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.     

Size and settling behaviour in male and female cypris larvae of the parasitic barnacle Sacculina carcini Thompson (Crustacea: Cirripedia: Rhizocephala)

Twenty-one broods from different externae of Sacculina carcini Thompson were cultured to the cypris stage. The size of the cyprids was measured and the larvae subjected to settling upon unparasitized crabs (Garcinus maenas (L.)) and small juvenile externae. The cyprids occur in two sizes that may appear singly or together in the same brood. Small cyprids are of the female sex that settle upon crabs and are infective, while large cyprids are of the male sex and only settle upon juvenile externae. These results are in agreement with other well-studied rhizocephalans.     

Probing the settlement signals of Amphibalanus amphitrite

To achieve their reproductive potential, barnacles combine tactile exploration of surface structural properties and integration of cellular signals originating from their antennular sensory setae within a developmentally defined, temporally narrow window of settlement opportunity. Behavioural assays with cyprids coupled with biometric analysis of scanning electron microscopy-acquired images in the presence of specific chemical compounds were used to investigate how settlement on a substratum is altered in response to the presence of these compounds. Impeding tactile exploration was shown which altered cellular signalling and/or induced malformation of anatomical features of the antennular sensory setae, which disrupted the settlement behaviour of the model barnacle species Amphibalanus amphitrite. It is concluded that surface exploration by the cyprids relies on mechanical and nociception-related and calcium-mediated signals while a protein kinase C signalling cascade controls the timely metamorphosis of the cyprids to sessile juveniles.     

The cypris larvae of the rhizocephalan barnacle Heterosaccus lunatus with particular reference to antennular morphology

SEM investigation of laboratory-reared cyprids of the parasitic barnacle Heterosaccus lunatus has revealed morphological differences between the male and female larvae, particularly the attachment organ of their antennules. The detailed features of the attachment organ (segment III) and segment IV for each sex are described and compared with those of other rhizocephalan cyprids, particularly Sacculina carcini , a reportedly closely related species. The absence of the large posterior sac from segment III in the H. lunatus male highlights the need for careful larval investigations on other sacculinid species. Settlement of female H. lunatus cyprids occurred on the gill lamellae of the host crab with the subsequent kentrogon stage taking the sacculinid form. The inclusion of H. lunatus in the Sacculinidae was therefore confirmed, based on the morphology of the cypris antennules and kentrogon.     

Cypris Ultrastructure,Metamorphosis and Sex in Seven Families of Parasitic Barnacles (Crustacea: Cirripedia: Rhizocephala)

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.     

藤壶金星幼虫附着变态机制

藤壶属节肢动物门(Arthropoda)甲壳亚门(Crustacea)蔓足下纲(Cirripedia)围胸总目(Thoracica), 具备特殊的形态结构、生活史和种群生态特征,是最主要的海洋污损生物。其幼虫阶段通常经历6期无节幼体和1期不摄食的金星幼虫,从浮游的金星幼虫附着变态成固着的稚体是藤壶生活史中的一个关键环节。外界化学和生物因子中成体提取物、水溶性信息素、足迹、神经递质、激素、生物膜等均影响藤壶金星幼虫的附着变态;内在因子即金星幼虫的生理状态(能量储量和年龄)决定了其对外界因子的反应程度。概括了近年来藤壶附着变态生理机制和分子机制研究的进展,可为深入了解藤壶金星幼虫附着变态机制提供参考,也为开发新型、高效、环保的防污剂提供理论指导。     

Cloning and expression of the engrailed.a gene of the barnacle Sacculina carcini

 Cirripedia (barnacles) constitute a crustacean monophyletic taxon which is very well defined by several synapomorphies. In particular, all cirripedes are composed of six thoracic segments, but are devoid of any complete abdominal segment. This body plan is preserved in the adult in non-parasitic groups, while the parasitic rhizocephalan cirripedes completely lose arthropodian segmentation at the adult stage. These traits make them a particularly favourable model for studying the formation and maintenance of segmental identity. For the above reasons, it seemed worthwhile to look at the segmentation gene engrailed in a cirripede. A complete engrailed.a cDNA was isolated from larvae of the rhizocephalan cirripede Sacculina carcini. Its expression was monitored during larval development by use of the monoclonal antibody MAb4D9 directed against the Drosophila homologous proteins. The Sacculina engrailed.a gene is expressed during the second and third larval stages in stripes within a posterior area corresponding to the presumptive trunk segments. Surprisingly, these stripes appear in a posterior to anterior sequence. Six engrailed.a stripes characterize the thoracic segments of the cirripedean ground plan. Received: 18 June 1998 / Accepted: 24 October 1998     

The Morphology of the Naupliar Stages of Sacculina carcini(Crustacea: Cirripedia: Rhizocephala)

A morphological study was carried out on the fournaupliar stages of Sacculina carciniusing mainly scanning electron microscopy. Frontal horns were present and throughout development the typical nauplius limbs remained simple and gnathobases were lacking. Such features are characteristic of other lecithotrophic barnacle nauplii. The presence of a vestigial ventral thoracic process was evident on the stage III nauplius and was even more prominent on the stage IV nauplius. These observations confirm that the rhizocephalan nauplius is close to the thoracican nauplius form and lend strong support for the retention of the Rhizocephala within the Cirripedia.     

