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

Metamorphosis in the Cirripede Crustacean Balanus amphitrite

Stalked and acorn barnacles (Cirripedia Thoracica) have a complex life cycle that includes a free-swimming nauplius larva, a cypris larva and a permanently attached sessile juvenile and adult barnacle. The barnacle cyprid is among the most highly specialized of marine invertebrate larvae and its settlement biology has been intensively studied. By contrast, surprisingly few papers have dealt with the critical series of metamorphic events from cementation of the cyprid to the substratum until the appearance of a suspension feeding juvenile. This metamorphosis is both ontogenetically complex and critical to the survival of the barnacle. Here we use video microscopy to present a timeline and description of morphological events from settled cyprid to juvenile barnacle in the model species Balanus amphitrite, representing an important step towards both a broader understanding of the settlement ecology of this species and a platform for studying the factors that control its metamorphosis. Metamorphosis in B. amphitrite involves a complex sequence of events: cementation, epidermis separation from the cypris cuticle, degeneration of cypris musculature, rotation of the thorax inside the mantle cavity, building of the juvenile musculature, contraction of antennular muscles, raising of the body, shedding of the cypris cuticle, shell plate and basis formation and, possibly, a further moult to become a suspension feeding barnacle. We compare these events with developmental information from other barnacle species and discuss them in the framework of barnacle settlement ecology

