Structure and mineralization of the spearing mantis shrimp (Stomatopoda; Lysiosquillina maculata) body and spike cuticles.

Stomatopoda is a crustacean order including sophisticated predators called spearing and smashing mantis shrimps that are separated from the well-studied Eumalacotraca since the Devonian. The spearing mantis shrimp has developed a spiky dactyl capable of impaling fishes or crustaceans in a fraction of second. In this high velocity hunting technique, the spikes undergo an intense mechanical constraint to which their exoskeleton (or cuticle) has to be adapted. To better understand the spike cuticle internal architecture and composition, electron microscopy, X-ray microanalysis and Raman spectroscopy were used on the spikes of 7 individuals (collected in French Polynesia and Indonesia), but also on parts of the body cuticle that have less mechanical stress to bear. In the body cuticle, several specificities linked to the group were found, allowing to determine the basic structure from which the spike cuticle has evolved. Results also highlighted that the body cuticle of mantis shrimps could be a model close to the ancestral arthropod cuticle by the aspect of its biological layers (epi- and procuticle including exo- and endocuticle) as well as by the Ca-carbonate/phosphate mineral content of these layers. In contrast, the spike cuticle exhibits a deeply modified organization in four functional regions overprinted on the biological layers. Each of them has specific fibre arrangement or mineral content (fluorapatite, ACP or phosphate-rich Ca-carbonate) and is thought to assume specific mechanical roles, conferring appropriate properties on the entire spike. These results agree with an evolution of smashing mantis shrimps from primitive stabbing/spearing shrimps, and thus also allowed a better understanding of the structural modifications described in previous studies on the dactyl club of smashing mantis shrimps.

Postembryonic development and differentiation of the midgut in the freshwater shrimp Neocaridina davidi (Crustacea, Malacostraca, Decapoda) larvae.

Neocaridina davidi is a freshwater shrimp that originates from Taiwan and is commonly bred all over the word. Like all decapods, which develop indirectly, this species has pelagic larvae that may differ entirely in their morphology and habits from adult specimens. To fill a gap of knowledge about the developmental biology of freshwater shrimps we decided to document the 3D-localization of the midgut inside the body cavity of larval stages of N. davidi using X-ray microtomography, and to describe all structural and ultrastructural changes of the midgut epithelium (intestine and hepatopancreas) which occur during postembryonic development of N. davidi using light and transmission electron microscopy. We laid emphasis on stem cell functioning and cell death processes connected with differentiation. Our study revealed that while the intestine in both larval stages of N. davidi has the form of a fully developed organ, which resembles that of adult specimens, the hepatopancreas undergoes elongation and differentiation. E-cells, which are midgut stem cells, due to their proliferation and differentiation are responsible for the above-mentioned processes. Our study revealed that apoptosis is a common process in both larval stages of N. davidi in the intestine and proximal region of the hepatopancreas. In zoea III, autophagy as a survival factor is activated in order to protect cells against their death. However, when there are too many autophagic structures in epithelial cells, necrosis as passive cell death is activated. The presence of all types of cell death in the midgut in the zoea III stage confirms that this part of the digestive tract is fully developed and functional. Here, we present the first description of apoptosis, autophagy and necrosis in the digestive system of larval stages of Malacostraca and present the first description of their hepatopancreas elongation and differentiation due to midgut stem cell functioning.

Ultraviolet B radiation affects epithelial cell morphology and ultrastructure in the hepatopancreas of the freshwater decapod Macrobrachium olfersii.

