One of the most complex life-cycles among trematodes: a description of Parvatrema margaritense (Ching, 1982) n. comb. (Gymnophallidae) possessing parthenogenetic metacercariae.

This study used light and electron microscopy to provide observations and morphometric details of the life-cycle of the gymnophallids (Trematoda, Digenea), Parvatrema margaritense (Ching, 1982) n. comb., the parthenogenetic metacercariae ('germinal sacs') of which were previously described by Ching (1982) as Cercaria margaritensis. The research was instigated by the discovery, on the Barents Sea coast, of a high prevalence of gymnophallid sporocysts and cercariae in the bivalve Turtonia minuta and an equivalent presence of distinctive gymnophallid metacercariae in the gastropod Margarites helicinus. Experiments and data obtained from naturally infected M. helicinus demonstrated that cercariae released from the bivalves invaded the gastropods to give rise to the metacercariae. Two generations (M1 and M2) of these parthenogenetic metacercariae were formed in the extrapallial cavities of their bivalve hosts and they, in turn, gave rise to a third generation (M3) which was shown to infect marine ducks such as the eider (Somateria mollissima). As only small numbers of cercariae are released from T. minuta, it was concluded that the inclusion of parthenogenetic metacercariae in the life-cycle is particularly significant. It allows each cercaria that infects M. helicinus to give rise to over 2000 invasive metacercariae. Evidence suggests that the parthenogenetic metacercariae are commensal rather that parasitic in the pallial cavities of their hosts. Implications of this for theories of early digenean evolution are discussed.

A study of the mechanisms by which the cercariae of Microphallus primas (Jag, 1909) Stunkard, 1957 penetrate the shore crab, Carcinus maenas (L).

This study established the mechanisms by which Microphallus primas cercariae penetrate the crab Carcinus maenas in which they form metacercarial cysts. Light and electron microscopy were used to investigate cercarial features and to follow the fate of cercariae released in close proximity to crabs. It was shown that cercariae were carried in respiratory currents into crabs' branchial chambers where each enveloped itself in a transparent penetration cyst on the gill lamellae. When cercariae were present the number of respiratory current reversals performed by crabs increased. Using an 'artificial branchial chamber' it was possible to observe how cercariae attached to crab gills during breaks in current flow that preceded each current reversal. Inside the penetration cysts the now tail-less larvae used their stylets to pierce holes through which they levered themselves into underlying haemolymph channels in the gills. Histochemical tests demonstrated that the penetration cysts were products of glands in the cercariae and that penetration of the crabs was achieved by mechanical means. The importance of crab respiratory current reversals to the success of cercarial penetration is discussed as it represents the exploitation by a parasite of a host behavioural response to an unrelated stimulus.

Evolutionary relationships within 'pygmaeus' group microphallids using genetic analysis and scanning electron microscopy.

There are four species of 'pygmaeus' microphallids, namely Microphallus pygmaeus, M. piriformes, M. pseudopygmaeus and M. triangulatus (Trematoda: Microphallidae) which are parasites of marine birds and their sporocysts give rise to transmissible metacercariae inside littoral gastropods (mostly littorines). Universally primed polymerase chain reaction (UP-PCR) showed no apparent pattern between genetic diversity of the metacercariae as estimated by genomic banding profiles and their geographic region or molluscan host species. At the same time UP-PCR product cross-hybridization showed that M. pseudopygmaeus and M. triangulatus are genetically very similar, indicating that these taxa represent one species complex. In contrast, M. pygmaeus and M. piriformes are genetically well separated from each other and also from the pseudopygmaeus-triangulatus complex. Scanning electron microscopy of ventral spines, and analyses of spine angles and the number of teeth per spine, showed that all species differed significantly from one another. It was concluded that M. piriformes represents the original western member of the 'pygmaeus' group. Microphallus pygmaeus probably diverged from M. piriformes as it progressively specialized for sea duck final hosts. Microphallus pseudopygmaeus and M. triangulatus diverged from each other and the piriformes-pygmaeus ancestral line relatively recently. Microphallus pseudopygmaeus specialized for adoption of a wide range of gastropod host species and M. triangulatus developed morpho-functional specialization associated with final host exploitation.

An ultrastructural study of reproduction in the parthenogenetic metacercariae of Cercaria margaritensis Ching, 1982 (Digenea: Gymnophallidae).

