Polymorphic parasitic larvae cooperate to build swimming colonies luring hosts.

Parasites have evolved a variety of astonishing strategies to survive within their hosts, yet the most challenging event in their personal chronicles is the passage from one host to another. It becomes even more complex when a parasite needs to pass through the external environment. Therefore, the free-living stages of parasites present a wide range of adaptations for transmission. Parasitic flatworms from the group Digenea (flukes) have free-living larvae, cercariae, which are remarkably diverse in structure and behavior.1,2 One of the cercariae transmission strategies is to attain a prey-like appearance for the host.3 This can be done through the formation of a swimming aggregate of several cercariae adjoined together by their tails.4 Through the use of live observations and light, electron, and confocal microscopy, we described such a supposedly prey-mimetic colony comprising cercariae of two distinct morphotypes. They are functionally specialized: larger morphotype (sailors) enable motility, and smaller morphotype (passengers) presumably facilitate infection. The analysis of local read alignments between the two samples reveals that both cercaria types have identical 18S, 28S, and 5.8S rRNA genes. Further phylogenetic analysis of these ribosomal sequences indicates that our specimen belongs to the digenean family Acanthocolpidae, likely genus Pleorchis. This discovery provides a unique example and a novel insight into how morphologically and functionally heterogeneous individuals of the same species cooperate to build colonial organisms for the purpose of infection. This strategy bears resemblance to the cooperating castes of the same species found among insects.5.

Cancer spares no one: First record of neoplasm in parasitic barnacles (Arthropoda: Rhizocephala).

Cancer-like neoplasms are extremely rarely present in arthropods, particularly in crustaceans. Thus, it is assumed that these animals have some efficient cancer-preventing mechanisms. However, several cases of cancer-like neoplasms are described in crustaceans, though only for the Decapoda. We identified a tumor in the parasitic barnacle Peltogaster paguri (Cirripedia: Rhizocephala), and described its histological structure. A spherical cell mass consisting mostly of roundish cells with big translucent nuclei, prominent nucleoli, and sparse chromatin, and of cells with condensed chromosomes, was found in the main trunk of the P. paguri rootlet system. Numerous mitoses were observed in this area. Such tissue organization is utterly uncharacteristic of the Rhizocephala. Based on acquired histological data, we assume that this tumor is a cancer-like neoplasm. This is the first report of a tumor identified in the rhizocephalans, as well as in non-decapod crustaceans as a whole.

Species complexes and life cycles of digenetic trematodes from the family Derogenidae.

The best way to study digenean diversity combines molecular genetic methods, life-cycle studies and elaborate morphological descriptions. This approach has been barely used for one of the most widespread digenean taxa parasitizing fish ­ the superfamily Hemiuroidea. Here, we applied the integrative approach to the hemiuroideans from the family Derogenidae parasitizing fish at the White and Barents Seas. Analysis of 28S, 18S, 5.8S rDNA, ITS2 and cox1 gene sequences from sexually adult worms (maritae) showed genetic heterogeneity for 2 derogenid species known from this area: Derogenes varicus and Progonus muelleri. Thus, 2 pairs of genetic lineages were found: DV1 and DV2, PM1 and PM2, respectively. Data from other regions indicate that 2 more lineages of D. varicus probably exist. Based on previous records from the White and Barents Seas, we hypothesized that the cercariae found in the moonsnails (family Naticidae) belong to the Derogenidae and may help to differentiate these lineages as species. According to our results, Cercaria appendiculata from Cryptonatica affinis matched DV1, similar nameless cercariae from Euspira pallida and Amauropsis islandica matched DV2, and Cercaria octocauda from C. affinis matched PM1. We provide new data on the structure of these cercariae and discuss the life-cycle pattern of the studied digeneans.

Life cycle truncation in Digenea, a case study of Neophasis spp. (Acanthocolpidae).

