Nemerteans as hosts for symbionts: a review

Nemerteans utilize other animals as hosts in a variety of well‐studied, symbiotic relationships, but relatively little is known concerning nemerteans as hosts for other organisms. Only three haplosporidians, one microsporidian, two gregarines, three ciliates, five mesozoans, one mite and one copepod have been identified to species from nemerteans, whereas 34 nemertean species (one Archinemertea, 17 Heteronemertea, 15 Hoplonemertea, one Bdellonemertea) have been recorded as hosts. The majority of recorded nemertean symbionts are endoparasitic protozoans and orthonectids. Among the Haplosporidia, there are three species of Haplosporidium. The hyperparasite, Nosemoides vivieri, from an unidentified gregarine, is the only known microsporidian associated with a nemertean. Within the phylum Apicomplexa, besides the two species of gregarines from three species of nemerteans, there are 11 other species of nemerteans with unidentified gregarines. One species of nemertean has been found with an unidentified coccidian parasite. ‘Sporozoan‐like’ parasites, however, are known from three other species of nemerteans, two of which are pelagic polystiliferans. Two endosymbiotic and one ectosymbiotic species of ciliates have been identified from three species of nemerteans. At least five species of mesozoans (Orthonectida) infect nine species of nemerteans. Unidentified cestode, trematode and nematode juveniles, using nemerteans as intermediate hosts, have been recorded rarely and have not been linked to any life cycles. An ectosymbiotic mite and a copepod from two species of nemerteans are likely incidental associates. The nature of the host–symbiont relationships has been rarely reported. Castration of hosts by haplosporidians and orthonectids, and tissue damage by an unidentified coccidian and some gregarines, are known to occur. The suitability of nemerteans as hosts for parasites, hypersymbioses and approaches for future studies of parasite prevalence and host–parasite relationships are discussed.


Introduction
Nemerteans are well-known symbionts on other organisms. For example, carcinonemertids are semiparasitic on brachyuran crabs and other crustaceans (Kuris 1993); bdellonemerteans, such as Malacobdella spp. live in the mantle chambers of bivalve molluscs (Gibson 1967(Gibson , 1968Kozloff 1991), and several worms associate with ascidians (Gibson 1974;Egan and Anderson 1979;Egan 1984) and sea anemones (Gibson 1986). On the other hand, we have scant knowledge of symbionts that use nemerteans as hosts. Gibson (1972) and Varndell (1980) provided brief summaries of known parasites and Lauckner (1980b) contributed an expanded review with illustrations of several species. Such symbionts are predominantly internal parasites, usually discovered in the process of examining histological sections used for specific taxonomic identifications. Major taxa to which known symbionts belong are the phyla Apicomplexa (coccidians and gregarines), Microsporida (microsporidians and haplosporidians), Ciliophora, Orthonectida, Platyhelminthes (cestodes and trematodes) and Nematoda. Nemerteans are either definitive or intermediate hosts in the life cycles of these taxa. The arthropods are also represented by a single species of ectosymbiotic arachnid (mite) and an ectosymbiotic crustacean (copepod). Very few experimental studies have been done on most of these relationships (Varndell 1980(Varndell , 1981aLanglet and Bierne 1989;Vernet and Fargette 1990;Haloti and Vernet 1994;Vernet and Haloti 1997), and many of the protozoan parasites have yet to be identified to species or higher taxa; most are known only incidentally from histological specimens of hosts.
I review species of nemerteans that harbour symbionts, the suitability of nemerteans as hosts, the roles of nemerteans as definitive or intermediate hosts and the effects of symbionts on their hosts. Future studies are suggested to develop a better understanding of these relationships. Table I lists known relationships of the Nemertea with the Microsporida through the Arthropoda. While most nemertean hosts have been identified, the identities of many symbionts have not been determined. Only three haplosporidians, one microsporidian, two gregarines, three ciliates, five orthonectids, one arachnid and one copepod have been specifically identified, whereas 33 nemertean hosts have been recorded (one Archinemertea, 17 Heteronemertea, 15 Hoplonemertea, one Bdellonemertea). Twelve of the hoplonemerteans belong to the suborder Monostilifera and two to the pelagic Polystilifera (Table II). Parasitic protozoans, previously referred to the phylum Sporozoa, are now within the Apicomplexa and are the most often recorded nemertean parasites. Members of this phylum are characterized by the presence of an apical complex, with complex life cycles alternating between sexual and asexual phases, some requiring intermediate hosts; constituents of most groups are intracellular while some (e.g. gregarines) are both intracellular and extracellular. Members of the class Sporozoea, such as malarial parasites, coccidians and gregarines, are probably the most well-known.

