella aged 20 days; numerous short microvilli are visible on acanthella’s tegument surface with vesicles separated from their tips, forming the cyst’s vesicular layer; in the superficial part of the vesicular layer, components of the cyst’s lamellar layer are visible. (b) – Acanthella aged 50 days; the number of microvilli and cyst components has increased; the lamellar layer is completely formed. (c) – Acanthella aged 50 days; electron-dense material (indicated by arrows) – the basis of the cyst’s future amorphous layer – is accumulated in the invaginates of the outer tegument membrane, forming a striated layer. (d) – Acanthella aged 60 days; electron-dense material has been excreted onto the tegument surface and, together with fragments of microvilli, forms the amorphous layer of cysts. (e) – Acanthella aged 60 days; a fully formed cyst at a higher magnification. Letter designations: al – amorphous layer of cyst; ll – lamellar layer of cyst; m – microvilli; sl – striated layer of tegument; t – tegument of acanthellae; vl – vesicular layer of cyst. Scale bar: a–d, 1 μm; e, 0.5 μm.
We observed the tri-layered structure of the cyst (referred to as a ‘capsule’ in the author’s edition) in a late acanthella of Arhythmorhynchus petrochenkoi from isopods, Asellus tschaunensis (Nikishin, Reference Nikishin1985), and also in Echinorhynchus gadi dissected from naturally infected amphipods, Eogammarus tschiushovi (unpublished data). In E. gadi, however, the lamellar layer was only found in certain regions of the cyst (Figure 2a), while it was absent from other regions (Figure 2b). Since this layer was recorded only by electron microscopic studies, it was impossible to determine the extent of the cyst’s regions that contained or lacked it.
Cyst surrounding a cystacanth of Echinorhynchus gadi from a naturally infected amphipod, Eogammarus tschiushovi (electron microscopy). (a) – Region of cyst consisting of three layers: lamellar, vesicular, and amorphous. (b) – Bi-layered region of the same cyst. Letter designations: al – amorphous layer of cyst; ll – lamellar layer of cyst; vl – vesicular layer of cyst. Scale bar: a, 1 μm; b, 2 μm.
Cystacanths of the acanthocephalan Corynosoma strumosum, which naturally infects amphipods Spinulogammarus ochotensis, were enclosed in a bi-layered cyst. Its amorphous (inner) layer was 0.15–0.18 μm thick and consisted of a fine-grained, electron-dense material permeated with fragments of degenerated microvilli. The outer vesicular layer, 1.5–1.8 μm in thickness, included vesicles, as well as fragments of microvilli and delicate fibres oriented parallel to the cyst’s surface. The number of such fibres markedly increased in the near-surface region of the layer. Morphologically, these fibres were identical to the fibres of the lamellar layer in the tri-layered cysts. Cystacanths of this species lacked the thick layer of glycocalyx on the tegument surface. However, an uneven layer of loose material of moderate electron density, similar to the material of the ‘normal’ glycocalyx, adjoined the inner layer of the cyst (Figure 3) (Skorobrekhova & Nikishin, Reference Skorobrekhova and Nikishin2019). It is important to emphasize that numerous fragments of microvilli were observed in the structure of both cyst layers in all the above-mentioned acanthocephalan species.
Cyst surrounding a cystacanth of acanthocephalan Corynosoma strumosum in a naturally infected amphipod, Spinulogammarus ochotensis (electron microscopy). Letter designations: al – amorphous layer of cyst; t – tegument; vl – vesicular layer of cyst. Scale bar: 2 μm.
No information about the cyst was provided in the description of the acanthocephalan Polymorphus minutus, which had developed in experimentally infected amphipods, Gammarus pulex. However, the authors noted a ‘membranous layer’ on the surface of acanthellae (Butterworth, Reference Butterworth1969), which was in fact a layer of microvilli, as evidenced by the author’s photograph 1C. However, microvilli were no longer present on the surface of a 115-day-old acanthella and a cystacanth (see photographs 4A, D) (Butterworth, Reference Butterworth1969). Other authors described a bi-layered cyst (which they referred to as an ‘envelope’) of 1.2–1.3 μm in thickness on the surface of a cystacanth of the same species from Echinogammarus stammeri (Dezfuli & Giari, Reference Dezfuli and Giari1999). The two layers in the cyst structure were described as a thicker vesicular layer, and a thin amorphous, electron-dense layer. Furthermore, in Figure 8 of their publication, a portion of the lamellar layer is noticeable on the outer surface of the cyst. The authors concluded that the cyst in general consisted of ‘granular and filamentous materials’, although they did not explain the origin of these ‘filaments’. In cystacanths of a similar species, Polymorphus strumosoides, which is also surrounded by a bi-layered cyst (referred to as a ‘capsule’ by the author), lamellar bodies and fragments of membranes likely fragments of microvilli – appeared as short tubules in the vesicular layer, along with small vesicles (Nikishin, Reference Nikishin1986).