Truncated life cycles may emerge in digeneans if the second intermediate host is eliminated, and the first intermediate host, the mollusc, takes up its role. To understand the causes of this type of life cycle truncation, we analyzed closely related species of the genus Neophasis (Acanthocolpidae) with three-host and two-host life cycles. The life cycle of Neophasis anarrhichae involves two hosts: wolffishes of the genus Anarhichas as the definitive host and the common whelk Buccinum undatum as the intermediate host. Neophasis oculata, a closely related species with a three-host life cycle, would be a suitable candidate for the comparison, but some previous data on its life cycle seem to be erroneous. In this study, we aimed to redescribe the life cycle of N. oculata and to verify the life cycle of N. anarrhichae using molecular and morphological methods. Putative life cycle stages of these two species from intermediate hosts were linked with adult worms from definitive hosts using ribosomal molecular data: 18S, ITS1, 5.8S-ITS2, 28S. These markers did not differ within the species and were only slightly different between them. Intra- and interspecific variability was also estimated using mitochondrial COI gene. In the constructed phylogeny Neophasis spp. formed a common clade with two other genera of the Acanthocolpidae, Tormopsolus and Pleorchis. We demonstrated that the first intermediate hosts of N. oculata were gastropods Neptunea despecta and B. undatum (Buccinoidea). Shorthorn sculpins Myoxocephalus scorpius were shown to act as the second intermediate and definitive hosts of N. oculata. The previous reconstruction of the two-host life cycle of N. anarrhichae was reaffirmed. We suggest that life cycle truncation in N. anarrhichae was initiated by an acquisition of continuous morphogenesis in the hermaphroditic generation and supported by a strong prey-predator relationship between A. lupus and B. undatum.

New type of xiphidiocercariae (Digenea: Microphalloidea) from South Vietnam.

We found unusual digenean intramolluscan stages, sporocysts and cercariae, in gastropods Sulcospira dautzenbergiana (Morelet) (Caenogastropoda: Pachychilidae) from Southern Vietnam and named them Cercaria cattieni 1. These cercariae have a stylet and thus belong to the Xiphidiata. However, such combination of characters as extremely large body size and I-shaped excretory bladder has not been found before in any other xiphidiocercariae. We obtained COI, ITS1, 5.8S + ITS2, and 28S rDNA sequences for C. cattieni 1. The latter allowed us to specify the phylogenetic position of the discovered cercariae: C. cattieni 1 falls within the superfamily Microphalloidea and is most closely grouped to Pachypsolus irroratus (Rudolphi, 1819) (Pachypsolidae), the sea turtle parasite. Information on the family Pachypsolidae is limited. Judging from the molecular phylogeny, C. cattieni 1 might be the larva of the Pachypsolidae, documented for the first time.

First elucidation of the life cycle in the family Brachycladiidae (Digenea), parasites of marine mammals.

Digeneans of the family Brachycladiidae are cosmopolitan parasites restricted to marine mammals. Their life cycles are unknown. Phylogenetically, Brachycladiidae are closely related to Acanthocolpidae, parasites of marine teleost fishes. Acanthocolpida typically possess three-host life cycles with gastropods of the superfamily Buccinoidea acting as the first intermediate hosts for most species, and either fishes or bivalves acting as the second intermediate hosts. A few species previously identified as Neophasis differ from other Acanthocolpidae in having naticid gastropods as first intermediate hosts, and both fishes and bivalves as second ones. We assumed that this may indicate an incorrect life cycle description and revised previous data on rediae and cercariae of Neophasis spp. from Cryptonatica affinis (Naticidae) and metacercariae from cardiid bivalves at the White Sea using molecular and morphological approaches. Sequence comparison showed that rediae and cercariae from C. affinis resembling some representatives of the genus Neophasis and metacercariae from bivalves resembling Neophasis oculata belong to the brachycladiid species Orthosplanchnus arcticus. Thus, the life cycle of O. arcticus proceeds as follows: seals serve as the definitive host, C. affinis as the first intermediate host and cardiid bivalves as the second. We found one more type of redia and cercaria in C. affinis which, by molecular evidence, also belongs to Brachycladiidae and is closely related to O. arcticus. Here we refer to them as Brachycladiidae gen. sp. 1 WS. We suggest that Brachycladiidae gen. sp. 1 WS may belong to either Orthosplanchnus or Odhneriella, with beluga whales possibly being the definitive host. Morphological features of O. arcticus and Brachycladiidae gen. sp. 1 WS cercariae are summarised and matched with published data on putatively brachycladiid cercariae. We compare and discuss the diversity of life cycle patterns among Brachycladiidae and Acanthocolpidae, and show that they differ not only in the type of definitive host, but also in both intermediate hosts.

Transatlantic discovery of Notocotylus atlanticus (Digenea: Notocotylidae) based on life cycle data.