General survey of relationships
Systematic discussion of relationships (Table I)

Haplosporidia
Haplosporidians are intracellular protozoan parasites found in a variety of fresh water and marine invertebrates; some are hyperparasitic in trematodes and nematodes that use marine molluscs and decapod crustaceans as hosts (Lee et al. 2000). Their life cycles are poorly understood and their identification relies chiefly on electron microscopical analysis of the morphological characteristics of mature spores. Three species of haplosporidians have been described from nemerteans, all belonging to the genus Haplosporidium.

Microsporida
Microsporidians are all intracellular protozoan parasites of hosts that include probably all invertebrate phyla and the five classes of vertebrates (Lee et al. 2000). Most life cycles are unknown and, like the haplosporidians, identification relies primarily on the morphology of mature spores, in this phylum, especially their elaborate internal structure. The only microsporidian associate of nemerteans is the hyperparasitic Nosemoides vivieri whose host is an unidentified gregarine parasitic in an unidentified nemertean (Vinckier et al. , 1971Vinckier, 1995) (Table III).

Apicomplexa (formerly Sporozoa)
The gregarines (subclass Gregarinasina) are primarily parasites of invertebrates, but also occur in lower vertebrates. They are generally conspicuous in sections of the digestive tract and body cavities due to their relatively large size. They have simple or complex life cycles. Gregarines are the most common apicomplexans to occur in nemerteans and have been found in 14 species of nemerteans (seven Heteronemertea, seven Hoplonemertea). All nemertean hosts, except the terrestrial Antiponemertes pantini and the freshwater Potamonemertes percivali, have been marine. Only two species of gregarines, however, have been specifically identified. Vinckier (1972Vinckier ( , 1973 Gallien, 1936 Lineus longissimus (Gunnerus, 1770) Gallien (1936); Stock (1985, new genus) Major taxa unknown Internal parasite Cephalurichus antarcticus Gibson, 1985Gibson (1985 Internal parasite Paraborlasia fueguina Serna de Esteban & Moretto, 1968Gibson (1985 Internal parasite Lineus bioculatus Sundberg & Gibson 1995 Sundberg and Gibson (1995) 1 Gibson's (1995) world list of species is a major reference to nemertean taxa. 2 Lee et al. (2000) is reference for this phylum and other major protozoan taxa. 3 Hyperparasite of unidentified gregarine. 4 In same host with orthonectid Intoshia linei. 5 In same host with gregarine Lecudina linei. 6 The polychaete Spio martinensis is also host for this species. 7 Kozloff (1992) could not assign this parasite with certainty to this genus.  dissertation). Arnoult and Vernet (2000) also reported finding this gregarine in the same host. In his study on the nutrition of Lineus ruber, Jennings (1960) noted that the intestines of ,75% of the worms examined were infected with the acephaline eugregarine Urospora nemertes (trophozoites in the lumen and intracellular stages in columnar cells). Distinct damage was caused to the epithelial cells of the hosts. Baseodiscus delineatus is also a host for U. nemertes.
Gregarines have been observed in 11 other nemerteans (seven hoplonemerteans and four heteronemerteans), but none of these parasites was identified. In three cases, authors were tentative in their characterization of the parasites as gregarines. Gibson (1979) found what appeared to be gregarines in the blood vessels and rhynchocoel fluid of Baseodiscus hemprichii, and noted that Punnett (1900) had misidentified them as nemertean ova. Cysts of an unidentified gregarine were found hyperparasitic in tissues of the egg predator Ovicides julieae, symbiotic in xanthid crabs from Australia (Shields 2001) (Table III). Roe (1976) provided prevalence data on what she considered to be (P. Roe, pers. comm. 2004) an unidentified gregarine from the hoplonemertean Paranemertes peregrina. Examination of feces for gregarine cysts in three populations of worms from Washington revealed a range in prevalence from 6.3 to 26.0%; examination of gut tissue from 17 worms showed 15 to be infected (88.2%).
Most coccidians (subclass Coccidiasina) are parasites of vertebrates. The first and only coccidian reported from a nemertean was found in the gut of two of seven specimens of Quasilineus lucidoculata examined by . Canning and Gibson (1990) subsequently described and illustrated the stages in the life cycle as revealed in histological sections of the worm. Because oocysts were unsporulated, it was not possible to give a generic diagnosis of this parasite. Evidence of widespread epithelial sloughing due to development of the merozoites and sexual stages was documented.
Sporozoan-like, unidentified parasites were reported by Brinkmann (1917) from the tissues of two species of pelagic polystiliferans, Nectonemertes primitiva and Parabalaenanemertes fusca. Given the relative frequency of gregarines in the tissues of other species of nemerteans, it is likely that these were also gregarines. Recent taxonomic studies on collections of pelagic nemerteans, however, have failed to reveal infections with parasitic protozoans (Norenburg and Roe 1998a, b;Roe and Norenburg 2001;P. Roe pers. comm. 2004).

