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Molecular and morphological methods are used to describe Lernaeodiscus rybakovi, a new rhizocephalan species parasitizing the porcellanid crab Pachycheles stevensii Stimpson, 1858, collected in Russian waters of the Sea of Japan. Molecular analysis of three species, including the new one, confirms the monophyly of the genus Lernaeodicus Muller, 1862 and its recent transfer to the family Peltogastridae. The main morphological features of the new species are also common with characters of the other species of the genus Lernaeodiscus Muller, 1862. Externa of L. rybakovi differs from the well studied Lernaeodiscus porcellanae from the E. Pacific by molecular markers, color and the absence of pronounced marginal lobes. Retinacula on the internal cuticle of L. rybakovi are found in the genus Lernaeodiscus for the first time. Some of the female hosts with adult externae were unusual in also carrying their own eggs on the pleopods, a rare situation among rhizocephalans. The complete larval development in the genus Lernaeodiscus is described here for the first time and includes five naupliar and one cypris instar. The main morphological features of Lernaeodiscus nauplii (the presence of flotation collar, morphology of the frontolateral horns and furcal spines, and the arrangement of dorsal setae on the shield head) are common with those of other known peltogastrid larvae, but also resemble nauplii of Peltogasterella in the presence of naupliar eyes and thin structure of the flotation collar. The sizes of male and female cyprids of L. rybakovi overlap slightly. In summer months, larval sex ratio is male-biased. We briefly review important larval characters in the Rhizocephala.

The association between anemonefish and anemone is a classical example of mutualism in coral reefs. Although mutualism is probably the key innovation that triggered the adaptive radiation o...

Molecular and morphological methods are used to describe the rhizocephalan Lernaeodiscus kasyanovi sp. nov. from Russian waters of the Sea of Japan, the second of two Lernaeodiscus species parasitizing the porcellanid crab Pachycheles stevensii Stimpson, 1858. Lernaeodiscus kasyanovi sp. nov. differs from the other species found on this host, Lernaeodiscus rybakovi Korn et al., 2020a, by molecular markers, by a smaller size, a lighter color, by the shape of receptacles, by the presence of marginal lobes in mature specimens, and by the retinacula type on the internal cuticle. Lernaeodiscus kasyanovi sp. nov. is the sister taxon to Lernaeodiscus porcellanae in the monophyletic genus Lernaeodiscus. Lernaeodiscus kasyanovi is rather rare compared to L. rybakovi, their prevalence on the porcellanid crab P. stevensii does not exceed 2%. It is the second example of two rhizocephalan species infesting the same brachyuran species in Peter the Great Bay.

Rhizocephalan cirripedes are a very unique group of parasites infecting decapod crustaceans, but apart from a few well-studied species, little is known on their ecology and impact on hosts. Here we report on the resultsof a 14-month study of infestations of the rhizocephalanParasacculina leptodiae in the rocky shore crab Leptodius exaratus along the shores of Kuwait in the Persian (Arabian) Gulf. Monthly samples along an intertidal gradient revealed a slightly higher prevalence of P. leptodiae in female (18%) compared to male crabs (11%) and marked differences in prevalence among the sampling sites. Crabs from more sheltered locations in Kuwait Bay showed lower prevalence of P. leptodiae compared to crabs from more exposed sites. Seasonal patterns were largely absent, but prevalence in female crabs showed some monthly variation depending on the site. Rhizocephalan prevalence was generally highest in both crab sexes at the lower shores. This possibly resulted from lower exposure of crabs to infective stages in the higher intertidal and movements of infected crabs to lower parts of the shore. Prevalence of ovigerous females significantly declined with increasing local parasite prevalence. This suggests that the well-known castrating effects of rhizocephalans on individual hosts can also affect local crab reproduction at the population level which has notbeen shown before. Our results indicate that the rhizocephalanP. leptodiae is a common parasite of the rocky shore crab L. exaratus along the shores of Kuwait, with potential effects on the crab’s population dynamics which warrants further study.

The barnacle Austrominius modestus, native of New Zealand and Australia was introduced to the South of England around 1943 and has since spread to most coastal waters in Western Europe, including the southern North Sea. Apart from an ephemeral incursion into the Danish Wadden Sea in 1978, since 2010 it has established permanent populations capable of reproduction along the North Sea coast of the Jutland peninsula, the Limfjord, the north-western Kattegat, and the Skagerrak. It has probably invaded Danish waters by pelagic larvae originating in the German Wadden Sea. The species has since spread to other Danish localities, likely following the prevailing currents, but other means, as for instance transport by vessels, are possible. The barnacle inhabits stones, stone-reefs, mollusk shells, and live shore crabs in shallow waters. Based on hydrographical data from its native and recently invaded areas, we predict its future distribution to extend to most coasts of continental Europe except for brackish waters (< 20 PSU), and the Arctic seas. The northernmost distribution limit may include the Lofoten Islands of Norway publishedVersion