The cyst surrounding acanthocephalans of the class Palaeacanthocephala has a structure similar to that described from members of the class Archiacanthocephala. On the surface of 12-day-old acanthellae of the acanthocephalan Moniliformis moniliformis that were injected as acanthors into the body cavity of cockroaches, Periplaneta americana, there was a layer of microvilli of up to 2 μm in length whose tips were expanded in the form of vesicles (Rotheram & Crompton, Reference Rotheram and Crompton1972). A vesicular cyst (referred to as ‘capsule’ by the authors) was described somewhat earlier from acanthellae of this species, while in cystacanths, the layer of vesicular material was already delimited by a narrow layer of amorphous, electron-dense material (Mercer & Nicholas, Reference Mercer and Nicholas1967).
Acanthellae of the acanthocephalan Centrorhynchus milvus from woodlice, Porcellionides pruinosus, developed microvilli approximately 1 to 1.5 μm in length on the tegument surface (Marchand & Grita-Timoulali, Reference Marchand and Grita-Timoulali1992). According to the authors, at the late acanthella stage, microvilli are detached from the tegument, fragmented, and form a primordium of the cyst wall that eventually consists of three layers: an inner, granular layer adjacent to the tegument surface (judging by the description, this layer is actually a glycocalyx typical of cystacanths), a middle, amorphous, electron-dense layer, and an outer, vesicular layer.
Noteworthy results were obtained in a study of acanthocephalans Pomphorhynchus laevis from naturally infected Echinogammarus stammeri. Numerous long microvilli were found on the surfaces of acanthellae and cystacanths of this species (Dezfuli et al., Reference Dezfuli, Bosi and Rossi1992). These densely arranged microvilli initially form a continuous layer, then increase in number and length (1.6–8 μm) during the development from acanthella into cystacanth. Small vesicles associated with microvilli were more numerous at the base of the layer. The authors assumed that these microvilli detached from the tegument and formed the cyst (referred to as ‘capsule’ or ‘membrane shell’ by the authors), but no images of such cyst are provided in their paper. It remains unclear whether all microvilli are separated from the tegument, because in the author’s photographs 13 and 14 of cystacanths, many microvilli retain connection with the tegument. Furthermore, photographs 8a and 11 show that the microvilli with vesicles connected to them are much shorter than the neighbouring ones, and it seems that at least some of the vesicles represent their expanded tips, which is a phenomenon suggesting the micro-apocrine secretion.
In conclusion of the performed analysis, it should be emphasized that in the above examples, the cyst formation occurs with the obligatory involvement of microvilli. In the vast majority of cases, either materials secreted by microvilli or fragments of microvilli are involved in the formation of the cyst’s vesicular layer. The material of the inner, electron-dense amorphous layer is released from invaginates of the outer membrane of the tegument at the late acanthella stage, as suggested, within a relatively short time. The source and mechanism of formation of the cyst’s lamellar layer remain largely unclear. In the case of Pomphorhynchus laevis cystacanths, the cyst is formed by whole microvilli, according to Dezfuli et al. (Reference Dezfuli, Bosi and Rossi1992). No layers resembling the vesicular and electron-dense amorphous ones were found.
