vasa-related genes and their expression in stem cells of colonial parasitic rhizocephalan barnacle Polyascus polygenea (Arthropoda: Crustacea: Cirripedia: Rhizocephala)

vasa (vas)-related genes are members of the DEAD-box protein family and are expressed in the germ cells of many Metazoa. We cloned vasa-related genes (PpVLG, CpVLG) and other DEAD-box family related genes (PpDRH1, PpDRH2, CpDRH, AtDRHr) from the colonial parasitic rhizocephalan barnacle Polyascus polygenea, the non-colonial Clistosaccus paguri (Crustacea: Cirripedia: Rhizocephala), and the parasitic isopodan Athelgis takanoshimensis (Crustacea: Isopoda). The colonial Polyascus polygenea, a parasite of the coastal crabs Hemigrapsus sanguineus and Hemigrapsus longitarsis was used as a model object for further detailed investigations. Phylogenetic analysis suggested that PpVLG and CpVLG are closely related to vasa-like genes of other Arthropoda. The rest of the studied genes form their own separate branch on the phylogenetic tree and have a common ancestry with the p68 and PL10 subfamilies. We suppose this group may be a new subfamily of the DEAD-box RNA helicases that is specific for parasitic Crustacea. We found PpVLG and PpDRH1 expression products in stem cells from stolons and buds of internae, during asexual reproduction of colonial P. polygenea, and in germ cells from sexually reproducing externae, including male spermatogenic cells and female oogenic cells.     

A Study of the Morphology and Biology of Thompsonia littoralis (Crustacea: Cirripedia: Rhizocephala)

Interna development within the intertidal tropical crab, Leptodius exaratus, requires up to 4.5 months; the roots become distributed from the CNS along the major nerves and, when the crab moults, results in emergence of numerous externae located symmetrically on the abdomen and bases of the thoracic appendages. As many externae drop off, their number is continuously reduced; the survivors reach sexual maturity and ovulate after 2.5 months and complete embryonic development after 2 more months. At the next moult a new and more numerous generation of externae appears, and this is repeated until the crab dies. Presence of externae significantly prolongs the host's normal intermoult period and minimizes its growth. The parasite has little effect on the host's sexual characters although females are normally sterilized. The cuticle of the externa is moulted once, leaving a distinct annulus around the stalk. Spermatogonia are presumably injected by male cyprids into the female externa and develop into sperm within one or more vesicular spermatogenic bodies in its mantle. The eggs are laid through a single oviduct into the mantle cavity where they become fertilized and develop into cyprids that are liberated at the disintegration of the externa.     

Effects of temperature and salinity on the larvae of two species of rhizocephalan (Crustacea: Cirripedia)

Summary

Responses of larvae of two rhizocephalan species to changes in seawater temperature and salinity were studied under laboratory conditions. Peltogasterella gracilis parasitizes the hermit crab Pagurus pectinatus, which occurs at stable salinity and gradually changing temperature in summer. Sacculina polygenea is a parasite of the crab Hemigrapsus sanguineus, which lives in the intertidal zone in summer where salinity and temperature can fluctuate during the day. The development of both species is comprised of five naupliar stages and the cyprid stage, and it was considered successful if more than 50% of the nauplii attained the cyprid stage. P. gracilis nauplii successfully developed at 12–20°C and 30–34‰, but at 22°C successful development occurred in a narrower salinity range (32–34‰). All nauplii died both at 25°C and in 26‰. S. polygenea nauplii successfully reached the cyprid stage at higher temperatures (18–25°C) and a wider salinity range (18–34‰) than P. gracilis nauplii, but at 12°C and 16‰ larval development of S. polygenea was suppressed. Under favorable conditions, naupliar development lasted 3.5 days in P. gracilis and 2–3 days in S. polygenea. The cyprids of both rhizocephalan species demonstrated a greater resistance to temperature and salinity changes than nauplii. However, P. gracilis cyprids were active in a narrower salinity range (16–34‰), as compared to S. polygenea cyprids (8–34‰). Under favorable conditions the cyprids of both species survived for 6 to 10 days.     