Background By comparing spatial geographical structures of host populations with that of their symbionts light can be shed on their biological interactions, and the degree of congruence between host and symbiont phylogeographies should reflect their life histories and especially dispersal mechanisms. Methods Here, we analyzed the genetic diversity and structure of a host, the blue swimming crab, Portunus pelagicus, and its symbiotic pedunculate barnacle Octolasmis angulata from six location sites representing three geographic regions (north, central and south) along the Vietnam coastline. High levels of congruence in their phylogeographic patterns were expected as they both undergo planktonic larval stages. Results Based on the COI mtDNA markers, O. angulata populations showed higher genetic diversity in comparison with their host P. pelagicus (number of haplotype/individuals, haplotype and nucleotide diversity are 119/192, 0.991 ± 0.002 and 0.02; and 89/160, 0.913 ± 0.02 and 0.015, respectively). Pairwise Fst and AMOVA analyses showed a more pronounced population structure in the symbiotic barnacle than in its crab host. The DAPC analyses identified three genetic clusters. However, both haplotype networks and scatter plots supported connectivity of the host and the symbiotic barnacle throughout their distribution range, except for low subdivision of southern population. Isolation by distance were detected only for the symbiont O. angulata (R2 = 0.332, P = 0.05), while dbMEM supported spatial structure of both partners, but only at MEM-1 (Obs. 0.2686, P < 0.01 and Obs. 0.2096, P < 0.01, respectively).

Infestation of Sacculina beauforti on an aquaculture species, mud crab Scylla olivacea is alarming due to its high prevalence and the extreme morphological changes in hosts. To further understand its pathological effect on growth and reproduction of S. olivacea, gonadal and hepatopancreatic histological changes of infected individuals were compared with healthy individuals. Also, the histological characteristics of S. beauforti's mature externa was described. Hepatopancreases of infected individuals were loosely packed and rootlets were observed in the intertubular spaces. Although hepatopancreatic tubule count was significantly lower, tubule diameters were unaffected. Gonads, however, were severely affected. No germ cells were found in the infected testes (except for remnants of spermatozoa), indicating the arrest of spermatogenesis. Rootlets were also present in testes of infected individuals. Ovarian tissues of infected females were severely damaged with no rigid cell structures. Gonadosomatic index (GSI) of infected males and females were lower, but the hepatosomatic index (HSI) were higher than their healthy counterparts. No significant differences in GSI and HSI values were observed among infected males and females. Multiple regression analysis revealed that carapace width, GSI and HSI are statistically important for the prediction of infection status. Given the high prevalence of S. beauforti infection and its disruptive effects on the morphology and internal physiology of S. olivacea, this study, albeit fundamental and incomprehensive, highlights to farmers and researchers the emergence of a castrating parasite and the urgency for the development of preventive measures and treatments for this disease in an economically important aquaculture species.

The blue swimming crab (Portunus pelagicus Linnaeus, 1758) is one of the commercially exploited crab fishery resources in Vietnam. This is the first study to provide a broad survey of genetic diversity, population structure and migration patterns of P. pelagicus along the Vietnamese coastline. The crab samples were collected from northern, central and southern Vietnam. Here, we used a panel of single nucleotide polymorphisms (SNPs) generated from restriction site-associated DNA sequencing (RADseq). After removing 32 outlier loci, 306 putatively neutral SNPs from 96 individuals were used to assess fine-scale population structure of blue swimming crab. The mean observed heterozygosity (Ho) and expected heterozygosity (He) per locus was 0.196 and 0.223, respectively. Pairwise Fst and hierarchical AMOVA supported significant differentiation of central and northern from southern populations (P

Barnacles and their allies (Thecostraca) are a biologically diverse, monophyletic crustacean group, which includes both intensely studied taxa, such as the acorn and stalked barnacles, as well as cryptic taxa, for example, Facetotecta. Recent efforts have clarified phylogenetic relationships in many different parts of the barnacle tree, but the outcomes of these phylogenetic studies have not yet been combined into a single hypothesis for all barnacles. In the present study, we applied a new “synthesis” tree approach to estimate the first working Barnacle Tree of Life. Using this approach, we integrated phylogenetic hypotheses from 27 studies, which did not necessarily include the same taxa or used the same characters, with hierarchical taxonomic information for all recognized species. This first synthesis tree contains 2,070 barnacle species and subspecies, including 239 barnacle species with phylogenetic information and 198 undescribed or unidentified species. The tree had 442 bifurcating nodes, indicating that 79.3% of all nodes are still unresolved. We found that the acorn and stalked barnacles, the Thoracica, and the parasitic Rhizocephala have the largest amount of published phylogenetic information. About half of the thecostracan families for which phylogenetic information was available were polyphyletic. We queried publicly available geographic occurrence databases for the group, gaining a sense of geographic gaps and hotspots in our phylogenetic knowledge. Phylogenetic information is especially lacking for deep sea and Arctic taxa, but even coastal species are not fully incorporated into phylogenetic studies.

