The cephalothoracic sucker of sea lice (Crustacea: Copepoda: Caligidae): The functional importance of cuticular membrane ultrastructure.

Sea lice adhere to the body surface of host fish with a cephalothoracic sucker. Caligus adheres to this substrate using legs 2 and 3, and the action of cephalothoracic muscles. Lunules, small, paired, anterior sucker-like structures, have a vital function in the initial step of adhering and contain a unique endocuticule containing elements that may behave like active matter and serve as the actuating mechanism. Cuticular membranes bordering the cephalothorax have a unique endocuticule with an undulating dorsal surface and a smooth ventral surface. A high-speed camera revealed that this undulation likely facilitates rapid automatic application of the sucker to the substrate. The cuticular membranes on the posterior margin of the first exopodal segment of leg 2 have a specialized endocuticle with tubules each surrounded by fine fibers. This reinforcement helps them to generate a posteriorly-directed jet of water. Opening-closing of these membranes is controlled by postero-anterior motion of the distal exopodal segments of leg 2. The outer cuticular membrane of leg 3 is simple, presumably effected by powerful extrinsic muscles. The consistency of sucker morphology within Caligus implies a highly stereotyped attachment behavior that is effective across a remarkable variety of fishes.

Evolutionary transformation of mouthparts from particle-feeding to piercing carnivory in Viper copepods: Review and 3D analyses of a key innovation using advanced imaging techniques.

BACKGROUND: Novel feeding adaptations often facilitate adaptive radiation and diversification. But the evolutionary origins of such feeding adaptations can be puzzling if they require concordant change in multiple component parts. Pelagic, heterorhabdid copepods (Calanoida) exhibit diverse feeding behaviors that range from simple particle feeding to a highly specialized form of carnivory involving piercing mouthparts that likely inject venom. We review the evolutionary history of heterorhabdid copepods and add new high-resolution, 3D anatomical analyses of the muscular system, glands and gland openings associated with this remarkable evolutionary transformation. RESULTS: We examined four heterorhabdid copepods with different feeding modes: one primitive particle-feeder (Disseta palumbii), one derived and specialized carnivore (Heterorhabdus subspinifrons), and two intermediate taxa (Mesorhabdus gracilis and Heterostylites longicornis). We used two advanced, high-resolution microscopic techniques - serial block-face scanning electron microscopy and two-photon excitation microscopy - to visualize mouthpart form and internal anatomy at unprecedented nanometer resolution. Interactive 3D graphical visualizations allowed putative homologues of muscles and gland cells to be identified with confidence and traced across the evolutionary transformation from particle feeding to piercing carnivory. Notable changes included: a) addition of new gland cells, b) enlargement of some (venom producing?) glands, c) repositioning of gland openings associated with hollow piercing fangs on the mandibles, d) repurposing of some mandibular-muscle function to include gland-squeezing, and e) addition of new muscles that may aid venom injection exclusively in the most specialized piercing species. In addition, live video recording of all four species revealed mandibular blade movements coupled to cyclic contraction of some muscles connected to the esophagus. These behavioral and 3D morphological observations revealed a novel injection system in H. subspinifrons associated with piercing (envenomating?) carnivory. CONCLUSIONS: Collectively, these results suggest that subtle changes in mandibular tooth form, and muscle and gland form and location, facilitated the evolution of a novel, piercing mode of feeding that accelerated diversification of the genus Heterorhabdus. They also highlight the value of interactive 3D animations for understanding evolutionary transformations of complex, multicomponent morphological systems.

Pheromone gland development and monoterpenoid synthesis specific to oviparous females in the pea aphid.

BACKGROUND: Aphids display "cyclic parthenogenesis," in which parthenogenetically and sexually reproducing morphs seasonally alternate in the aphid annual life cycles. There are various characteristics that differ between asexual viviparous and sexual oviparous females. In oviparous females, swollen cuticular structures (~ 10 µm in diameter), called "scent plaques," are scattered on the surface of hind tibias, and secrete monoterpenoid sex pheromones. However, the developmental processes of the pheromone glands and the biosynthetic pathways of monoterpenoid pheromones have yet to be elucidated. RESULTS: Comparisons of the developmental processes that form hind tibias between sexual and parthenogenetic females revealed that, in sexual females, the epithelial tissues in proximal parts of hind tibias become columnar in fourth instar nymphs, and circular pheromone glands with Class 1 gland cells appear in adults, although they do not appear in parthenogenetic females. Furthermore, by comparing the expression levels of genes involved in the mevalonate pathway, which is required for monoterpenoid synthesis, we show that genes that encode the downstream enzymes in the pathway are highly expressed in hind tibias of sexual females. CONCLUSION: Glandular tissues of scent plaque are differentiated from the fourth instar in sexual females, while parthenogenetic females lack the glandular cells. Only the downstream steps of the mevalonate pathway appear to occur in scent plaques on hind tibias of sexual females, although the upstream steps may occur somewhere in other body parts.