Digenean parasites feature a series of stages with a distinct appearance, reproduction mode, and lifestyle that together constitute their well-known, complex life cycle. Species descriptions of Digenea have always been based on one of these stages-the marita, or sexually reproducing adult in the final host. However, in some cases, data on the life cycle are essential for the differential diagnosis of closely related species. Here, we present the case of Notocotylus atlanticus, where different stages of its life cycle were discovered for the first time since the species description, and across the Atlantic. We used a material from a naturally infected intertidal marine snail, Ecrobia ventrosa, and several waterfowl species and also carried out infection experiments. For morphological studies, we employed light microscopy, SEM, and CLSM; molecular data obtained include sequences of ITS1 and 28S rRNA gene. We demonstrate that N. atlanticus adult worm morphology is barely sufficient to distinguish it from several other species. Cercariae morphology and identity of the first intermediate hosts provide crucial additional information. According to our preliminary phylogenetic reconstructions, two notocotylid lineages are associated with two major gastropod lineages-the Caenogastropoda and the Heterobranchia. The traditional character to identify notocotylid genera (structure of ventral organs) fails to explain the phylogeny and thus requires reassessment. Further reliable morphological, life cycle and molecular data on other species are likely to reveal more patterns in notocotylid systematics, host specificity, and evolution.

Possible progenesis in Neophasis anarrhichae (Nicoll, 1909) Bray, 1987 in the White Sea.

Neophasis anarrhichae (Nicoll, 1909) Bray, 1987, unlike the majority of acanthocolpid digeneans, has an abbreviated two-host life cycle. The reproduction of rediae, development of cercariae, and their transformation into unencysted metacercariae occur within the only intermediate host, the whelk Buccinum undatum. Normally, the metacercariae develop into sexual adults (maritae) and egg production starts when the infected whelk is eaten by a wolffish Anarhichas lupus. In the White Sea, we have found three cases of infection of B. undatum by progenetic metacercariae of N. anarrichae. These metacercariae had a fully developed and functioning hermaphroditic reproductive system, and eggs were found in their uterus. Most eggs observed in the histological sections were abortive, but some contained embryos at early stages of development. The progenetic metacercariae were similar in their morphometric characteristics to the sexual adults from the wolffish, the main differences being the size of the ovary and eggs. In order to confirm progenesis, and thus a facultative one-host life cycle in N. anarrichae, we need to prove that the eggs from metacercariae are viable.

Morphological framework for attachment and locomotion in several Digenea of the families Microphallidae and Heterophyidae.

Digenea usually use ventral sucker for sustainable attachment within intestine of their definitive vertebrate host. However, if the ventral sucker is absent or poorly developed, the means of attachment are unclear. We investigated attachment and locomotion in such digeneans: three species of the family Microphallidae (Microphallus piriformes, M. pygmaeus, and Levinseniella brachysoma) and two species of the family Heterophyidae (Cryptocotyle concava and C. lingua). Their tegumental spines and musculature were described with use of fluorescent actin staining, confocal microscopy, and scanning electron microscopy. Locomotion of living worms was observed and recorded. Wide serrated tegumental spines probably play the main role in attachment. Their firm contact with the host mucosa may be provided by the action of the ventral concavity-when the entire body or its part acts as a sucker. Dorsoventral muscle bundles act like radial musculature of the sucker generating negative pressure in the ventral concavity. The solid layer of longitudinal muscle fibers on the ventral body surface provides support for the bottom of the ventral concavity. In all microphallids, a U-shaped arrangement of body wall musculature (mostly originating from longitudinal fibers) outlines posterior part of the ventral concavity ridge. In all the studied species, tegumental spines, body wall musculature, and dorsoventral muscle bundles are better developed in the forebody which moves more actively than the hindbody.

Ventral concavity and musculature arrangement in notocotylid maritae (Digenea: Notocotylidae).

Maritae of family Notocotylidae have no ventral sucker, an organ that serves for attachment in many other digeneans. Instead, attachment is accomplished through the ventral concavity which is formed by the whole body. Musculature providing such an attachment was previously described only in part, and specific patterns of body-wall and internal muscle arrangement in notocotylids have never been studied before. In this paper we describe muscle system organization in maritae of three notocotylid species by means of fluorescent actin staining and confocal microscopy. A very special U-shaped pattern of body-wall longitudinal muscle fibres is found to support the ventral concavity. Together with very prominent dorsoventral musculature this pattern must play a key role in actions of ventral concavity. Other details concerning the muscle system are provided, including the musculature of ventral protrusions, oral sucker, walls of digestive tract and reproductive organs. We discuss functionality of the musculature and also make comparisons with cercariae of this family.