Crustaceans play essential roles as food sources for global coastal communities, and their fisheries and aquaculture support the economic growth of many coastal countries. Thus, the understanding of diseases in crustaceans, especially those with high economic values, are important for their cure and prevention. Among other parasites that parasitise crustaceans, parasitic barnacles (Rhizocephala) are considered the most intriguing, both in terms of their unique life cycle and the detrimental changes they induced upon their hosts such as host sterilisation, moult inhibition (anecdysis), growth reduction, feminisation of male individuals, and behaviour alteration. In addition to discussing in detail the morphological, physiological and behavioural changes caused by rhizocephalan infestations, this review also shows that rhizocephalans are found in most economically important crustacean species around the world. Based on their reported adverse effects on their hosts, their potential implications on various aspects of crustacean aquaculture including broodstock selection, culture and fattening, soft-shell crab production and breeding programs are discussed. Although treatment or cure for rhizocephalan infestation has yet to be found, several recommendations are provided to enable accurate identification and handling of infected hosts and exposed aquaculture facilities in order to minimize losses and prevent parasite outbreaks.

Anelasma squalicola is a barnacle found attached to deep-water lantern sharks of the family Etmopteridae and is the only known cirriped on fish hosts. While A. squalicola is equipped with mouth and thoracic appendages (cirri), which are used for suspension feeding in conventional barnacles, its attachment device (peduncle) appears to have evolved into a feeding device, embedded into the tissue of its host. Here we demonstrate, through comparisons of the feeding apparatuses between A. squalicola and conventional suspension-feeding barnacles, that mouthparts and cirri of A. squalicola are highly reduced, and incapable of suspension-feeding activities. We show that in conventional suspension-feeding barnacles strong symmetries exist within these vital trophic structures. In A. squalicola strong asymmetries are widespread, indicating that those structures have been uncoupled from natural selection. The digestive tract is consistently empty, suggesting that feeding via cirri does not occur in A. squalicola. In addition, comparisons of stable isotope ratios (δ13C and δ15N) between A. squalicola, its shark host, and a conventional suspending feeding barnacle indicate that A. squalicola is taking nutrition directly from its host shark and not from the surrounding water. Our results strongly indicate that this barnacle has abandoned suspension feeding and now solely relies on obtaining nutrition from its host by a de novo evolved feeding mechanism. publishedVersion

Rhizocephalan barnacles have been reported to parasitize a wide range of king crab species (Lithodidae). So far all these parasites have been assigned to a single species, Briarosaccus callosus Boschma, 1930, which is assumed to have a global distribution. Here we investigate Briarosaccus specimens from three different king crab hosts from the fjord systems of Southeastern Alaska: Lithodes aequispinus Benedict, 1895, Paralithodes camtschaticus (Tilesius, 1815), and Paralithodes platypus (Brandt, 1850). Using molecular markers and by morphological comparison we show that Briarosaccus specimens from these three commercial exploited king crabs are in fact morphologically distinct from B. callosus, and further represent two separate species which we describe. The two new species, Briarosaccus auratum n. sp. and B. regalis n. sp., are cryptic by morphological means and were identified as distinct species by the use of genetic markers (COI and 16S). They occur sympatrically, yet no overlap in king crab hosts occurs, with B. auratum n. sp. only found on L. aequispinus, and B. regalis n. sp. as parasite of the two Paralithodes hosts.

The ecologically important shore crab Carcinus maenas is commonly infected in its native range by the rhizocephalan Sacculina carcini. However, several aspects of this host-parasite interaction are poorly understood. Here, we analyse data from approximately 60000 Danish crabs to unravel factors governing infection patterns in time and space, and according to host sex and size. Female crabs were more frequently infected (12.6%) than males (7.9%). Sites with high salinity supported the highest infection prevalence. Infection prevalence peaked in summer (10 to 15%) and winter (20 to 35%) due in part to emergence of virginal externae in summer (main outbreak) and autumn (minor outbreak) preceded by peaks in crabs with lost externa (scars). Younger externae and scars dominated among males, whereas adult externae were most frequent among females. Infection prevalence increased with size in females but decreased in males, and modified (feminized) males showed lower scar frequency than unmodified ones. Modified males occurred frequently among the smaller size classes, whereas unmodified males dominated the larger size classes. Externa size was positively related to host size in both genders (same linear relationship). Molecular analyses suggested that hosts below 16 mm in carapace width do not become infected. Dissections of infected hosts revealed marked reduction of ovaries, whereas testes were unaffected by sacculinization. Our study demonstrates great spatio-temporal variation in infection prevalence mainly related to the parasite's life history. S. carcini appears capable of infecting all host sizes except the smallest. Owing to incomplete feminization of males, infections are rapidly lost from the larger and highly profitable male hosts.