Anatomy of virgin and mature externae of Loxothylacus texanus,parasitic on the dark blue crab Callinectes rathbunae (Crustacea: Cirripedia: Rhizocephala: Sacculinidae)

Rhizocephalan parasites are dioecious organisms, in that one or several dwarf males are implanted into the external part of the female parasite soon after it emerges from the interior of the host animal. The structure of the female externa and its resident males is crucial for understanding both the reproductive biology and the taxonomy of these specialized parasites. We use scanning electron microscopy and histological methods to study the anatomy of juvenile and the mature externae of the rhizocephalan barnacle Loxothylacus texanus parasitizing the blue crab Callinectes rathbunae. We put emphasis on the implantation of males and the histology of the female reproductive organs. In the virgin externae, male cyprids attach around a cuticular hood covering the mantle aperture, which is partially blocked by a plug of cuticle so only trichogon larvae, not cyprids, can access the mantle cavity. This resembles the situation known from Sacculina carcini. The mature externa is characterized by a visceral mass that contains the ovary, paired colleteric glands, a single male receptacle, but paired receptacle ducts. The proximal attachment of the visceral mass is located at some distance from the basal stalk, as is characteristic for the genus Loxothylacus. The internal anatomy of the mature externa of L. texanus is in most features similar to that seen in other species of the Sacculinidae, which comprises the majority of rhizocephalan species. However, the single receptacle creates a situation where the two implanted males cannot be kept separate as in most other rhizocephalans, but pass through spermatogenesis in a common chamber. This may have unknown effects on the reproductive biology such as male–male competition. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

A new peltogastrid rhizocephalan parasitising a hermit crab from the Japanese coast: a second species of Dipterosaccus Van Kampen & Boschma, 1925 (Crustacea: Cirripedia)

A new rhizocephalan species of the genus Dipterosaccus Van Kampen & Boschma, 1925, is described from the host hermit crab Calcinus vachoni Forest on the coasts of mainland Japan and the Ryukyu Archipelago. Dipterosaccus shiinoi n. sp. differs from the previously described species, D. indicus Van Kampen & Boschma, 1925, which is redescribed, principally in the morphology of the mantle aperture and the disposition of the receptacle ducts. The distribution ranges of the two species partly overlap and they are sympatric at the same reef site near Okinawa Island. Their differentiation as two distinct species was confirmed by partial sequences of the COI gene. The monophyly of Dipterosaccus is supported by high Bayes posterior possibility and bootstrap values.     

Speciation,phenotypic plasticity,or ontogeny,the case of the genus Galkinius (Pyrgomatidae,Cirripedia, Crustacea)

Barnacles of the genus Galkinius occupy a large spectrum of host corals, making it one of the least host‐specific genera within the Pyrgomatidae. Molecular analyses show that within the genus Galkinius there are highly supported clades, suggesting that the genus Galkinius is a complex of evolutionarily significant units (ESUs). The morphology of the opercular valves has been used as the basis for the separation of species of Galkinius. In this study, morphological variability was found both between specimens within ESUs extracted from different host species and between specimens extracted from the same colony. Identifications based on the opercular valves cannot therefore be assigned to different species despite being genetically distinguishable. It is proposed that in many cases the differences between valve morphology of different species of Galkinius are the outcome of ontogeny. Allometric growth of the valves has resulted in differences in the proportions of the parts of the valve. © 2015 The Linnean Society of London     

A rhizocephalan (Crustacea; Cirripedia) infestation of the deep-sea galatheid Munida sarsi (Crustacea; Decapoda), the effects on the host and the influence of depth upon the host-parasite relationship