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 cypris larvae of the parasitic barnacle Heterosaccus lunatus (Crustacea, Cirripedia, Rhizocephala): some laboratory observations

Heterosaccus lunatus parasitizes the portunid crab, Charybdis callianassa in Moreton Bay, Australia. With the host crabs maintained at 22.5 degrees C this sacculinid rhizocephalan released larval broods every 6-7 days. During July-August 1996 and particularly August 1999 such broods showed the change-over from male only larvae in the early broods to females only in the later broods. As the host crabs were maintained under similar aquarium conditions in both years it is concluded that the light/dark cycle is the principal cue triggering this larval sex reversal. Oogenesis in the parasite externa is somehow controlled to produce two different sized ova - male larvae develop from large ova and females from small ova. A working hypothesis outlining how sex is probably determined for the larvae of sacculinids is erected. H. lunatus is considered the ideal sacculinid for the further experimental work necessary to verify the proposed sex-determining mechanism and its control processes. Measurements of the maximum swimming speeds of H. lunatus male and female cyprids showed the larger males to be the faster in absolute terms (27.95 compared with 17.60 mm s(-1), respectively), however, the calculated relative speeds were almost identical at approximately 90 body lengths s(-1). Settlement experiments confirmed that female H. lunatus cyprids settle only on the gills of C. callianassa; these cyprids needed to be at least 2 days old before they were able to settle.     

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.     

Larval Development in the Rhizocephalan Barnacle Sacculina pilosella

We studied, under laboratory conditions, the larval development of a rhizocephalan barnacle Sacculina pilosellaVan Kampen et Boschma, 1925, which parasitizes the kelp crab Pugettia quadridens(de Haan) in Vostok Bay, Sea of Japan. It is shown that at 22–23°C, the whole cycle of larval development takes about 3 days. The larvae of S. pilosellaare lecithotrophic; their development, like in other rhizocephalans, comprises five naupliar instars. Like the larvae of all sacculinids, the nauplii of S. pilosellahave no flotation collar. In their structure, the larvae of S. pilosellaare similar to the nauplii of the typical sacculina, S. carcini(elongated body outline, long furcal branches, and weakly pronounced segmentation of the abdomen). On the other hand, the characteristic outgrowth inbetween the furcal branches that is characteristic of stages IV and V in S. carciniand S. polygeneais absent in the larvae of S. pilosella.The first seta on the antennula of S. pilosellacompletely disappears only at stage IV; however, at stage III, it is already significantly reduced. No morphological differences have been revealed between male and female larvae of S. pilosellaexcept certain size differences.     

Host specificity of <Emphasis Type="Italic">Sacculina carcini</Emphasis>, a potential biological control agent of the introduced European green crab Carcinus maenas in California

The European green crab, Carcinus maenas, is an introduced marine predator established on the west coast of North America. We conducted laboratory experiments on the host specificity of a natural enemy of the green crab, the parasitic barnacle Sacculina carcini, to provide information on the safety of its use as a possible biological control agent. Four species of non-target, native California crabs (Hemigrapsus oregonensis, H. nudus, Pachygrapsus crassipes and Cancer magister) were exposed to infective larvae of S. carcini. Settlement by S. carcini on the four native species ranged from 33 to 53%, compared to 79% for green crabs. Overall, cyprid larvae tended to settle in higher numbers on individual green crabs than on either C. magister or H. oregonensis. However, for C. magister this difference was significant for soft-shelled, but not hard-shelled individuals. Up to 29% of the native crabs arrested early infections by melanizing the rootlets of the parasite. Most native and green crabs settled on by S. carcini became infected, especially when settled on by >3 cyprids. Infected green crabs died at more than twice the rate of uninfected green crabs. In contrast to green crabs, all infected native crabs died without producing an externa (reproductive sac). At high settlement intensities, infected native crabs frequently exhibited neurological symptoms (twitching, loss of movement) before death. These results indicate that use of S. carcini as a biological control agent could result in the death of native crabs. The magnitude of this effect would be proportional to the density of infected green crabs in the environment and the probability that cyprids would contact native crabs in the wild. Potential benefits of biological control should be assessed in relation to these potential non-target effects.     