Summary The barnacle Anelasma squalicola is a marine epibiont found on members of the species-rich, deep-sea lantern shark family Etmopteridae ( Figure 1 A) but is unlike any other epibiotic thoracian barnacles [1] . While many barnacle species are associated with various marine animals including turtles and whales, with the exception of Anelasma these all retain a filter-feeding lifestyle and have a commensal relationship with their host; despite often being deeply embedded in the dermis, no other species has been reported as feeding on its host. Although Anelasma is fully equipped with cirri (thoracic appendages), these are no longer used for filter feeding [1] . Instead, Anelasma embeds a stalk with root-like structures into the flesh of the shark ( Figure S1 C in Supplemental Information, published with this article online) that it uses to parasitize its host. Here, we show that specimens of Anelasma sampled from all over the world show very little genetic differentiation, suggesting that this innovation coincided with a rapid worldwide expansion.

All available biological, ultrastructural, and molecular data support the conclusion that the cirripedian suborders, Rhizocephala and Thoracica, are closely related. Williamson’s speculations (Williamson, D. I., 2014. The origin of barnacles (Thecostraca, Cirripedia). Crustaceana,87: 755-765) that the so-called hybridogenesis can explain how Rhizocephala evolved, rest on a selective choice of other authors’ data and an ignorance of solid facts derived from decades of comparative anatomical and molecular studies.

Rhizocephala, a group of parasitic castrators of other crustaceans, shows remarkable morphological adaptations to their lifestyle. The adult female parasite consists of a body that can be differentiated into two distinct regions: a sac-like structure containing the reproductive organs (the externa), and a trophic, root like system situated inside the hosts body (the interna). Parasitism results in the castration of their hosts, achieved by absorbing the entire reproductive energy of the host. Thus, the ratio of the host and parasite sizes is crucial for the understanding of the parasite’s energetic cost. Using advanced imaging methods (micro-CT in conjunction with 3D modeling), we measured the volume of parasitic structures (externa, interna, egg mass, egg number, visceral mass) and the volume of the entire host. Our results show positive correlations between the volume of (1) entire rhizocephalan (externa + interna) and host body, (2) rhizocephalan externa and host body, (3) rhizocephalan visceral mass and rhizocephalan body, (4) egg mass and rhizocephalan externa, (5) rhizocephalan egg mass and their egg number. Comparing the rhizocephalan Sylon hippolytes, a parasite of caridean shrimps, and representatives of Peltogaster, parasites of hermit crabs, we could match their different traits on a reconstructed relationship. With this study we add new and significant information to our global understanding of the evolution of parasitic castrators, of interactions between a parasitic castrator and its host and of different parasitic strategies within parasitic castrators exemplified by rhizocephalans. publishedVersion

Screening of mud crab genusScyllawas conducted in four locations (Marudu Bay, Lundu, Taiping, Setiu) representing Malaysia.Scylla olivaceawith abnormal primary and secondary sexual characters were prevalent (approximately 42.27% of the local screenedS. olivaceapopulation) in Marudu Bay, Sabah. A total of six different types of abnormalities were described. Crabs with type 1 and type 3 were immature males, type 2 and type 4 were mature males, type 5 were immature females and type 6 were mature females. The abdomen of all crabs with abnormalities were dented on both sides along the abdomen’s middle line. Abnormal crabs showed significant variation in their size, weight, abdomen width and/or gonopod or pleopod length compared to normal individuals. The mean body weight of abnormal crabs (type 1–5) were higher than normal crabs with smaller body size, while females with type 6 abnormality were always heavier than the normal counterparts at any given size. Sacculinid’s externa were observed in the abdomen of crabs with type 4 and type 6 abnormalities. The presence of embryos within the externa and subsequent molecular analysis of partial mitochondrial COI region confirmed the rhizocephalan parasite asSacculina beauforti. Future in-depth descriptions of the life cycle and characteristics ofS. beaufortiare recommended as it involves a commercially important edible crab species and the effect on human health from the consumption of crabs is of crucial concern.