Parallel Saltational Evolution of Ultrafast Movements in Snapping Shrimp Claws.

How do stunning functional innovations evolve from unspecialized progenitors? This puzzle is particularly acute for ultrafast movements of appendages in arthropods as diverse as shrimps [1], stomatopods [2], insects [3-6], and spiders [7]. For example, the spectacular snapping claws of alpheid shrimps close so fast (∼0.5 ms) that jetted water creates a cavitation bubble and an immensely powerful snap upon bubble collapse [1]. Such extreme movements depend on (1) an energy-storage mechanism (e.g., some kind of spring) and (2) a latching mechanism to release stored energy quickly [8]. Clearly, rapid claw closure must have evolved before the ability to snap, but its evolutionary origins are unknown. Unearthing the functional mechanics of transitional stages is therefore essential to understand how such radical novel abilities arise [9-11]. We reconstructed the evolutionary history of shrimp claw form and function by sampling 114 species from 19 families, including two unrelated families within which snapping evolved independently (Alpheidae and Palaemonidae) [12, 13]. Our comparative analyses, using micro-computed tomography (microCT) and confocal imaging, high-speed video, and kinematic experiments with select 3D-printed scale models, revealed a previously unrecognized "slip joint" in non-snapping shrimp claws. This slip joint facilitated the parallel evolution of a novel energy-storage and cocking mechanism-a torque-reversal joint-an apparent precondition for snapping. Remarkably, these key functional transitions between ancestral (simple pinching) and derived (snapping) claws were achieved by minute differences in joint structure. Therefore, subtle changes in form appear to have facilitated wholly novel functional change in a saltational manner. VIDEO ABSTRACT.

How reversible is development? Contrast between developmentally plastic gain and loss of segments in barnacle feeding legs.

Segmented organisms and structures have fascinated biologists since William Bateson first described homeotic transformation and recognized the fundamental evolutionary significance of segmental organization. On evolutionary time scales, segments may be lost or gained during major morphological transitions. But how segment loss compares to gain on developmental time scales remains mysterious. Here, we examine the ease of reverse development (opposite to normal growth) by comparing developmentally plastic leg segment loss versus gain in individual barnacles transplanted between different water flow conditions. Plastic segment addition occurred rapidly (one to two molts) and exclusively near the limb base. In contrast, developmentally plastic segment loss-the first observation in any arthropod-took much longer (>10 molts) and, remarkably, occurred throughout the leg (23% of losses occurred mid-limb). Segment loss was not a simple reversal of segment addition. Intersegmental membranes fused first, followed by elimination of duplicate tendons and gradual shortening (but not loss) of duplicate setae. Setal loss, in particular, may impose a severe developmental constraint on arthropod segment fusion. This asymmetric developmental potential (time lag of phenotypic response)-plastic segment addition (amplified normal development) is faster and more orderly than segment loss (reverse development)-adds a new dimension to models of developmental plasticity because the cost of making a developmental mistake in one direction will be greater than in the other.

Mesoscale morphology at nanoscale resolution: serial block-face scanning electron microscopy reveals fine 3D detail of a novel silk spinneret system in a tube-building tanaid crustacean.

BACKGROUND: The study of morphology is experiencing a renaissance due to rapid improvements in technologies for 3D visualization of complex internal and external structures. But 3D visualization of the internal structure of mesoscale objects - those in the 10-1000 µm range - remains problematic. They are too small for microCT, many lack suitable specific fluorescent markers for confocal microscopy, or they require labor-intensive stacking and smoothing of individual TEM images. Here we illustrate the first comprehensive morphological description of a complete mesoscale biological system at nanoscopic resolution using ultra-modern technology for 3D visualization - serial block-face scanning electron microscopy (SBF-SEM). The SBF-SEM machine combines an in-chamber ultramicrotome, which creates a serial array of exposed surfaces, with an SEM that images each surface as it is exposed. The serial images are then stacked automatically by 3D reconstruction software. We used SBF-SEM to study the spinneret (thread-producing) system of a small, tube-dwelling crustacean that weaves tubes of silk. Thread-producing ability is critical for the survival of many small-bodied animals but the basic morphology of these systems remains mysterious due to the limits of traditional microscopy. RESULTS: SBF-SEM allowed us to describe - in full 3D - well-resolved components (glands, ducts, pores, and associated nerves and muscles) of the spinneret system in the thoracic legs and body segments of Sinelobus sp. (Crustacea, Peracarida, Tanaidacea), a tube-building tanaid only 2 mm in body length. The 3D reconstruction by SBF-SEM revealed at nanoscale resolution a unique structure to the gland and duct systems: In each of three thread-producing thoracic segments, two separate ducts, derived from two separate glands located in the body, run through the entire leg and merge at the leg tip just before the spinneret pore opening. We also resolved nerves connecting to individual setae, spines and pores on the walking legs, and individual muscles within each leg segment. CONCLUSIONS: Our results significantly expand our understanding of the diversity of spinneret systems in the Crustacea by providing the first well-resolved view of spinneret components in the peracarid crustacean order, Tanaidacea. More significantly, our results reveal the great power of SBF-SEM technology for comprehensive studies of the morphology of microscopic animals.