Cyst formation without the involvement of microvilli
Somewhat different results were obtained in a study of developing acanthocephalans of the genus Acanthocephalus. A single-layered, 1.1–1.3 μm thick cyst (referred to as ‘envelope’ by the authors), composed of moderately dense granular and filamentous material was found in cystacanths of Acanthocephalus clavula from naturally infected amphipods, Echinogammarus stammeri (Dezfuli et al., Reference Dezfuli, Bosi and Ross1994). However, the cystacanths of acanthocephalans Acanthocephalus anguillae that parasitized isopods Asellus aquaticus were already surrounded by a bi-layered, 1.1–1.6 μm thick cyst (or ‘envelope’) with its structure consisting of an outer layer composed of a ‘granular and filamentous’ material and an inner electron-dense ‘filamentous’ material (Dezfuli, Reference Dezfuli2000). In both species, neither microvilli nor their fragments were found in the cyst structure. When studying the histogenesis in acanthocephalans Acanthocephalus tenuirostris from an experimentally infected intermediate host (isopod, Asellus hilgendorfi) we found that the cystacanth’s cyst in this species is bi-layered, as is the case in many other acanthocephalans. Moreover, we found that acanthellae of this species lack microvilli on the tegument surface throughout the period of their residence in the intermediate host (Davydenko & Nikishin, Reference Davydenko and Nikishin2024). Accordingly, the mechanism of cyst formation differs from that described above. Even in early acanthella, large vesicles of up to 2.2 μm in diameter and containing amorphous material of moderate electron density and few electron-dense granules, are formed in its tegument with the involvement of a granular endoplasmic reticulum (Figures 4a–d). These vesicles move to the striated layer of the tegument, merge with invaginates of the outer cytoplasmic membrane, and release their contents into the invaginates, which are then excreted onto the tegument surface where they become incorporated in the vesicular (outer) layer of the future cyst (Figure 4c). The material of the amorphous (inner) layer of the cyst is released later (at the mid and late acanthella stages) through invaginates of the tegument’s outer plasma membrane (Figure 4d, e). The encystment process is completed at the cystacanth stage, when a thick glycocalyx layer forms on the parasite’s tegument surface (Figure 4e).
Formation of cysts around acanthella of Acanthocephalus tenuirostris in experimentally infected isopods, Asellus hilgendorfi (electron microscopy). (a, b) – Early acanthella; invaginates of the tegument’s outer membrane forming the striated layer are expanded; material released through their orifices (arrows) forms a future cyst; large vesicles with heterogeneous contents are observed in the tegument. (c) – Mid acanthella; cyst consisting of two layers – vesicular and amorphous; large vesicles migrate to the tegument surface, come into contact with invaginates of the outer membrane, and release their contents through them. (d) – Late acanthella; the number of vesicles decreases, and invaginates of the tegument’s outer membrane are narrowed. (e) – Cystacanth; a thick layer of glycocalyx forms on the tegument surface; the cyst is completely formed, and vesicles are absent (according to Davydenko & Nikishin, Reference Davydenko and Nikishin2023). Letter designations: al – amorphous layer of cyst; c – cyst; g – glycocalyx; sl – striated layer of tegument; t – tegument; v –vesicles; vl – vesicular layer of cyst. Scale bar: a–d, 2 μm; e, 1 μm.
Conclusion
In all the cases studied, cystacanths of the acanthocephalan classes Palaeacanthocephala and Archiacanthocephala were surrounded by a cyst (envelope) formed by the parasite itself during development in an intermediate host.
Although the cyst structure has an externally uniform appearance, the data considered in this review suggests the existence of at least three mechanisms of cyst formation in developing acanthellae of acanthocephalans. Two of these are characterized by the presence of microvilli, whose length and density vary between species. In most of the acanthellae studied, microvilli are short or medium in length; their apical parts, expanded in the form of small vesicles, are detached from the proximal parts and become incorporated in the cyst’s vesicular layer (micro-apocrine secretion). Simultaneously, dense material, accumulated in invaginates of the outer plasma membrane of the tegument, is released onto the parasite’s surface at the late acanthella stage. Together with microvilli detached from the tegument, this forms the inner layer of the cyst. The formation of the surface, lamellar layer of the cyst remains unclear: this layer is presumably modified or degraded at the final stage of cyst formation. The above-described process illustrates the first mechanism of cyst formation, characteristic of cystacanths of the genera Filicollis, Polymorphus, and Arhythmorhynchus.
Acanthellae of the acanthocephalan Pomphorhynchus laevis are distinguished by very long and densely arranged microvilli (Dezfuli et al., Reference Dezfuli, Bosi and Rossi1992). According to the authors, these microvilli are detached from the tegument at the cystacanth stage and form a cyst. However, there is no data on any secretion products found in its composition, including material of the amorphous layer. We attribute such features to the second form of organization of the microvilli border. Nevertheless, the mechanism of cyst formation requires further clarification.
The third form of cyst formation is characteristic of acanthocephalans of the genus Acanthocephalus, and is distinguished by the lack of microvilli on the tegument surface of acanthellae throughout the period of their development in the intermediate host. In these acanthellae, the material forming both layers of the cyst (vesicular and amorphous) is sequentially released immediately from invaginates of the tegument’s outer membrane.
As the authors suggest, the above-described variety of patterns of cyst organization and formation around acanthocephalan acanthellae is far from complete, and thus requires further primarily experimental study of other acanthocephalan species.
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Competing interests
The authors declare no competing interests.
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