Three species of rhizocephalan barnacle were found to be infesting 293 specimens of Munida sarsi Huus from the Porcupine Sea-bight (49–52°N, 11–14°W): Tortugaster boschmai, Lernaeodiscus ingolfi and, by far the most common, Triangulus munidae . Each species was found to have its own preferred abdominal segment site of emergence and particular effect on both male and female host pleopod structure. Triangulus munidae , however, caused a series of pleopod structure forms which led to the definition of a range of pleopod 'Types' for male and female hosts, including a peculiar masculinization of the female hosts' pleopods. The hormonal mechanisms behind these modifications are discussed.
Depth was found to have an influence upon the host-parasite relationship, there being both an increase in the percentage infestation of M. sarsi and a decrease in the effects of T. munidae on the host's pleopod structure with an increase in depth. At extremes of their bathymetric ranges, the host becomes more susceptible to infestation, while the effect of the parasite on its host breaks down.     

Control region sequences indicate that multiple externae represent multiple infections by Sacculina carcini (Cirripedia: Rhizocephala)

The rhizocephalan barnacle, Sacculina carcini, is a common parasite of the European shore crab, Carcinus maenas, in which it causes significant detrimental physical and behavioral modifications. In the vast majority of cases, the external portion of the parasite is present in the form of a single sac‐like externa; in rare cases, double or even triple externae may occur on the same individual host. Here, we use a highly variable DNA marker, the mitochondrial control region (CR), to investigate whether multiple externae in S. carcini represent infection by multiple parasites or asexual cloning developed by a single parasite individual. Sequences for multiple externae from C. maenas hosts from the Danish inlet, Limfjorden, and from the mud flates at Roscoff, France, were compared. In almost all cases, double or triple externae from an individual host yielded different haplotypes. In the few cases where identical haplotypes were identified from externae on a multiple‐infected host, this always represented the most commonly found haplotype in the population. This indicates that in Sacculina carcini, the presence of multiple externae on a single host reflects infection by different individual parasites. A haplotype network of CR sequences also suggests a degree of geographical partitioning, with no shared haplotypes between the Limfjorden and Roscoff. Our data represent the first complete CR sequences for a rhizocephalan, and a unique gene order was also revealed. Although the utility of CR sequences for population‐level work must be investigated further, the CR has proved a simple to use and highly variable marker for studies of S. carcini and can easily be applied to a variety of studies in this important parasite.     

Morphogenesis and Variability of Cuticular Structures in the Genus lbla (Crustacea, Cirripedia)

The cuticular structures of adult Ibla cumingi Darwin, I. quadrivalvis (Cuvier) and of the nauplius stages I-IV of I. cumingi have been examined, mainly by scanning but also by transmission electron microscopy. The various kinds of cuticular structures present have been listed and their morphogenetic differentiation is elucidated. The effect of size on the structures has been investigated. Irrespective of the cause of the size difference (sexual dimorphism, stage of growth, stage of ontogenetical development), the cuticular structures in the smaller individuals are simpler (lower level of complexity), they are arranged less regularly and they show a more primitive type of distribution than in the larger individuals. In the deviated males, the cuticular structures vary, but not in the fully developed females and hermaphrodites. It is, therefore, suggested that the males may be a young phylogenetic stage. The function of the cuticular structures has also been discussed.     

TEM studies on the lattice organs of cirripede cypris larvae (Crustacea, Thecostraca, Cirripedia)

 Lattice organs consist of five pairs of sensory organs situated on the dorsal carapace in cypris larvae of the Crustacea Cirripedia. The lattice organs in cypris larvae of Trypetesa lampas (Acrothoracica) and Peltogaster paguri (Rhizocephala) represent the two main types found in cirripedes, but only minor differences exist at the TEM level. Each lattice organ is innervated by two bipolar, primary receptor cells. The inner dendritic segment of each receptor cell carries two outer dendritic segments. The outer dendritic segments contain modified cilia with a short ciliary segment (9×2+0 structure). Two sheath cells envelop the dendrite except for the distal ends of the outer dendritic segments. This distal end enters a cavity in the carapace cuticle and reaches a terminal pore situated at the far end of the cavity. The cuticle above the cavity is modified. In both species the epicuticle is partly perforated by numerous small pores and the underlying exocuticle is much thinner and less electron dense than the regular exocuticle. Lattice organs very probably have a chemosensory function and are homologous with the sensory dorsal organ of other crustacean taxa. Accepted: 18 August 1998     