Morphogenesis and evolution of annuli in arthropod appendages

A widely used distinction among articles that usually composed an arthropod appendage is the one between true articles and annuli. This distinction is often claimed to be based on the anatomy of the muscular system, true articles have intrinsic musculature while annuli do not. Annuli are also usually considered a subdivision of a true article. Recently, it has also been noted that annuli tend to be produced later during development. Observations on development of Drosophila appendages also seem to support a basic difference between the process that produce true article and the one that produce annuli. In the present project I studied selected aspects of article anatomy and development, in order to understand: a) which are (if present) the developmental similarities among annuli of different appendages and different arthropods, and b) which are (if present) the developmental differences between annuli and true articles. I decided to focalise the research on two topics: a) the relationships between muscles, muscle insertions and joints, and b) the mechanism of annulation in flagellar structures (terminal part of an appendage composed of only annuli) and its relationship with growth at the cellular level. According to the definitions of true articles and annuli given above, the anatomy of the muscular system is the most important aspect. For the most studied true articles, those of insect leg, there is evidence of a close developmental relationship between the development of the arthrodial membrane cells (epidermal cells that produce the joint) and muscle insertions. However some variation is expected as annuli are supposed to be joint without any muscle insertion. Parts of appendages composed of only annuli often show indeterminate postembryonic increasing in the number of annuli. The mechanism by which new annuli are produced has been studied only in few species or groups, and only for the antennae. Where both the mechanism of article production and the overall growth have been studied, a close relationship between the two was noted, but little is known about the development of the epidermis (cellular division, differentiation and apoptosis) during segmentation. Different models have been employed to study the relationships between muscles, muscle insertions and joints and these are: the naupliar appendages (first antennae and exopod of both second antennae and mandibles) of the cirriped crustacean Balanus improvisus Darwin, 1854, the exopod of the naupliar second antennae of the branchiopod crustacean Artemia sp., the antennae of the centipede Lithobius forficatus (Linnaeus, 1758) and the rami of the pleopods of the malacostracan crustacean Gammarus roeselii Gervais, 1835. In these models the segmentation, the muscular system and the postembryonic changes have been studied. Literature on naupliar appendages anatomy and postembryonic development has also been reviewed in detail. There are some muscles running parallel to the proximo-distal axis throughout the first antennae and the exopod of both second antennae and mandibles in the nauplii of B. improvisus. These muscles have insertions on every joint. The exopod of both second antennae and mandibles increase in article number during naupliar development and new joints have new intermediate insertion of already present muscles. Very similar conditions are usually found in the naupliar appendages of other crustaceans. Unexpected results have been obtained on the exopod of naupliar second antennae of Artemia. The exopod has 8-10 natatory setae (number with individual variation) on the posterior-ventral side, which have some cuticular folds at their base, resembling a joint; on the opposite side there are 8-14 (number with individual variation) cuticular folds. Number and position of setae and cuticular folds do not match and thus complete joints are lacking. Three muscles are present within the exopod; they run parallel to the proximo-distal axis and have insertions at the base of a seta (for the two muscles that are on that side) or on a cuticular fold (for the single muscle that run on that side). Since setae and cuticular folds do not match, there is mismatch also in the muscular insertions of the two sides. In the antennae of L. forficatus there are four muscles that run parallel to the proximo-distal axis throughout their length. These muscles have an insertion on each joint. The rami of the pleopods of G. roeselii have two muscles that run parallel to the proximo-distal axis throughout their length, with insertion on each joint. Thus, even if the articles of the antennae of Lithobius are usually considered true articles and those of the naupliar exopod of second antennae and mandibles of Balanus (and other crustaceans) as well as those of the rami of the pleopods of Gammarus are usually considered annuli, there is no difference on the presence/absence of muscular insertions. Anatomical differences are present in the structure of the muscular insertion (tendon matrix) and of the joint (extent of arthrodial membrane). All the appendages originally studied here or those discussed in the review that increase in article number during postembryonic development produce new joints with new intermediate insertions of already present muscles. The mechanism of annulation in flagellar structures has been studied in detail in the flagellum of the second antennae of isopod crustaceans. Asellus aquaticus (Linnaeus, 1758) has been the main species studied, with observations on both normal postembryonic development and regeneration; other species studied have been Idotea chelipes (Pallas, 1766), Lirceus fontinalis Rafinesque-Schmaltz, 1820 e Sphaeroma serratum (Fabricius, 1787). Most of the flagellum of A. aquaticus is composed of "quartets": four articles units where each article has a specific setal distribution pattern. New articles and quartets are produced during the whole life, in the proximal part of the flagellum: the first article divides and produces articles that, relatively independently from each others, divides three more times producing a quartet. During regeneration the mechanism is identical, although there are some difference in the relative development of different quartets, irrespectively of the amputation point. In L. fontinalis (Asellidae) most of the flagellum is composed of couples of articles, each one of which bearing setae correspondent to those of two articles of an A. aquaticus quartet. The mechanism of production is also very similar, but articles produced by the first one divide just once. In L. fontinalis some variability is, anyhow, present and it is sometimes possible to observe three articles units (an article produced by the first one divided twice) and even four article units identical to those of A. aquaticus. In S. serratum (Sphaeromatidae) most of the flagellum has articles with subequal setal pattern; the mechanism of new article production involves the division of the first article and one further division of the articles produced by it. In I. chelipes (Idoteidae) most of the flagellum has articles with subequal setal pattern; the mechanism of new article production involves, unlike S. serratum, the division of the first article only. The mechanism of annulation in flagellar structures and its relationship with growth at the cellular level has been studied in two models (already used for other observations previously described): the flagellum of the second antennae of A. aquaticus and the rami of the pleopods of G. roeselii. The pleopodal rami of G. roeselii increase their article number for the whole life. New articles are produced in the proximal part, by division of the first article only. In this structure, as well as in the second antennal flagellum of A. aquaticus, mitotic figures are found only in the proximal part and going distally, to "older" parts, nuclei becomes more spaced and longer. Thus, in both the models studied there is a proximal proliferative zone; cells produced there are then moved distally by the production of new cells and they go through a shape change. How this process is related to the diversity of the segmentation mechanism is not currently understood. The production of joints and muscle insertions are developmentally correlated processes. Evidence for it was already available for the articles (except tarsomeres) of insect leg, but I have shown in this thesis this is also true for other arthropod appendages, since new joints produced during postembryonic development have also new muscle insertions, if a muscle is present. Joints without any muscle insertion can occur in arthropod appendages, but these have either no muscle passing through or just tendon(s); the occurrence of joints without any muscle insertion but with muscle(s) passing through is currently very doubtful. Thus, the traditional distinction between true article and annuli based on the presence/absence of intrinsic musculature is wrong; articles usually considered annuli may have muscle insertion. Functionally, however, this distinction is still valid, since articles with intermediate insertions of muscles parallel to the proximo-distal axis can not move the appendage independently from other articles as the other true articles (equipped with intrinsic and antagonist muscles confined within them) can do. The naupliar antennal exopod of Artemia also provide evidence that joints and muscle insertions are developmentally correlated processes. In this model there are not complete joints, but just "partial" cuticular folds, but also these (which are probably derived from a complete joint) have muscle insertions. A general difference in the timing of expression of true articles and annuli was previously noted and has been here discussed in some deep. A difference in timing exists, but it is not between true articles and annuli (if defined by the presence/absence of muscle insertions) but between articles with independent movements and articles with movements not independent to each others. Also this ontogenetic difference is connected with the different functional morphology of these articles. Flagellar structure also exhibits similarities in their postembryonic development, and these similarities are connected to the presence of a specific proximal "growth zone" (a zone where both new articles are produced and mitoses are localized). The phylogenetic distribution of this growth zone is discussed and it is here proposed to be an ancestral condition for the postembryonic development of (first) antennae and rami of postantennulary appendages of, at least, mandibulate (myriapods, insects and crustaceans) arthropods