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. publishedVersion

Summary In his monograph on Cirripedia from 1851 [1], Darwin pointed to a highly unusual, plateless, and most likely parasitic barnacle of uncertain phylogenetic affinity. Darwin’s barnacle was Anelasma squalicola, found on deep-water sharks of the family Etmopteridae, or lantern sharks [2]. The barnacle is uncommon and is therefore rarely studied. Recent observations by us have shown that they occur at an unusually high prevalence on the velvet belly lantern shark, Etmopterus spinax, in restricted fjord areas of western Norway. A phylogenetic analysis based on ribosomal DNA data (16S, 18S, and 28S) from 99 selected barnacle species, including all available pedunculate barnacle sequences from GenBank, shows that A. squalicola is most closely related (sister taxon) to the pedunculate barnacle Capitulum mitella. Both C. mitella and species of Pollicipes, situated one node higher in the tree, are conventional suspension feeders from the rocky intertidal. Our phylogenetic analysis now makes it possible to establish morphological homologies between A. squalicola and its sister taxon and provides the evolutionary framework to explain the unprecedented transition from a filter-feeding barnacle to a parasitic mode of life.

Parasitic barnacles (Cirripedia: Rhizocephala) are highly specialized parasites of crustaceans. Instead of an alimentary tract for feeding they utilize a system of roots, which infiltrates the body of their hosts to absorb nutrients. Using X-ray micro computer tomography (MicroCT) and computer-aided 3D-reconstruction, we document the spatial organization of this root system, the interna, inside the intact host and also demonstrate its use for morphological examinations of the parasites reproductive part, the externa. This is the first 3D visualization of the unique root system of the Rhizocephala in situ, showing how it is related to the inner organs of the host. We investigated the interna from different parasitic barnacles of the family Peltogastridae, which are parasitic on anomuran crustaceans. Rhizocephalan parasites of pagurid hermit crabs and lithodid crabs were analysed in this study. publishedVersion

The fish ectoparasites Branchiura (Crustacea) display two different ways of attachment to the fish surface as adults: the first maxillae are either hooks (Dolops) or suction discs (Argulus, Chonopeltis, and Dipteropeltis). In larval Argulus foliaceus the first maxillae are hooks. With the first molecular phylogeny of the Branchiura as a background, the present paper discusses the evolutionary scenarios leading to hooks versus suction discs. Specific homologies exist between larval Argulus foliaceus hooks and adult Dolops ranarum hooks. These include the presence of a comparable number of segments/portions and a distal segment terminating in a double structure: a distal two-part hook (in Argulus) or one hook and an associate spine-like structure (in Dolops). In the phylogenetic reconstruction based on three molecular markers (mitochondrial 16S rRNA, nuclear 18S and 28S rRNA), Dolops ranarum is found to be in a sister group position to all other Branchiura, which in this analysis include six Argulus and one Chonopeltis sequences. Based on the molecular phylogeny a likely evolutionary scenario is that the ancestral branchiuran used hooks (on the first maxilla) for attachment, as seen in Dolops, of which the proximal part was subsequently modified into suction discs in Argulus and Chonopeltis (and Dipteropeltis). The sister group relationship of the Branchiura and Pentastomida is confirmed based on the most comprehensive taxon sampling until now. No evidence was found for a branchiuran in-group position of the Pentastomida.

Within parasitic barnacles of the family Sacculinidae, the genus Heterosaccus is the third largest, with 12 species infesting various brachyuran hosts throughout the world. As part of an effort to reconstruct rhizocephalan phylogeny we performed an analysis of four species of Heterosaccus and of selected sacculinid and non-sacculinid rhizocephalans. We used both molecular sequence data (16s rDNA and 18s rDNA) and morphological characters from an SEM analysis of the cypris larvae. Using Bayesian methods we obtained a highly supported tree in which the four species of Heterosaccus form a monophylum, whereas both the genus Sacculina and the family Sacculinidae are polyphyletic. The sistergroup to Heterosaccus is a clade consisting of the sacculinids Loxothylacus panopaei and the "classical" rhizocephalan Sacculina carcini. The molecular results found support in cypris morphology, where we identified two distinct synapomorphies for Heterosaccus, both present in male cyprids only: A large flap extending posteriorly from the third antennular segment, and the male-specific aesthetasc on the third segment being reduced to a rudiment or lacking completely. Female cyprids didn't show generic level apomorphies but resembled those of other sacculinid species. No morphological synapomorphies were identified between Heterosaccus, L. panopaei and S. carcini. While larval characters proved to be informative, we conclude that future studies on rhizocephalan taxonomy must rely primarily on molecular data, both to provide an overall phylogenetic framework and to assure an accurate identification of species for biogeographical and other biological purposes.