Immunocytochemical studies on the naupliar nervous system of Balanus improvisus (Crustacea, Cirripedia, Thecostraca)

The nervous system of nauplii of the crustacean taxon Cirripedia was analysed in the species Balanus improvisus Darwin, 1854 using for the first time immunocytochemical staining against serotonin, RFamide and α-tubulin in combination with confocal laser scanning microscopy. This approach revealed a circumoesophageal neuropil ring with nerves extending to the first and second antennae and to the mandibles, all features typical for Crustacea. In addition, RFamidergic structures are present in the region of the thoraco-abdomen. A pair of posterior nerves and a pair of lateral nerves run in anterior-posterior direction and are connected by a thoracic nerve ring and a more posteriorly situated commissure. A median nerve is situated along the ventral side of the thoraco-abdomen. The innervation of frontolateral horns and the frontal filaments are α-tubulin-positive. Several pairs of large neurons in the protocerebrum, along the circumoesophageal connectives and in the mandibular ganglion stain only for serotonin. Due to the almost complete absence of comparable data on the neuroanatomy of early (naupliar) stages in other Crustacea, we include immunocytochemical data on the larvae of the branchiopod, Artemia franciscana Kellogg, 1906 in our analysis. We describe several characteristic neurons in the brains of the nauplius larvae of both species which are also found in decapod larvae and in adult brains of other crustaceans. Furthermore, our data reveal that the naupliar brain of cirripedes is more complex than the adult brain. It is concluded that this ontogenetic brain reduction is related to the sessile life style of adult Cirripedia.     

Energy budget for the larval development ofElminius modestus (Crustacea: Cirripedia)

Biomass (CHN), respiration rate and food uptake were estimated for the larval development ofElminius modestus at three temperatures (12, 18, 24°C). Mean values of dry weight, elemental composition and energy equivalents increased exponentially with the development from nauplius II to VI. Dry weight, elemental composition and energy content exhibited the highest values at 18°C. Respiration rates increased with the larval stages expressed by a power function, but increased logarithmically with the dry weight of the larvae. The cypris larvae showed a reduced respiration rate compared with nauplius VI. The ingestion rate was measured at a concentration of 100 cells ofSkeletonema costatum μl⁻¹. At 12 and 18°C ingestion rates increased exponentially and at 24°C by a logarithmic function. The fittings were used to estimate the energy budget ofE. modestus during larval development. The energy content of the larvae increased during the development from nauplius II to VI by a factor of 21 at 12°C, 25 at 24°C and 31 at 18°C. The estimated energy content of the freshly metamorphosed barnacle is 100 mJ (12°C), 130 mJ (24°C) and 150 mJ (18°C). The assimilation- (A/I) and gross growth efficiencies (K₁) increased strongly during the development from nauplius II to VI (A/I: 6–14% in nauplius II to 50–90% in nauplius VI; K₁: 4% in nauplius II to 75% in nauplius VI). The net growth efficiency (K₂) showed a relatively constant level ranging between 57 and 83%.     

Sex determination in the androdioecious barnacle Scalpellum scalpellum (Crustacea: Cirripedia)