Functional transformation series and the evolutionary origin of novel forms: evidence from a remarkable termite defensive organ.

The origins of evolutionary novelties are often deeply puzzling. They are generally associated with new functions that were absent in ancestors. The new functional configuration should arise via intermediate stages without any loss of function or impediment to the whole organism during the transitions. Therefore, understanding of the functional configurations of transitional states can shed light on how novel forms arise. Here we infer the evolutionary origin of a highly specialized termite defensive organ "nasus" where different functions overlap in different structural configurations at intermediate evolutionary stages to ensure that each phase is functional. Soldiers of a nasutitermitine termite use reconfigured mandibular muscles to squirt a viscous secretion from a nozzle-like head projection (the nasus). This contrasts sharply with the primitive defensive strategy where mandibles are used to bite. MicroCT observations of soldiers of Nasutitermes takasagoensis and of species with the ancestral state (Hodotermopsis sjostedti, Embiratermes neotenicus) revealed three different yet fully functional configurations in the transition from ancestral to novel state: (i) elevated hydrostatic pressure induced by contraction of mandibular muscles when biting gently oozes secretion from a gland; (ii) direct pressure on an enlarged gland arises from expansion of the mandibular muscles when biting; (iii) squirting in a piston-like manner by an inflated gland enveloped by highly modified mandibular muscles. Even a structure as exotic as the nasus therefore appears to have evolved with no loss of function at any stage. Such a functional approach, holds much promise for understanding the evolutionary origin of seemingly preposterous novel forms.

Male claspers in clam shrimps (Crustacea, Branchiopoda) in the light of evolution: a case study on homology versus analogy.

Male "clam shrimps" possess highly modified first (and second) trunk limbs for clasping the carapace of females during copulation. Claspers are present in all three clam shrimp taxa (Laevicaudata, Spinicaudata, and Cyclestherida) but despite striking similarities in their morphology and function, the matter of their homology is controversial. In this study, we address the question of the homology and evolution of these structures by comparing the developmental transformation of an unspecialized trunk limb into a clasper. In addition, we study the musculature and the nervous system in trunk limbs and claspers using confocal laser scanning microscopy. We establish that most (but not all) of the various parts of the claspers are homologous between clam shrimp taxa. We suggest that a single pair of claspers was already present in the ground pattern of Diplostraca, probably most comparable to those in Cyclestherida. The claspers, therefore, do not represent a case of analogy.

The lunule of caligid copepods: an evolutionarily novel structure.

Nearly half of the genera of the family Caligidae possess an evolutionarily novel structure called the "lunule" on the ventral surface of the frontal plate. Lunules are paired cup-like suckers that assist in securing attachment of the copepod parasite to its host. Although present in genera such as Caligus and Pseudocaligus, lunules are absent in other caligid genera such as Lepeophtheirus as well as in more primitive caligiforms such as members of the families Trebiidae and Dissonidae. We compared the morphology and development of the anterior margin of the frontal plates between two caligids, Pseudocaligus fugu and Lepeophtheirus sekii, and a more basal caligiform, Dissonus heronensis (a dissonid), using scanning electron, transmission electron, and laser confocal microscopes. Our observations suggest that the lunules originated as a modification of the marginal membranes of the ancestral frontal plates. We also demonstrated the presence of an anlagen cell population for the lunule and marginal membrane in the developing frontal plate. These primordial cells can be detected as early as the first stage of the chalimus phase. Based on these observations, an evolutionary scenario for the lunule is proposed based on cytological evidence. This case study enhances our understanding of "evolutionary novelty," which is a main focus of contemporary evolutionary developmental biology.

Developmental process of musculoskeletal integration in ostracod antenna.

The functional morphology of arthropod appendages shows remarkable diversity. Plausible functional integrations, particularly between muscles and the exoskeleton, must be achieved in these diverse morphologies. This study provides an insight into the evolutionary pathway of diversified appendages from a functional point of view. The musculoskeletal structure and development of antennae in five species of Cypridocopina were compared. The muscle and skeletal systems are integrated in several ways: The integration in Propontocypris attenuata occurs during various stages of the molting growth, whereas that in Fabaeformiscandona breuili occurs during the myogenesis. These two types of developmental processes have notable similarities, despite their occurrence during different developmental phases. From the overview of the molecular phylogeny presented by earlier studies, it is suggested that the integrated musculoskeletal system has reappeared repeatedly in cypridoid lineages as an atavism. This study demonstrates how arthropod appendages evolve without losing the integrity of the functional whole.