Musculature arrangement and locomotion in notocotylid cercariae (Digenea: Notocotylidae) from mud snail Ecrobia ventrosa (Montagu, 1803).

Cercariae of digenean family Notocotylidae are characterized by a set of morphological traits which make them easily distinguishable from any other. One of the key features is absence of ventral sucker. This affects basic ways of locomotion and attachment. To understand how these functions are fulfilled we studied musculature arrangement in cercariae of two species by means of fluorescent-phalloidin staining and confocal microscopy. We used Cercaria Notocotylidae sp. No. 11 and 12 Deblock, 1980 from mud snails Ecrobia (=Hydrobia) ventrosa. Information on gross morphology (especially body-tail junction) and basic behavioural patterns of these cercariae is also updated. Major special features of musculature are associated with the ventral concavity: extreme development of dorsoventral muscle fibres and formation of annular arrangement of longitudinal muscle fibres on the ventral side. Additional body-wall and internal muscle bundles in the anterior region are also specific for notocotylid cercariae and seem to play important role in twisting movements during substratum testing. Musculature of dorsal adhesive pockets, oral sucker and tail is also described. These results are discussed in relation to observed locomotory patterns.

Somatic musculature in trematode hermaphroditic generation.

BACKGROUND: The somatic musculature in trematode hermaphroditic generation (cercariae, metacercariae and adult) is presumed to comprise uniform layers of circular, longitudinal and diagonal muscle fibers of the body wall, and internal dorsoventral muscle fibers. Meanwhile, specific data are few, and there has been no analysis taking the trunk axial differentiation and regionalization into account. Yet presence of the ventral sucker (= acetabulum) morphologically divides the digenean trunk into two regions: preacetabular and postacetabular. The functional differentiation of these two regions is already evident in the nervous system organization, and the goal of our research was to investigate the somatic musculature from the same point of view. RESULTS: Somatic musculature of ten trematode species was studied with use of fluorescent-labelled phalloidin and confocal microscopy. The body wall of examined species included three main muscle layers (of circular, longitudinal and diagonal fibers), and most of the species had them distinctly better developed in the preacetabuler region. In majority of the species several (up to seven) additional groups of muscle fibers were found within the body wall. Among them the anterioradial, posterioradial, anteriolateral muscle fibers, and U-shaped muscle sets were most abundant. These groups were located on the ventral surface, and associated with the ventral sucker. The additional internal musculature was quite diverse as well, and included up to twelve separate groups of muscle fibers or bundles in one species. The most dense additional bundles were found in the preacetabular region and were connected with the suckers. CONCLUSIONS: Previously unknown additional somatic musculature probably provides the diverse movements of the preacetabular region, ventral sucker, and oral sucker (or anterior organ). Several additional muscle groups of the body wall (anterioradial, posterioradial, anteriolateral fibers and U-shaped sets) are proposed to be included into the musculature ground pattern of trematode hermaphroditic generation. This pattern is thought to be determined by the primary trunk morphofunctional differentiation into the preacetabular and the postacetabular regions.

Muscle system of Diplodiscus subclavatus (Trematoda: Paramphistomida) cercariae, pre-ovigerous, and ovigerous adults.

The musculature of cercariae, pre-ovigerous, and ovigerous adults of Diplodiscus subclavatus was studied by means of TRITC-conjugated phalloidin staining of filamentous actin and confocal scanning laser microscopy. The body wall appears to include four muscle layers as follows: circular, outer longitudinal, diagonal, and inner longitudinal. Two layers of longitudinal muscle fibers are arranged in different modes due to the secondary transformed paramphistomid body construction. The organization of the acetabulum turned out to be more complex than ever described, with a radial layer, two layers of circular, two layers of meridional, an additional starry layer of muscle fibers, as well as a few separate muscle layers of the accessory sucker. Within the pharynx, I found a group of alar muscle fibers, never described before for any paramphistomids, and some morphological features which were not considered to be characteristic for D. subclavatus (namely--the middle semicircular layer and the transverse muscle fibers in the pre-sphincteric space). No significant reorganizations of the somatic musculature occur throughout the development from the cercaria to the ovigerous adult worm, so the metamorphosis goes in the manner of completion. The cercarial tail includes a layer of circular muscle fibers and a longitudinal muscle layer beneath. The latter consists of two medial longitudinal bundles of smooth muscle fibers and two lateral longitudinal bands of obliquely striated muscle fibers which are partially divided in halves.