Segmental mismatch in crustacean appendages: the naupliar antennal exopod of Artemia (Crustacea, Branchiopoda, Anostraca)

Based on traditional techniques and confocal laser scanning microscopy for external morphology, and immunohistochemistry for the muscular system, we describe here the segmental features of the antennal exopod of Artemia nauplii. Two kinds of serial elements are present, i.e. setae (with cuticular folds at their base) and ringlets (serially arranged sclerites separated by joint-like cuticular folds not extending to form complete rings around the appendage). The two series are usually not in register. The cuticular folds of the setae and of the ringlets are also sites of intermediate insertions of the three exopod muscles: as the two tegumentary structures are discordant in periodicity, this is also mirrored in the pattern of muscle insertions on the two sides of the appendage. Similar cases of segmental mismatch are known for the trunk of several arthropods, but segmental mismatch along the appendages has received very little attention. The occurrence of segmental mismatch in the naupliar appendages of both extant and fossil crustaceans is reviewed and it is suggested here to be a primitive feature of the exopods of both second antennae and mandibles. Problems in the interpretation of morphological evidence are discussed, also in relation to development and evolution of segmentation of naupliar appendages

Multi-scale relationships between numbers and size in the evolution of arthropod body features

Size-related changes of form in animals with periodically patterned body axes and post-embryonic growth discontinuously obtained throughout a series of moulting episodes cannot be accounted for by allometry alone. We address here the relationships between body size and number and size of appropriately selected structural units (e.g., segments), which may more or less closely approximate independent developmental units, or unitary targets of selection, or both. Distinguishing between units fundamentally involving one cell only or a small and fixed number of cells (e.g., the ommatidia in a compound eye), and units made of an indeterminate number of cells (e.g., trunk segments), we analyze and discuss a selection of body features of either kind, both in ontogeny and in phylogeny, through a review of current literature and meta-analyses of published and unpublished data. While size/number relationships are too diverse to allow easy generalizations, they provide conspicuous examples of the complex interplay of selective forces and developmental constraints that characterizes the evolution of arthropod body patterning

Segmental pattern formation following amputation in the flagellum of the second antennae of Asellus aquaticus (Crustacea, Isopoda)

Regeneration of the second antennae of Asellus aquaticus is described here following amputations along the antennal flagellum. The process involves the frequent resorption (loss of the distalmost joint remained on the amputated antenna) and the regular apicalization of the new terminal article. In the distal part of the flagellum, resorption occurs only when less than 70% of the original article length is left. For amputations performed in the proximal meristematic region, where new articles are normally produced, the new terminal article may also divide, sometimes producing articles with abnormal setal pattern; instead, articles that would normally divide may fail to do so if they are the nearest proximal neighbour of the new terminal article. Outcome of the increased production of new articles from the meristematic region is a regenerated antenna with a number of flagellomeres close to that shown by the undamaged controlateral one. Similarities and differences in respect to the processes occurring after amputation in the antennal peduncle, as well as in other arthropod limbs, are discussed. These differences may help with understanding general properties of the regeneration process, such as the distinction between epimorphosis and morphallaxis and the relationship between normal development and regeneration