Ciliophora
Only three species of ciliates are known from the Nemertea; two of these are associated with Malacobdella grossa. The stomatous Thigmophrya annella lives within the gut of this worm as does the astomatous Orchitophrya malacobdellae. Fenchel (1965) speculated that T. annella might have evolved from a species of Thigmophrya that lived in the mantle chamber of bivalves known to harbour M. grossa. Jennings (1968) found O. malacobdellae in four of 175 worms (2.3%) examined. Duboscq (1925) described a new suctorian, Ophryodendron prenanti, from the integument of the archinemertean Cephalothrix linearis. The nature of these symbioses is unknown, but given the fact that these ciliates have not penetrated the body cavity of the host, and given their symbiotic nature in other host taxa, it seems likely that these are relatively benign associations.

Orthonectida
Orthonectids are mesozoans that parasitize the tissues and body cavities of marine invertebrates (e.g. turbellarians, nemerteans, polychaetes, bivalve mollusks, ophiuroids, etc.). The microscopic, ciliated, dioecious adults reproduce outside of their host and ciliated larvae infect new hosts where they become amoeboid, syncytial plasmodia, reproducing asexually. Kozloff (1992Kozloff ( , 1994, however, believes that the ''plasmodium'' is not a part of this life cycle, but rather hypertrophied, infected, host cells. The life cycles of the orthonectids are poorly known as are those of the other mesozoan phylum, Rhombozoa, parasitic in the excretory organs of Cephalopoda. Infections with orthonectids may cause host sterilization (Stunkard 1982). All of the species from nemerteans apparently belong to the family Rhopaluridae (Stunkard 1982), but there are still uncertainties concerning family designations (Kozloff 1992). Intoshia linei lives in the connective tissue and muscle layers of Lineus ruber, L. viridis and Ramphogordius sanguineus. Rhopalura sanguinea similarly infects R. sanguineus. These orthonectids may coexist with gregarines in the same hosts (Table I). Kozloff (1992) expressed difficulty in confirming the generic characteristics of Rhopalura intoshi and R. pelseneeri as well as R. pelseneeri var. vermiculicola. The only apparent record of orthonectids from nemerteans in North America is a species of Rhopalura from a single specimen of Amphiporus ochraceus collected by Meinkoth (1956) near Woods Hole, Massachusetts. These parasites were found inside the worm and swimming in the water of a whole-mount preparation. Additional worms (n5171) were without infections. Meinkoth (1956) noted that the parasites were similar to R. metchnikovi or Intoshia linei.
Intoshia linei is probably the best-studied species of orthonectid parasitizing nemerteans. Anatomical descriptions of adults based on light and scanning electron (SEM) microscopy were done by Kozloff (1992) and Haloti and Vernet (1993). Grafting experiments with parasitized tissues from Lineus ruber and L. viridis have been used to investigate the nemerteans' immune responses of these hosts to I. linei (Langlet and Bierne 1989;Vernet and Fargette 1990;Haloti et al. 1992;Haloti and Vernet 1994). Surprisingly, successful grafts from infected L. ruber into nonparasitized hosts resulted in a decrease in the parasites during the first 2 months, but was followed by a rapid increase in the parasite load during the next 2 months. This indicated that the immune response to the orthonectids occurred but was not strong enough to eliminate the parasites (Langlet and Bierne 1989).