How androdioecy (coexistence of hermaphrodites and males) is maintained is still poorly understood. Therefore, sex determination was studied in the androdioecious barnacle Scalpellum scalpellum L. First, 247 cypris larvae from seven broods were investigated for sexual dimorphism in larval morphology and found to be all identical. Second, experiments with cyprids showed that males and hermaphrodites differ distinctly in morphology as soon as 4–5 days after settlement. Third, 14 252 cyprids were allowed to settle on the bottom of their culture cages, and all surviving larvae developed into hermaphrodites and none into dwarf males. Fourth, larvae settled in hermaphrodite receptacles (i.e. future males) were removed at increasing intervals after settlement to study if the male and hermaphrodite sexual expressions are fixed or plastic. All larvae became dwarf males if allowed to stay there for more than 8 h after settlement. But if removed within 3 h after settlement, half of them developed into hermaphrodites. We conclude that an environmental sex determination mechanism operates in S. scalpellum. Together with a 1:1 hermaphrodite/male ratio observed in previously reported experiments offering a free choice of settlement, we suggest that all larvae are potential hermaphrodites, but only 50% can settle in hermaphrodite receptacles and yield males.     

The monophyletic origin of a remarkable sexual system in akentrogonid rhizocephalan parasites: A molecular and larval structural study

We use sequences from the nuclear ribosomal genes, 18S and 28S to analyze the phylogeny of the Rhizocephala Akentrogonida including two species, Clistosaccus paguri and Chthamalophilus delagei, that are critical for understanding rhizocephalan evolution but have not previously been part of a molecularly based study. In addition we use light and scanning electron microscopy to compare the cypris larvae of C. paguri, Sylon hippolytes and two species of the family Thompsoniidae, since this larval stage offers a suite of characters for analyzing the evolution of these otherwise highly reduced parasites. The Rhizocephala Akentrogonida form a monophyletic group nested within a paraphyletic “Kentrogonida”. C. paguri and S. hippolytes are sistergroups confirming the monophyly of the Clistosaccidae that was originally based on similarities in the cypris larvae. We find numerous LM and SEM level similarities between the two species, many of which appear to be correlated with their specialized sexual system, where male cyprids use an antennule to implant cells into the virgin female parasite. Some of these traits are also found in cyprids of the thompsoniid species. We conclude that the special cypris morphology and the implantation of males by antennular penetration was present in the stem species to the Thompsoniidae and the Clistosaccidae and emphasize the power of larval characters in rhizocephalan systematics. C. delagei is a sister group to Boschmaella balani and the two are nested deep within the Akentrogonida. This confirms the monophyly of the Chthamalophilidae and falsifies the theory that C. delagei should represent the most primitive extant rhizocephalan. Instead, chthamalophilid rhizocephalans represent some of the most highly advanced members of the parasitic barnacles.     

The taxonomic position of rhizocephalan crustaceans of the genus Parthenopea Kossmann, 1874 (Cirripedia: Rhizocephala) with validation of a new family, Parthenopeidae fam. nov

Data on the external morphology are reviewed and new information is provided about the structure of the first naupliar instar of Parthenopea subterranea Kossmann, a rare and poorly known species of rhizocephalan barnacles. The taxonomic position of P. subterranea, which has so far been uncertain, is discussed. A new family, Parthenopeidae fam. nov., is erected to include a single genus, Parthenopea, with a single species, P. subterranea. The distinctive characters of the new family are described.     

Phylogeny and evolution of life history strategies of the parasitic barnacles (Crustacea, Cirripedia, Rhizocephala)

The barnacles (Crustacea, Cirripedia) consist of three well-defined orders: the conventional filter-feeding barnacles (Thoracica), the burrowing barnacles (Acrothoracica), and the parasitic barnacles (Rhizocephala). Thoracica and Acrothoracica feed by catching food particles from the surrounding seawater using their thoracic appendages while members of Rhizocephala are exclusively parasitic. The parasite consists of a sac-shaped, external reproductive organ situated on the abdomen of its crustacean host and a nutrient-absorbing root system embedded into the heamolymph of the host. In order to resolve the phylogenetic relationship of the order Rhizocephala and elucidate the evolution of the different life history strategies found within the Rhizocephala, we have performed the first comprehensive phylogenetic analysis of the group. Our results indicate that Rhizocephala is monophyletic with a filter-feeding barnacle-like ancestor. The host-infective stage, the kentrogon larva, inserted in the lifecycle of the rhizocephalan suborder, Kentrogonida, is shown to be ancestral and most likely a homologue of the juvenile stage of a conventional thoracican barnacle. The mode of host inoculation found in the suborder Akentrogonida, where the last pelagic larval stage directly injects the parasitic material into the heamolymph of the host is derived, and has evolved only once within the Rhizocephala. Lastly, our results show that the ancestral host for extant rhizocephalans appears to be the anomuran crustaceans (Anomura), which includes hermit crabs and squat lobsters.     

