First recorded occurrence of the parasitic barnacle (Anelasma squalicola) on a Greenland shark (Somniosus microcephalus) in the Canadian Arctic.

A solitary Anelasma squalicola specimen was collected from the cloaca of a Greenland shark (Somniosus microcephalus), the first time this association has been recorded. The specimen's identity was confirmed through morphological and genetic assessment (mitochondrial markers: COI and control region). A. squalicola is a species typically associated with deep-sea lantern sharks (Etmopteridae) and, until the present observation, had never been observed at a sexually mature size in the absence of a mating partner. Given the reported negative effects of this parasite on its hosts, monitoring Greenland sharks for additional cases is recommended.

Eyes, Vision, and Bioluminescence in Deep-Sea Brisingid Sea Stars.

AbstractSea stars are a major component of the megabenthos in most marine habitats, including those within the deep sea. Being radially symmetric, sea stars have sensory structures that are evenly distributed along the arms, with a compound eye located on each arm tip of most examined species. Surprisingly, eyes with a spatial resolution that rivals the highest acuity known among sea stars so far were recently found in Novodinia americana, a member of the deep-sea sea star order Brisingida. Here, we examined 21 species across 11 brisingid genera for the presence of eyes; where eyes were present, we used morphological characteristics to evaluate spatial resolution and sensitivity. This study found that eyes were present within 43% of the examined species. These brisingid eyes were relatively large compared to those of other deep-sea sea stars, with a high number of densely packed ommatidia. One of the examined species, Brisingaster robillardi, had more than 600 ommatidia per eye, which is the highest number of ommatidia found in any sea star eye so far. Combined, the results indicate that brisingid eyes are adapted for spatial resolution over sensitivity. Together with results showing that many brisingids are bioluminescent, this relatively high spatial resolution suggests that the group may use their eyes to support visually guided intraspecific communication based on bioluminescent signals. Phylogenetic analysis indicated that the common ancestor of brisingids had eyes (P = 0.72) and that eyes were lost once within the clade.

Genomic Adaptations to an Endoparasitic Lifestyle in the Morphologically Atypical Crustacean Sacculina carcini (Cirripedia: Rhizocephala).

The endoparasitic crustacean Sacculina carcini (Cirripedia: Rhizocephala) has a much simpler morphology than conventional filter-feeding barnacles, reflecting its parasitic lifestyle. To investigate the molecular basis of its refined developmental program, we produced a draft genome sequence for comparison with the genomes of nonparasitic barnacles and characterized the transcriptomes of internal and external tissues. The comparison of clusters of orthologous genes revealed the depletion of multiple gene families but also several unanticipated expansions compared to non-parasitic crustaceans. Transcriptomic analyses comparing interna and externa tissues revealed an unexpected variation of gene expression between rootlets sampled around host midgut and thoracic ganglia. Genes associated with lipid uptake were strongly expressed by the internal tissues. We identified candidate genes probably involved in host manipulation (suppression of ecdysis and gonad development) including those encoding crustacean neurohormones and the juvenile hormone binding protein. The evolution of Rhizocephala therefore appears to have involved a rapid turnover of genes (losses and expansions) as well as the fine tuning of gene expression.

Adding pieces to the puzzle: insights into diversity and distribution patterns of Cumacea (Crustacea: Peracarida) from the deep North Atlantic to the Arctic Ocean.

The Nordic Seas have one of the highest water-mass diversities in the world, yet large knowledge gaps exist in biodiversity structure and biogeographical distribution patterns of the deep macrobenthic fauna. This study focuses on the marine bottom-dwelling peracarid crustacean taxon Cumacea from northern waters, using a combined approach of morphological and molecular techniques to present one of the first insights into genetic variability of this taxon. In total, 947 specimens were assigned to 77 morphologically differing species, representing all seven known families from the North Atlantic. A total of 131 specimens were studied genetically (16S rRNA) and divided into 53 putative species by species delimitation methods (GMYC and ABGD). In most cases, morphological and molecular-genetic delimitation was fully congruent, highlighting the overall success and high quality of both approaches. Differences were due to eight instances resulting in either ecologically driven morphological diversification of species or morphologically cryptic species, uncovering hidden diversity. An interspecific genetic distance of at least 8% was observed with a clear barcoding gap for molecular delimitation of cumacean species. Combining these findings with data from public databases and specimens collected during different international expeditions revealed a change in the composition of taxa from a Northern Atlantic-boreal to an Arctic community. The Greenland-Iceland-Scotland-Ridge (GIS-Ridge) acts as a geographical barrier and/or predominate water masses correspond well with cumacean taxa dominance. A closer investigation on species level revealed occurrences across multiple ecoregions or patchy distributions within defined ecoregions.