Platyhelminthes
From a fragmented specimen of Cerebratulus lacteus, collected at Beaufort, North Carolina, Hunter (1950) recovered 120 unencysted, plerocercoid larvae (order Tetraphyllidea). Characteristics of the scolices suggested that these cestodes belonged to the genus Echeneibothrium Van Beneden, 1850, species of which are exclusive intestinal parasites of their definitive hosts, elasmobranch fishes. As plerocercoid larvae are usually found in the tissues of teleost, cephalopod or crustacean intermediate hosts, it remains to be determined how nemerteans might be involved in tetraphyllidean life cycles (McDermott 2001).
There are a few records of trematode metacercariae occurring in nemertean tissue, but their specific identity has not been determined (McIntosh 1873(McIntosh /1874Gibson 1972).

Nematoda
A single specimen of the terrestrial Geonemertes philippinensis harboured large numbers of unidentified, coiled, encapsulated nematodes in its muscles, parenchyma and intestinal epithelium (Gibson and Moore 1998). Worms were 15-16 mm in diameter and some were 180-220 mm long. These larval worms were presumably employing G. philippinensis as an intermediate host in an unknown life cycle.

Arthropoda
Two species of arthropod ectosymbionts have been recorded from nemerteans. Laubier (1960) described a new species of mite, Parhalixodes travei, from a larva and adult male collected from the body of Cerebratulus hepaticus. Gallien (1936) described the only known crustacean from a nemertean, the copepod Pseudanthessius nemertophilus, living on the body of Lineus longissimus. Stock (1985) later created the new genus, Nemerthessius, for this species. Nothing is known concerning relationships of these two symbionts with their nemertean hosts, if indeed they are any more than incidental occurrences of free-living organisms.

Major taxa unknown
At least three encysted, unknown, microscopic parasites have been reported in the tissues of heteronemerteans from the southern hemisphere. In all cases, nondescript cysts could not be identified with certainty.

General discussion and conclusions
Several questions arise concerning the meagre literature on symbionts of nemerteans: (1) what are the probabilities of finding parasites in nemerteans compared to other invertebrates? (2) How suitable are nemerteans as host for parasites? (3) What is the status of nemerteans as intermediate hosts in parasitic life cycles? (4) Can we learn more about the parasites of nemerteans?

Probabilities of finding parasites
The heavy mucus covering the external surfaces and the burrowing habit of many species of nemerteans makes them extremely unlikely hosts for ectosymbionts and, to date, this seems to be the case (Lauckner 1980b). In general, the same may be said for turbellarians as hosts for epibionts (Lauckner 1980a). As far as internal parasites are concerned, a few orthonectid infections have been detected from external signs on the surface of some worms (Langlet and Bierne 1989), but the opaque and dense body of most nemerteans obscures the revelation of most internal parasites. However, parasites certainly gain access to the interior of nemerteans. Thus, the time-consuming examination of histological sections for the purpose of locating protozoan and metazoan endoparasites needs to continue, but this process certainly sets a lower limit on the number of worms that can be examined, compared to other types of hosts for which sectioning is not required. Added to this is the burden of fixation for electron microscopy or molecular analysis, and species identifications become insurmountable. However, the chance revelation of plerocercoid larvae in a fragmenting Cerebratulus lacteus by Hunter (1950) suggests that such observations, along with whole animal dissections, might be rewarded with the discovery of greater prevalences of encysted larval metazoans. Jennings and Gibson (1968) made squashes of fresh Malacobdella grossa for detecting the presence of haplosporidians, and Roe (1976) was able to detect gregarines in Paranemertes peregrina by examining the gut tissue. Isolating nemerteans and examining the feces for gregarine cysts (Roe 1976), or looking for emerging stages of orthonectid parasites (Meinkoth 1956), may prove fruitful. The latter is reminiscent of the well-known parasitological technique isolating large numbers of snails in shell vials for determining the prevalence of infections with digenetic trematodes by examining those releasing cercarial larvae.