Synaptonemal polycomplexes in spermatocytes of the gooseneck barnacle,Pollicipes polymerus Sowerby (Crustacea: Cirripedia)

Polycomplexes are described for the first time in spermatocytes of a cirripede crustacean, Pollicipes polymerus Sowerby. Synaptonemal complexes of regular tripartite construction are seen from zygotene to mid-pachytene. Although some of the synaptonemal complexes are disrupted at late pachytene and may degenerate at this stage, some persist and by diplotene may form polycomplexes by the bending and self-fusion of their lateral elements. These polycomplexes are still encompassed by chromosomes and consist of four dense plates and intercalated central elements and transverse fibers. Other polycomplexes with five or six dense plates, all of which are considerably wider than lateral elements of mid-pachytene synaptonemal complexes, are also seen in diplotene nuclei. These may be attached to a chromosome at only one end or may be in the nucleoplasm, free of chromosomal involvement except for fine fibrous connectives. No polycomplexes are seen in meiotic cells after diplotene and their fate is unknown. It is suggested that poly-complexes serve as sequestra for synaptonemal material which could prevent normal chromosomal disjunction.     

The distribution and molecular diversity of the Eastern Atlantic and Mediterranean chthamalids (Crustacea, Cirripedia)

The three chthamalids Chthamalus stellatus , C. montagui and Euraphia depressa are common inhabitants of the intertidal zone in the Eastern Atlantic, Mediterranean Sea and Black Sea. In this study, we investigated the occurrence of these barnacles in a wide range of their distribution. Population divergences of these two species have been inferred using three molecular markers — internal transcribed spacer (ITS), elongation factor 1α (EF-1α) and cytochrome oxidase subunit I (COI). ITS sequences of C. stellatus were identical throughout the species range, whereas ITS sequences of C. montagui indicated that the Black Sea and Mediterranean populations are isolated from the Atlantic population. The COI and EF-1α sequences were the most variable and informative. They indicated a high genetic divergence between Atlantic, Mediterranean and Black Sea populations for C. montagui . In addition significant genetic structure was found among the populations of C. stellatus based on EF-1α but not COI. Interestingly, our molecular dating analysis correlated the pattern of diversification in C. montagui to major geological changes that occurred in the Mediterranean during the end of the Messinian and Pleiocene periods. We suggest that palaeohistory shaped the divergences between Chthamalus populations that have probably been maintained by current hydrographic conditions. Finally, COI phylogenetic analysis placed the genus Euraphia within the Chthamalus clade, suggesting the need for a taxonomic revision of Euraphia . This study represents the most detailed phylogeographical analysis of intertidal Mediterranean species to date, and shows that geological events have strongly shaped the current diversity pattern of this fauna.     

Biodiversity and Biogeography of Chthamalid Barnacles from the North-Eastern Pacific (Crustacea Cirripedia)

The biogeography and ecology of the species of Chthamalus present on the west coast of America are described, using data from 51 localities from Alaska to Panama, together with their zonation on the shore with respect to that of other barnacles. The species present were C. dalli, Pilsbry 1916, C. fissus, Darwin, 1854, C. anisopoma Pilsbry 1916 and four species in the C. panamensis complex. The latter are C. panamensis Pilsbry, 1916, C. hedgecocki, Pitombo & Burton, 2007, C. alani nom. nov. (formerly C. southwardorum Pitombo & Burton, 2007) and C. newmani sp. nov.). These four species were initially separated by enzyme electrophoresis. They could only be partially separated by DNA bar coding but may be separated using morphological characters.