Growth and regeneration of the second antennae of Asellus aquaticus (Isopoda) in the context of arthropod antennal segmentation

The production of new articles in the flagellum of the second antennae of Asellus aquaticus was studied in both undamaged and regenerating antennae. Segmentation is an iterative process in two phases: a) the first proximal flagellomere (the meristematic article) repeatedly gives off distally a new primary article; b) each primary article divides into four secondary articles (a quartet). To a certain extent, production and development of different quartets are independent processes. Evidence is provided that the formation of new articles and their setae are partly decoupled. During regeneration from the preferred breakage point (the so-called \u2018autotomy plane\u2019), the flagellum is generated by the same mechanism of two-phase segmentation. The regenerated flagellum has a normal segmental composition, except for the tip (the apical complex), which has four flagellomeres rather than the normal five. The similar segmental pattern observed also in other malacostracan crustaceans and in insects, supports a close phylogenetic relationship among the two groups; if the latter proves not to hold, that similarity would provide an example of parallel evolution of developmental mechanism. The difference between \u2018true\u2019 articles and annulations, defined on the structure of the muscular system, is discussed on the basis of comparative developmental data. In general, annulations are produced more sequentially, compared with the almost simultaneous emergence of true articles

Appendage loss and regeneration in arthropods: a comparative view

Evidence for loss and regeneration of arthropod appendages is reviewed and discussed in terms of comparative developmental biology and arthropod phylogeny. The presence of a preferential breakage point is well documented for some, but not all, lineages within each of the four major groups - chelicerates, myriapods, crustaceans and hexapods. Undisputed evidence of true autotomy, however, is limited to isopods, decapods and some basal pterygotes, and claimed for other groups. Regeneration of lost appendages is widespread within arthropods, even if not present or documented in some groups. During regeneration, growth and differentiation of epidermis, nerves, muscles and tracheae are to some extent mutually independent, thus sometimes failing to reproduce their usual developmental interactions, with obvious consequences on the reconstruction of the lost part of the appendage. In the regeneration of appendages composed of \u2018true segments\u2019, all the segments the animal is able to regenerate are already present (with extremely rare exceptions) following the first post-operative molt, whereas the regeneration of flagellar structures is often accomplished in steps, e.g., the first regenerate may show a reduced number of flagellomeres. Lack of autotomy is likely to be the plesiomorphic condition in arthropods, a condition maintained in the Myriochelata (myriapods plus chelicerates). Autotomy evolved within the Pancrustacea, perhaps close to the origin of a Malacostraca-Hexapoda clade, and was subsequently lost by some lineages, e.g., the Hemipteroidea and the endopterygote insects. A diaphragm reducing the risk of hemorrhage at the preferred breakage point of the appendage is generally associated with autotomizing appendages, but this anatomical specialization has been lost in some groups, including one (the Dictyoptera) where autotomy is still present

An integrated approach to biological monitoring in the Lagoon of Venice

Within the course of researches for the identification of qualified keystone species, and the development of reliable methodologies for the biological monitoring of the environmental quality and the human impact on the ecosystems, we have developed an integrated approach that may provide indications of possible environmental alterations that affect biological processes at different temporal scales. Through the analysis of respiratory protein polymorphisms, measures of developmental instability, and the evaluation of genetic variation within and between populations, it is possible to get a picture of the environmental variations at short, middle and long term scale. To test this method, we chose Carcinus aestuarii as keystone species. This decapod crustacean, broadly diffused in the Venetian lagoon and along the cost of the Adriatic sea, seems to be optimal for comparing populations from different local contexts