Suitability of nemerteans as hosts
Considering the method for finding endoparasites in nemerteans and the limited literature, nemerteans may not be unusual hosts for haplosporidians, microsporidians and gregarines. Three haplosporidians and the hyperparasitic microsporidian Nosemoides vivieri, from an unidentified gregarine, have been found in at least four species of nemerteans. Gregarines have been shown to parasitize at least 14 species of nemerteans (seven heteronemerteans and seven hoplonemerteans) (Table I). Hyperparasitism is most likely overlooked by those examining histological sections. Orthonectids, on the other hand, utilize nemerteans for their early development, releasing males and females to the aquatic environment; these and the infective larvae may be collected and identified through the careful culture and handling of potentially infected hosts. At least five species of orthonectids are known from nine species of nemerteans. One of these orthonectids, Intoshia linei, lacks host specificity, infecting three species of nemerteans (Table I).  (Kem 1985(Kem , 2000 that must be tolerated by potentially invasive larvae. The burrowing nature of many nemerteans presents a further problem for the establishment of infective larval stages. Turbellarians, however, have similar characteristics and are known to harbour a variety of metacercariae linked with described life cycles (Lauckner 1980a). It seems reasonable that nemerteans may eventually be found to harbour these trematodes (Lauckner 1980a). The coracidium larvae of cestodes, however, are generally ingested by arthropod or molluscan intermediate hosts.

Nemerteans as intermediate hosts
On the other hand, infective orthonectid larvae (one host cycle) penetrate, not only nemerteans, but also numerous mucus-producing turbellarians, polychaetes and molluscs that are susceptible to parasite invasion (Kozloff 1992).
For the completion of cestode or trematode life cycles, it is expected that the intermediate host falls prey to definitive or paratenic hosts. The palatability of nemerteans as prey for a variety of animals has been reviewed (McDermott 2001). While there is evidence that nemerteans are consumed by some fishes, birds and other invertebrate predators, there is no substantial evidence that they are important prey, and there is experimental evidence that nemerteans are violently rejected by some predators (Sundberg 1979;McDermott 1984McDermott , 1993Heine et al. 1991). It appears that toxic mucus is the distasteful element.

Host resistance
Little is known concerning innate (cellular) or acquired resistance in nemerteans that might limit the establishment, growth and development of invasive larvae, but nemertean grafting studies have shed some light on resistance to orthonectids (Langlet and Bierne 1989;Haloti and Vernet 1994).

Hypersymbiosis
There are five hypersymbiotic relationships involving nemerteans (Table III), three of which involve the commensal of bivalves, Malacobdella grossa, host for two endosymbiotic ciliates and a haplosporidian. There is little biological information on the nature of these relationships except for the parasite Haplosporidium malacobdellae (Jennings and Gibson 1968).

Future studies
To learn more about the importance of nemerteans as hosts for parasites, it will be necessary to continue searching through histological sections, but we must also employ whole animal dissections to find encysted metazoans. Isolation of worms for fecal examinations and detection of liberated life history stages of parasites may be useful. Experimental exploration of the susceptibility of nemerteans to cercarial invasion or coracidial ingestion could prove enlightening and may be accomplished with relative ease as these parasitic life-history stages are readily manipulated. Experimentation on the life cycles of parasites and susceptibility of nemerteans to infections, however, requires finding populations of nemerteans with high prevalences of infections -and this is a major challenge.