The evolutionary history of Branchiopoda (Crustacea) traditionally has attracted considerable interest due to the diversity of the group. Recently molecular methods have been applied to the study of branchiopod systematics with some success, but central questions, such as the phylogenetic position of Laevicaudata and Notostraca, and the intrinsic cladoceran phylogeny, remain unanswered. We examined the phylogeny of Branchiopoda by using two genes, mitochondrial 16S rRNA and nuclear 28S rRNA, which previously have seen little use for inferring branchiopod phylogeny. The number of ingroup taxa included was 42, representing all eight extant branchiopod orders. The data were analyzed using parsimony, maximum likelihood, and Bayesian Inference of phylogeny. Some higher-level taxa were supported in all analyses of the combined data: Phyllopoda, Cladoceromorpha, Cladocera, and Gymnomera. Other higher-level taxa were not supported in any trees: Diplostraca and Conchostraca. A case is made for a possible diplostracan ingroup position of Notostraca based on our data and on previously published molecular and morphological evidence. The recent discovery of a Devonian branchiopod, which is morphologically an intermediate between a notostracan and a 'conchostracan', is congruent with a diplostracan ancestry of Notostraca.

Scanning electron microscopy, light microscopy, and histology were used to study growth in species of the pedunculate barnacle genus Octolasmis (O. angulata, O. cor, O. californiana, O. mulleri). These species are epizoic in the gill chamber of portunid crabs and have highly reduced capitular shell plates, with large areas of general cuticle in between. The external integument grows by means of a system of narrow growth zones, one encircling the peduncle and a Y-shaped system on either side of the capitulum. Growth is by a regular series of molts, but shedding of old cuticle and production of new layers is entirely restricted to the growth zones. Just prior to ecdysis, the new cuticle lies in a highly folded fashion beneath the old cuticle that is about to be shed. At ecdysis, the old cuticle breaks along the margins of the growth zones and the resulting scars remain as a system of “ecdysial lines” along either side of the zone. Once exposed after ecdysis, the new cuticle remains as a part of the permanent external integument. The growth zones divide the externa into five cuticular areas, two on the peduncle and three on the capitulum. The calcareous shell plates (carina, paired scuta, and, when present, paired terga) all lie within the capitular regions and the ecdysial lines pass across, not around, these mineralized areas. The number, relative spacing, and topology of the ecdysial lines form a record of the growth history of the specimen. These and other growth patterns demonstrate that size increase is due to the formation of new cuticle by molting in the growth zones, while expansion of the shell plates by mineralization follows only after production of the new cuticle. Thus, although specialized, growth in Octolasmis still complies with the general crustacean model, complicated only by the mineralization of parts of the capitular cuticle into shell plates. The results are compared with the very scarce information on molting in other barnacles. We argue that at least the circular peduncular growth zone is omnipresent in the Cirripedia Thoracica. J. Morphol. 260:154–164, 2004. © 2004 Wiley-Liss, Inc.

The Rhizocephala is a group of extremely reduced parasitic crustaceans, that exclusively parasitize other Crustacea. In the family Sacculinidae, the external sac-like part (externa) of the adult parasite contains the reproductive apparatus and is attached beneath the abdomen of the host crab. Hosts with more than one externa may occur and are in most cases believed to have arisen from multiple cyprid larvae. However, in three species of the genus Sacculina, multiple externae have been shown to originate by asexual reproduction from a single parasitic cypris larva. We present a phylogenetic analysis of ten species of Sacculina and outgroups based on partial sequences from the cytochrome oxidase 1 (CO1) and the entire 18s rDNA gene. A separate parsimony analysis from the 18s rDNA and CO1 genes resulted in two trees with almost identical topologies. Both genes strongly support a monophyletic, asexually reproducing clade and fail to support a monophyletic Sacculina genus. As a consequence we have est...

Rhizocephala is a group of crustaceans that exclusively parasitizes other crustaceans. It is taxonomically placed within the class Cirripedia, the barnacles, with which it shares a unique larval type, the cyprid. The main objective of the cyprid is to find and irreversibly attach to a suitable substratum and initiate metamorphosis. In the presumed sister group to Rhizocephala, the true barnacles or Thoracica, metamorphosis leads to a juvenile filter-feeding version of the adult organism. In Rhizocephala the female cyprid settles on the integument of a crustacean and undergoes metamorphosis into a kentrogon that possesses a hollow cuticular-tube structure, the stylet, which penetrates the integument of the host and acts as a guide tube for the prospective internal parasite. The first, hitherto unknown endoparasitic stage of a rhizocephalan, the vermigon, was recently discovered (Glenner and Hoeg [1995] Nature 377:147–150) and its migration through the hemolymph of the host, as well as its internal development, was described in Glenner et al. ([2000] Mar Biol 136:249–257). The present article provides detailed information on kentrogon and vermigon formation, the injection process, and the succeeding developmental stages up to the stage of the earliest primordium reported from the literature. The anlage of the ovary is traced back to the free-swimming cypris stage and it is implied that the mesoderm and ectoderm of the endoparasite are already differentiated in the cyprid. J. Morphol. 249:43–75, 2001. © 2001 Wiley-Liss, Inc.