Comparative population genetics of swimming crab host (Portunus pelagicus) and common symbiotic barnacle (Octolasmis angulata) in Vietnam.

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).

From tides to nucleotides: Genomic signatures of adaptation to environmental heterogeneity in barnacles.

The northern acorn barnacle (Semibalanus balanoides) is a robust system to study the genetic basis of adaptations to highly heterogeneous environments. Adult barnacles may be exposed to highly dissimilar levels of thermal stress depending on where they settle in the intertidal (i.e., closer to the upper or lower tidal boundary). For instance, barnacles near the upper tidal limit experience episodic summer temperatures above recorded heat coma levels. This differential stress at the microhabitat level is also dependent on the aspect of sun exposure. In the present study, we used pool-seq approaches to conduct a genome wide screen for loci responding to intertidal zonation across the North Atlantic basin (Maine, Rhode Island, and Norway). Our analysis discovered 382 genomic regions containing SNPs which are consistently zonated (i.e., SNPs whose frequencies vary depending on their position in the rocky intertidal) across all surveyed habitats. Notably, most zonated SNPs are young and private to the North Atlantic. These regions show high levels of genetic differentiation across ecologically extreme microhabitats concomitant with elevated levels of genetic variation and Tajima's D, suggesting the action of non-neutral processes. Overall, these findings support the hypothesis that spatially heterogeneous selection is a general and repeatable feature for this species, and that natural selection can maintain functional genetic variation in heterogeneous environments.

Ecological Load and Balancing Selection in Circumboreal Barnacles.

Acorn barnacle adults experience environmental heterogeneity at various spatial scales of their circumboreal habitat, raising the question of how adaptation to high environmental variability is maintained in the face of strong juvenile dispersal and mortality. Here, we show that 4% of genes in the barnacle genome experience balancing selection across the entire range of the species. Many of these genes harbor mutations maintained across 2 My of evolution between the Pacific and Atlantic oceans. These genes are involved in ion regulation, pain reception, and heat tolerance, functions which are essential in highly variable ecosystems. The data also reveal complex population structure within and between basins, driven by the trans-Arctic interchange and the last glaciation. Divergence between Atlantic and Pacific populations is high, foreshadowing the onset of allopatric speciation, and suggesting that balancing selection is strong enough to maintain functional variation for millions of years in the face of complex demography.

Genome-wide SNP analyses reveal population structure of Portunus pelagicus along Vietnam coastline.

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<0.01). Population structure analyses revealed that P. pelagicus in the south is a separate fisheries unit from the north and center. Contemporary migration patterns supported high migration between northern and central populations and restricted genetic exchange within the southern population. In contrast, historic gene flow provides strong evidence for single panmictic population. The results are useful for understanding current status of P. pelagicus in the wild under an environment changing due to natural and anthropogenic stresses, with implications for fisheries management.

Towards a barnacle tree of life: integrating diverse phylogenetic efforts into a comprehensive hypothesis of thecostracan evolution.

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.

Pancrustacean Evolution Illuminated by Taxon-Rich Genomic-Scale Data Sets with an Expanded Remipede Sampling.

The relationships of crustaceans and hexapods (Pancrustacea) have been much discussed and partially elucidated following the emergence of phylogenomic data sets. However, major uncertainties still remain regarding the position of iconic taxa such as Branchiopoda, Copepoda, Remipedia, and Cephalocarida, and the sister group relationship of hexapods. We assembled the most taxon-rich phylogenomic pancrustacean data set to date and analyzed it using a variety of methodological approaches. We prioritized low levels of missing data and found that some clades were consistently recovered independently of the analytical approach used. These include, for example, Oligostraca and Altocrustacea. Substantial support was also found for Allotriocarida, with Remipedia as the sister of Hexapoda (i.e., Labiocarida), and Branchiopoda as the sister of Labiocarida, a clade that we name Athalassocarida (="nonmarine shrimps"). Within Allotriocarida, Cephalocarida was found as the sister of Athalassocarida. Finally, moderate support was found for Hexanauplia (Copepoda as sister to Thecostraca) in alliance with Malacostraca. Mapping key crustacean tagmosis patterns and developmental characters across the revised phylogeny suggests that the ancestral pancrustacean was relatively short-bodied, with extreme body elongation and anamorphic development emerging later in pancrustacean evolution.

De novo innovation allows shark parasitism and global expansion of the barnacle Anelasma squalicola.

The barnacle Anelasma squalicola is a marine epibiont found on members of the species-rich, deep-sea lantern shark family Etmopteridae (Figure 1A) 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 S1C 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.