The parthenogenetic metacercarial stages of the gymnophallid trematode Cercaria margaritensis are found in the extrapallial cavity of the subtidal prosobranch mollusc Margarites helicinus. The primary metacercariae (M1) produce second-generation metacercariae (M2) which become independent and give rise to M3 metacercariae which are infective to the definitive host, the common eider (Somateria mollissima). This study used transmission electron microscopy to follow the development of M2 inside M1 organisms and M3 inside M2 organisms. The process is similar in both cases with embryos developing from individual cells from the parent body walls. In each case the brood sac was divided into brood chambers by multilaminated cells and both M2 and M3 embryos developed inside embryonic membranes that originated from specialized blastomeres. The tegument of M2 and M3 embryos developed in a similar manner underneath the embryonic membrane. Both the multilaminated cells and the embryonic membranes possessed features that indicated that they are involved in transport of nutrients. It is suggested that the continuous nature of M2 and M3 embryo development may well be similar to that postulated for ancestral digeneans.

Use of an in ovo technique to provide adults of Levinseniella sp. no. 17 that were identified as Levinseniella minuta (Trematoda: Microphallidae).

Metacercarial cysts identified as Levinseniella sp. no. 17 (Deblock, 1980) were found in the mud snail Hydrobia ulvae. These larval trematodes had never been allied to a known species of adult worm. They were chemically excysted and cultured for 120 hr on the chorioallantoic membranes of chicken embryos. The resulting adult worms were observed by light and scanning electron microscopy and details of their anatomy and topography were compiled. Their morphometric measurements were compared with those of adult worms taken from the scientific literature. A very high degree of similarity was discovered between anatomical details of the worms produced in this study and those provided for Levinseniella minuta. It was concluded that Levinseniella sp. no. 17 is the larval stage of L. minuta.

The structure and formation of metacercarial cysts in the trematode family Microphallidae travassos 1920.

This study deals with the formation of the metacercarial cysts of four microphallid trematodes, Maritrema subdolum, M. arenaria, Levinseniella brachysoma and Microphallus claviformis. The first observable cyst was present around Maritrema arenaria 18 h p.i. (post-infection). The other species had not developed a cyst by day 8 p.i. but their cysts were apparent by day 16 p.i. These were bi-layered and that of M. subdolum was thicker than those of L. brachysoma and Microphallus claviformis of the same age. The structure of older cysts varied substantially between the four species. Microphallus claviformis and Maritrema subdolum cysts were fully formed at 30 days p.i. Like those of M. arenaria they were bi-layered, the outer layer (up to 3 microm thick) being electron-dense and the inner one (up to 7 microm thick) being less electron-dense. The cysts of fully formed L. brachysoma metacercariae were much more complex, composed of four layers, one of which was divisible into three sub-layers. It was concluded that the outer cyst layer was the product of secretory granules which were previously identified in cercarial tegument. The inner, thicker layer was derived from several sources. These included small tegument vesicles produced over the entire surface of the metacercariae, larger fragments of tegument released from the anterio-ventral region and material liberated from the metacercarial excretory bladder. This heterogeneous material accumulated in the cyst lumen for some time before becoming polymerized to form the thick inner layer or layers of the metacercarial cysts.

In ovo cultivation of Microphallus primas (Trematoda: Microphallidae) metacercariae to ovigerous adults and the establishment of the life-cycle in the laboratory.

This investigation reports for the first time the establishment of a trematode life-cycle in the laboratory using in ovo cultivation of the adult stage. Microphallus primas metacercarial cysts were removed from Carcinus maenas crabs and chemically excysted. Using a modification of the technique developed by Fried (1962) the released metacercariae were grown in fertile hen's eggs to ovigerous adult flukes. These were removed from the under surface of the chorio-allantoic membrane of chick embryos 5 days post-infection and the fluke eggs recovered were used to infect laboratory-reared Hydrobia ulvae snails. When challenged against parasite-free crabs the cercariae released from the experimentally infected snails gave rise to metacercarial cysts that were again identified as M. primas.

An alternative method for the culture of Diplostomum spathaceum (Trematoda) in chick embryos.

Metacercariae of Diplostomum spathaceum were maintained in the allantoic cavities of chick embryos for 15 days. Some embryos had 0.2 ml chicken serum added to the allantoic cavity each day. Although the level of development varied considerably, worms from embryos with added serum developed hind bodies that were substantially larger than those of parasites maintained without added serum. There was no evidence that any worm ingested blood, and only 1 individual, from the serum-augmented group, became ovigerous.