The parasitic barnacles, Rhizocephala, are unique in Crustacea by having an entirely endo-parasitic phase inserted into their lifecycle. A cypris larva, remarkably similar to the cypris of conventional acorn and goose barnacles (Thoracica), settles on the crustacean host and develops an infective stage, the kentrogon, underneath the exuviae of the cypris. The kentrogon penetrates the integument of the host by a hollow cuticle structure, the stylet, and injects the parasitic material into the hemocoelic fluid of the host. Although advanced stages of the internal development have been found and described several times, the nature of the originally injected parasitic material has remained obscure for decades. Recently, however, it was shown that the parasitic material was injected by the kentrogon in the form of a motile, multi-cellular and vermiform body. The present study demonstrates that the vermiform stage is an instar which forms the only and direct link between the kentrogon and the maturing internal parasite. The vermiform instar, or vermigon, is at all times clothed in a cuticle, contains several types of cells, including epidermis and the anlage of the later ovary, and stays intact while growing into the internal parasites with rootlets.

We present a cladistic analysis of the Cirripedia Thoracica using morphological characters and the Acrothoracica and Ascothoracida as outgroups. The list of characters comprised 32 shell and soft body features. The operational taxonomic units (OTUs) comprised 26 well-studied fossil and extant taxa, principally genera, since uncertainty about monophyly exists for most higher ranking taxonomic units. Parsimony analyses using PAUP 3.1.1 and Hennig86 produced 189 trees of assured minimal length. We also examined character evolution in the consensus trees using MacClade and Clados. The monophyly of the Balanomorpha and the Verrucomorpha sensu stricto is confirmed, and all trees featured a sister group relationship between the `living fossil' Neoverruca and the Brachylepadomorpha. In the consensus trees the sequential progression of `pedunculate' sister groups up to a node containing Neolepas also conforms to current views, but certain well-established taxa based solely on plesiomorphies stand out as paraphyletic, such as Pedunculata (=Lepadomorpha), Eolepadinae, Scalpellomorpha and Chthamaloidea. The 189 trees differed principally in the position of shell-less pedunculates, Neoverruca, the scalpelloid Capitulum, and the interrelationships within the Balanomorpha, although the 50% majority rule consensus tree almost fully resolved the latter. A monophyletic Sessilia comprising both Verrucomorpha and Balanomorpha appeared among the shortest trees, but not in the consensus. A tree with a monophyletic Verrucomorpha including Neoverruca had a tree length two steps longer than the consensus trees. Deletion of all extinct OTUs produced a radically different tree, which highlights the importance of fossils in estimating cirripede phylogeny. Mapping of our character set onto a manually constructed cladogram reflecting the most recent scenario of cirripede evolution resulted in a tree length five steps longer than any of our shortest trees. Our analysis reveals that several key questions in cirripede phylogeny remain unsolved, notably the position of shell-less forms and the transition from `pedunculate' to `sessile' barnacles. The inclusion of more fossil species at this point in our understanding of cirripede phylogeny will only result in even greater levels of uncertainty. When constructing the character list we also identified numerous uncertainties in the homology of traits commonly used in discussing cirripede evolution. Our study highlights larval ultrastructure, detailed studies of early ontogeny, and molecular data as the most promising areas for future research.

The Rhizocephala are considered to be monophyletic due to several synapomorphies in the ontogeny of the cndoparasitic phase. The various types of metamorphosis described in the Rhizocephala are discussed and compared to metamorphosis in the Cirripedia Thoracica and Acrothoracica. In males and females of the suborder Kentrogonida. the cyprid settles and metamorphoses into a new instar, in males the trichogen and in females the infective kentrogon. The kentrogon goes through yet another. incomplete moult associated with the development of the stylet. Within the three kentrogonidan families. the Iernaeodiscid-peltogastrid type of kentrogon differs from the sacculinid type in the mode of attachment to the host. in the complexity of internal anatomy. in the position and penetration of the stylet, and in whether or not the cyprid carapace must be shed prior to penetration of the stylet. In the Akentrogonida metamorphosis never results in a new instar. Where observed (Clistosaccidae and Thompsoniidae). both male and female cyprids settle and penetrate into their substrate (female parasite or new host) with one of the antennules. Using the antennule as a syringe. male cyprids inject spermatogonia while female cyprids injects embryonic cells developing into an endoparasite. By comparison with metamorphosis in the Cirripedia Thoracica and Acrothoracica it is concluded that the presence of a metamorphic moult leading to a post-cyprid instar is plesiomorphic and that the trichogon and kentrogon are homologous with the first metamorphosed juvenile in these outgroups. The abbreviated ontogeny in the Akentrogonida without metamorphic moult and post-cyprid larval instars is considered apomorphic. This contradicts the long-held supposition that the Akentrogonida are the most‘primitive’Rhizocephala and dovetails with new information that this suborder contains many advanced traits. Within the Kentrogonida. the lernacodiseid-peltogastrid type of kentrogon is considered more plesiomorphic than the sacculinid type, which resembles the clistosaccidthompsoniid type in having the antennules involved in the penetration process. The homologization of the kentrogon with a juvenile barnacle indicates that presence of a kentrogon is plesiomorphic within the Rhizocephala and that the Kentrogonida is paraphyletic.