How whale and dolphin barnacles attach to their hosts and the paradox of remarkably versatile attachment structures in cypris larvae

How larvae of whale and dolphin epibionts settle on their fast-swimming and migrating hosts is a puzzling question in zoology. We successfully reared the larvae of the whale and dolphin barnacle Xenobalanus globicipitis to the cyprid stage. We studied the larval developmental ecology and antennular morphology in an attempt to assess whether an epibiotic lifestyle on this extreme substratum entails any unique larval specializations. Morphological parameters were compared with five other barnacle species that also inhabit extreme substrata. We found no larval specializations to a lifestyle associated with marine mammals. The external morphology of the antennules in Xenobalanus cyprids is morphologically similar to species from strikingly different substrata. We found variation only in the structures that are in physical contact with the substratum, i.e., the third segments carrying the villi-covered attachment disc. The third segments of the Xenobalanus cyprid antennules are not spear-shaped as in the stony coral barnacles, which are here used to penetrate the live tissue of their hosts. The presence of a cyprid cement gland implies that Xenobalanus uses cement protein when attaching to its cetacean host. Naupliar instars developed outside of the mantle cavity, indicating dispersal is planktonic. Our results militate against the idea that the cyprids settle during ocean migrations of their hosts. We suggest cyprids settle during coastal aggregations of the cetacean hosts. We conclude that the ecological success of barnacles has ultimately depended on a larva that with little structural alteration possesses the ability to settle on an amazingly wide array of substrata, including cetaceans.

     

On the morphology of antennular sensory and attachment organs in cypris larvae of the deep‐sea vent/seep barnacles,Ashinkailepas and Neoverruca

Barnacle cypris larvae show high morphological variation in the organs used in search of and attaching to a substratum. This variation may represent adaptation to the habitat of the species. Here, we studied SEM level morphologies of cypris antennular sensory and attachment organs in a deep‐sea vent endemic species (Neoverruca sp.) and a vent/seep inhabiting species (Ashinkailepas seepiophila). We compare them with three species from other environments. The antennular morphologies of Neoverruca sp. and A. seepiophila were similar, which is consistent with recent molecular studies showing a close relationship of the two species. The setation pattern of the antennules was very conservative among species from various environments. In contrast, striking differences were observed in the structure of the attachment organ (the third antennular segment). Neoverruca sp. and A. seepiophila had no velum or a skirt surrounding the attachment disc on the third segment, while other cirripede cyprids almost always have either of these structures. In addition, both cyprids of A. seepiophila and Neoverruca sp. had the attachment disc angled toward the substratum, whereas it faces distally in cyprids from hard bottom inhabiting barnacles. We suggest that both velum/skirt and the angle of the attachment disc play an important role, when the antennules are contacting the substratum during surface exploration. Differences in attachment organ structures may be highly adaptive, enabling cirripede species to enter new habitats during evolution. J. Morphol. 277:594–602, 2016. © 2016 Wiley Periodicals, Inc.     

THE ACTION OF COPPER IN ANTIFOULING PAINTS

Experiments on the number of barnacle cyprids found on plane surfaces and on the surface of small pits coated with compositions containing different amounts of cuprous oxide show that the presence of copper does not deter the cyprids from attempting to settle. Copper-oxide paints do not inhibit settlement appreciably; their efficiency as antifouling agents is caused by the toxic action of copper on cyprids and young spat after initial attachment.
The cementing of the shell to the substratum in young barnacles which have survived metamorphosis appears to be hindered by the presence of copper paints so that they are easily dislodged.     

The Phylogenetic Position of the Rhizocephala: Are They Truly Barnacles?

Incorporation of the Rhizocephala in the Cirripedia, reflecting the traditional view that these parasites evolved from a setose feeding barnacle, has recently been challenged in favour of rhizocephalans being the sister group to all other Thecostraca or a scenario where they evolved from a free-living, ‘precirripede’ ancestor. Adult morphology is useless in discussing the monophyly of the Cirripedia, since rhizocephalan adults are too reduced to furnish any phylogenetic evidence. But numerous, detailed similarities in nauplii and cyprids of the Thoracica, Acrothoracica and Rhizocephala as well as the ultrastructure of their sperm are synapomorphic relative to other Thecostraca and indicate that these three orders form a monophylum. There is evidence that the stylet in the rhizocephalan kentrogon is homologous to an element in the ancestral mouth field. If so, the Rhizocephala probably evolved before setose feeding was adopted, and constitute the sister group to the Acrothoracica and Thoracica. This conclusion is based on frail evidence so the term Cirripedia should be retained to comprise the Rhizocephala, Thoracica, and Acrothoracica. These three orders all possess remarkably similar cyprids, adapted to accomplish irreversible settlement by cement secretion and initiate metamorphosis, so their last common ancestor was most probably a permanently sessile organism.     

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.