Population dynamics and development of the rhizocephalan Sacculina carcini, parasitic on the shore crab Carcinus maenas.

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.

Infestation of parasitic rhizocephalan barnacles Sacculina beauforti (Cirripedia, Rhizocephala) in edible mud crab, Scylla olivacea.

Screening of mud crab genus Scylla was conducted in four locations (Marudu Bay, Lundu, Taiping, Setiu) representing Malaysia. Scylla olivacea with abnormal primary and secondary sexual characters were prevalent (approximately 42.27% of the local screened S. olivacea population) 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 as Sacculina beauforti. Future in-depth descriptions of the life cycle and characteristics of S. beauforti are 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 bigger, the better? Volume measurements of parasites and hosts: Parasitic barnacles (Cirripedia, Rhizocephala) and their decapod hosts.

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.

On a New Species of Parasitic Barnacle (Crustacea: Rhizocephala), Sacculina shiinoi sp. nov., Parasitizing Japanese Mud Shrimps Upogebia spp. (Decapoda: Thalassinidea: Upogebiidae), Including a Description of a Novel Morphological Structure in the Rhizocephala.

The rhizocephalan Sacculina shiinoi sp. nov. parasitizes three species of Upogebia in Japan. It is described morphologically and compared with another Upogebia parasite, Sacculina upogebiae Shiino, 1943 from Japan and Korea. These two species are the only sacculinids that parasitize mud shrimps. DNA analyses clearly show the two species to be separate and not closely related. The cuticle differs in being provided with close-set, branched, and spiny excrescences in S. shiinoi, while it lacks excrescences, but forms small scales in S. upogebiae. In S. upogebiae, the bulbous sperm-producing part and the narrow receptacle duct are separated by a compartmentalized mid portion, which is missing in S. shiinoi. A ridge, having a thickened, fluffy cuticle with a U-shaped course, passes across the visceral mass between the two receptacle openings in S. shiinoi. Such a structure has never been described in other rhizocephalans, and its function is uncertain.

Caught in the act: phenotypic consequences of a recent shift in feeding strategy of the shark barnacle Anelasma squalicola (Lovén, 1844).

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.

Two new cryptic and sympatric species of the king crab parasite Briarosaccus (Cirripedia: Rhizocephala) in the North Pacific.

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.

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.

On the origin of a novel parasitic-feeding mode within suspension-feeding barnacles.

In his monograph on Cirripedia from 1851, 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. 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.

Metamorphosis in balanomorphan, pedunculated, and parasitic barnacles: a video-based analysis.

Cypris metamorphosis was followed using video microscopy in four species of cirripeds representing the suspension-feeding pedunculated and sessile Thoracica and the parasitic Rhizocephala. Cirripede metamorphosis involves one or more highly complex molts that mark the change from a free cypris larva to an attached suspension feeder (Thoracica) or an endoparasite (Rhizocephala). The cyprids and juveniles are so different in morphology that they are functionally incompatible. The drastic reorganization of the body implicated in the process can therefore only commence after the cyprid has irreversibly cemented itself to a substratum. In both Megabalanus rosa and Lepas, the settled cyprid first passes through a quiescent period of tissue reorganization, in which the body is raised into a position vertical to the substratum. In Lepas, this is followed by extension of the peduncle. In both Lepas and M. rosa, the juvenile must free itself from the cypris cuticle by an active process before it can extend the cirri for suspension feeding. In M. rosa, the juvenile performs intensely pulsating movements that result in shedding of the cypris carapace ~8 h after settlement. Lepas sp. sheds the cypris cuticle ~2 days after settlement due to contractile movements of the peduncle. In Lepas anserifera, the juvenile actively breaks through the cypris carapace, which can thereafter remain for several days without impeding cirral feeding. Formation of the shell plates begins after 1-2 days under the cyprid carapace in Lepas. In M. rosa, the free juvenile retains its very thin cuticle and flexible shape for some time, and shell plates do not appear until sometime after shedding of the cypris cuticles. In Sacculina carcini, the cypris settles at the base of a seta on the host crab and remains quiescent and aligned at an angle of ~60° to the crab's cuticle. The metamorphosis involves two molts, resulting in the formation of an elongated kentrogon stage with a hollow injection stylet. Due to the orientation of the cyprid, the stylet points directly towards the base of the crab's seta. Approximately 60 h after settlement the stylet penetrates down one of the cyprid antennules and into the crab. Almost immediately afterwards the unsegmented vermigon stage, preformed in the kentrogon, passes down through the hollow stylet and into the crab's hemocoel in a process lasting only 30 s. In S. carcini, the carapace can remain around the metamorphosing individual without impeding the process.