A bridge between original and novel states: ontogeny and function of "suction discs" in the Branchiura (Crustacea).

The emergence of novel structures in the course of evolution faces an explanatory problem, leaving the gap from the ancestral structures difficult to bridge. This difficulty is caused by the lack of intermediate stages. Branchiurans are ectoparasitic crustaceans which use a pair of "suction discs" to attach to their host. These structures are modified first maxillae. During ontogeny, the first maxillae transform from a normal cephalic appendage to the specialized suction disc. However, supposedly ancestral branchiurans lack the suction discs in the adults and the first maxilla remains a normal appendage throughout. We describe the muscular arrangements in the developing first maxillae in Argulus coregoni. The suction discs originate as a fusion of the first and second podomeres. The sucker muscles of the suction discs are homologous to the muscles that insert in the second podomere at the early larval stages. The developmental process of the suction disc can be seen as a "recapitulation" of the evolutionary process. We thus show how the first maxilla can maintain not just the biological role but also a functional continuity during the evolution of the novel structure. From this example it is obvious that the intermediate stages of the emerging novelty, if present in the ontogeny, can help solve at least some of the enigmatic appearances of novel structures.

Ontogeny and function of the fifth limb in Cypridocopain ostracods.

The exoskeleton of arthropods undergoes reformation at every molting. Accordingly, external morphology can metamorphose through molting. In some crustaceans, the function of appendages is modified through ontogeny. These morphological modifications require accordant modification of the correlation between different body parts because the morphological function depends on the combined correlation between different parts. In the case of crustacean morphology, exoskeleton and muscles are correlated to each other. The functional morphology of the fifth limb of cypridoid ostracods transforms from "walking leg + mouthparts (+ possibly respiratory parts)" to "mouthparts + respiratory parts + grasping hook (in males only)" through ontogeny. In this study, the three-dimensional structures of the exoskeleton and muscular systems were observed by confocal laser-scanning microscopy in some species of suborder Cypridocopina. The muscular system is reportedly not changed by the ontogeny of appendages in females, but it does change in males. Furthermore, regional cell proliferation, which was detected previously, represented the causal factor of exoskeletal modification. I therefore conclude that the enlarged endite in the female fifth limb is produced by exoskeletal modification based on regional cell proliferation, rather than by a change in the muscular system. In contrast, modification in the male requires a change in the muscular system in addition to exoskeletal modification.

Homology and evolution of the antenna in podocopid ostracods from the perspective of aesthetascs.

Antennal podomere homology has not been well documented in podocopid ostracods. Difficulties associated with describing this homology are compounded by the occurrence of specialised podomeres in both cytheroids and bairdioids. Our research establishes the existence of two kinds of aesthetascs shared among multiple higher taxa. Overgrowth "t-setae" are present in males in Cytheroidea, Cypridocopina and Darwinuloidea, and "aesthetasc yc" is found in both sexes in Cytheroidea and Bairdioidea. Homology of the antennal podomeres among all podocopid superfamilies was determined by using the chaetotaxy of these aesthetascs, leading to a description of evolutionary modifications of the podocopid antenna, which suggests that changes in function of the articulation were prompted by the temporal demands of copulatory behavior in each lineage.

Origin of the novel chemoreceptor Aesthetasc "Y" in Ostracoda: morphogenetical thresholds and evolutionary innovation.

The morphology and developmental processes of the two types of ostracod chemoreceptors, the Aesthetasc "Y" and the "Grouped setae," were compared. Cypridoidea and Pontocypridoidea, belonging to Cypridocopina, have a large baseball bat-like seta as an autapomorphic character on the second antenna, whereas most ostracod taxa with plesiomorphic characters bear "Grouped setae" consisting of multiple setae on the second antenna. Their budding positions, morphology, and ontogenetic changes were compared, and our deduction is that the Aesthetasc "Y" originated from "Grouped setae-like" organ in the Paleozoic. The morphogenetic processes in the molting period of these chemoreceptors were compared at the cellular level. The observations suggest that the "Grouped setae" are formed by hypodermal cells and share sheath cells corresponding to those of the Aesthetasc "Y" as a common constraint in the molting process of setae. We conclude that modification of the morphogenetic processes in the molting period of the "Grouped setae" gave rise to the Aesthetasc "Y" as a novel organ in the evolutionary pathway of the Ostracoda.