Metamorphosis and early juvenile development was followed in the laboratory in Balanus amphitrite and B. improvisus (family Balanidae) and in Semibalanus balanoides and Elminius modestus (family Archaeobalanidae) from cyprid settlement until 24 h after ecdysis. Stages of development were studied in vivo and with scanning electron microscopy. Events in metamorphosis and early juvenile development are very similar in all four species and can be interpreted in terms of a highly modified moult. Notably, there was no indication whatsoever of the “amorphous decorticated settler” previously described from metamorphosis of B. amphitrite. The shape of a juvenile barnacle with cirri and incipient shell plates is apparent immediately after the shedding of the cypris carapace, and rudimentary peduncle can be distinguished below the developing wall plates. A basal row of cuticular hairs encircles the peduncle in all species except S. balanoides. These hairs seem to serve a restraining function during early development, when the juvenile barnacle is only attached by the initially secreted cyprid cement. Similarly situated hairs are also present in very young juveniles of the lepadomorph Scalpellum scalpellum. In Semibalanus balanoides nothing indicates that the rostrum originates from the fusion of two latera as previously claimed, since this plate is single as soon as it can be distinguished.

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 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.

RHIZOCEPHALANS are barnacles (Cirripedia), but are extremely specialized for parasitic life on decapod crustaceans. A cypris larva settles and develops into a new instar, the kentrogon, which inoculates the host with the parasite. The very early primordial parasite has been argued to consist solely of embryonic stem cells or even eggs1, but the true nature of this unknown stage has remained a puzzle for more than a century2. We present data from in vitro experiments on the rhizocephalan Loxothylacm panopaei documenting that, unlike previous postulations, the recently injected parasite is not naked embryonic cells, but has the form of a motile, vermiform body, enclosed in an acellular sheath. After a period of maturation the vermiform body splits up into a number of naked and independently moving cells, which in our in vitro experiments disperse by amoeboid movements. This suggests that, in vivo, the cells disperse in the haemolymph of the host crab, where each has the potential to develop into an adult parasite, although in most cases only one will succeed.

New insight into the life cycle leads us to present a scenario for the evolution of the Rhizocephala. We base it on a homology between the rhizocephalan kentrogon and the first juvenile stage of conventional barnacles and on the discovery of the vermigon, the earliest internal parasitic phase. We suggest that the urrhizocephalan was a filter feeder attached to the gills of decapod hosts. The stylet of the kentrogon evolved from the peduncle of the epibiont filter feeder. The vermigon is ontogenetically a part of the stylet, which suggests that the base of the peduncle evolved into the internal parasite. Indirect establishment could have evolved if parasites accidentally detached from their attachment to the overlying cuticle and drifted elsewhere within the host. Those that reached the ventral blood sinus in the abdomen and emerged in the abdominal brood chamber would have a selective advantage if they received the same treatment from the host as a brooded egg mass. This scenario could easily lead to the conseuent development of full-fledged host control and castration by the parasitic barnacle.

Cypris morphology in the Rhizocephala differs both between the sexes of individual species and between the species. The large aesthetasc on the third antennular segment is unique to male cyprids of the Lernaeodiscidae, Peltogasteridae and Sacculinidae (suborder Kentrogonida) and Mycetomorpha vancouverensis (suborder Akentrogonida). A fourth segmental aesthetasc, common to both sexes, is much longer in males than in females. A spinous process on the attachment disc is another male-specific character, but is absent altogether in the Lernaeodiscidae. It is argued that these aesthetascs help locate the settlement targets. Except in Mycetomorpha vancourverensis, aesthetascs have been secondarily lost in cyprids of the suborder Akentrogonida, and there are no other morphological means for separating the male and female settling stage. In cyprids of Sylon hippolytes (Sylonidae) and of Mycetomorpha vancouverensis and Thompsonia sp. (Akentrogonidae) the long and slender antennules are probably used in penetration without formation of a kentrogon, as already reported for Clistosaccus paguri (Clistosaccidae). Cyprids of Sylon hippolytes and Clistosaccus paguri are almost morphologically identical.