The hepatopancreas is the digestive organ of crustaceans, and plays important roles also in the synthesis and secretion of sexual hormones, immunological defenses and xenobiotic detoxification. Although the importance of this organ in crustaceans cannot be underestimated, the effects of ultraviolet B (UVB) radiation on hepatopancreas are poorly understood. Moreover, Macrobrachium prawns, have a transparent carapace, which make them more susceptible to UVB radiation, since their internal organs, such as hepatopancreas, are easily reached by solar radiation. Therefore, we aimed to evaluate UVB radiation toxicity on the morphology and morphometry of hepatopancreatic epithelial cells, and to investigate these UVB effects in subcellular compartments of the ecologically-important freshwater decapod, Macrobrachium olfersii. Hepatopancreas from the UVB-irradiated group showed a granular cytoplasm, with non-defined cell limits. Morphometric analyses revealed that the UVB-irradiated group exhibited a higher frequency of fibrillar (F-cell), resorptive (R-cell) and midget (M-cell), and decreased the blister-like (B-cell). It was also observed increased vacuole frequencies and increased F-, B- and R-cell volumes in the UVB-irradiated group. In addition, it was observed increased B-cell vacuolar volumes and decreased R-cell vacuolar volumes. Ultrastructural alterations occurred in subcellular compartments in F- and R-cells, e.g. loss of mitochondrial crests, morphologically compatible with mitochondrial fission, rough endoplasmic reticulum cisternae dilation, dilation of Golgi lamellar sacs, and increased vacuole and concentric membrane formation in the UVB-irradiated group. Our data showed that the hepatopancreas is an important target of UVB radiation, as demonstrated by a series of organ-specific morphological and morphometric impairments. Therefore, cell damage caused by UVB radiation can compromise metabolic functions in epithelial cells from the hepatopancreas, potentially affecting absorption, secretion and digestion processes, vitellogenin synthesis, immune responses and xenobiotic detoxification.

Functional cytology of the hepatopancreas of decapod crustaceans.

This article reviews the morphogenesis, morphology, histology, ultrastructure, and structural-functional relationships of the hepatopancreas, the main metabolic organ of the Decapoda. The hepatopancreas develops in early larval stages from a pair of lateral lobes of the midgut anlage. In adults, it consists of hundreds of blindly ending tubules that are enveloped by a muscle net consisting of longitudinal and circular fibers. Stem cells at the distal ends of the tubules give rise to three ultrastructurally different epithelial cell types, the R-, F-, and B-cells. Histochemistry, immunohistochemistry, in situ hybridization, and monitoring of ultrastructural changes under different experimental conditions allowed the attribution of functions to these cell types. R-cells serve for the absorption and metabolization of nutrients, storage of energy reserves and minerals, synthesis of lipoproteins for export to other organs, detoxification of heavy metals, and excretion of uric acid. F-cells synthesize digestive enzymes and blood proteins involved in oxygen transport and immune defense. They also detoxify some heavy metals and probably organic xenobiotics. B-cells are assumed to produce and recycle fat emulsifiers. The hepatopancreas tubules lack nerves. The presence of scattered M-cells with putative endocrine function in the epithelium suggests that the hepatopancreas is mainly hormonally controlled. M-cells probably represent a self-perpetuating cell lineage independent from E-cells. The interstitium between the tubules contains connective tissue, arterioles, hemolymph with circulating hemocytes, and fixed phagocytes that eliminate pathogens. The hepatopancreas is histologically and ultrastructurally uniform throughout the Decapoda, despite their broad variety in body size, morphology, life style, and ecology. However, in a few cavernicolous and deep-sea shrimps parts of the hepatopancreas are transformed into large oil storing and bioluminescent compartments. Within the malacostracan crustaceans, the hepatopancreas of the Decapoda is most similar to the digestive gland of the Euphausiacea, supporting close taxonomic relationship of these two taxa.

The crustacean cuticle does not record chronological age: New evidence from the gastric mill ossicles.

A proposed method to determine chronological age of crustaceans uses putative annual bands in the gastric mill ossicles of the foregut. The interpretation of cuticle bands as growth rings is based on the idea that ossicles are retained through the moult and could accumulate a continuous record of age. However, recent studies presented conflicting findings on the dynamics of gastric mill ossicles during ecdysis. We herein study cuticle bands in ossicles in four species of commercially important decapod crustaceans (Homarus gammarus, Nephrops norvegicus, Cancer pagurus and Necora puber) in different phases of the moult cycle using dissections, light microscopy, micro-computed tomography and cryo-scanning electron microscopy. Our results demonstrate that the gastric mill is moulted and ossicles are not retained but replaced during ecdysis. It is therefore not plausible to conclude that ossicles register a lifetime growth record as annual bands and thereby provide age information. Other mechanisms for the formation of cuticle bands and their correlation to size-based age estimates need to be considered and the effect of moulting on other cuticle structures where 'annual growth bands' have been reported should be investigated urgently. Based on our results, there is no evidence for a causative link between cuticle bands and chronological age, meaning it is unreliable for determining crustacean age.

Constant morphological patterns in the hemolymph vascular system of crayfish (Crustacea, Decapoda).

We present a study of the hemolymph vascular system of the marbled crayfish, Procambarus fallax f. virginalis, the only crayfish species known to be parthenogenetic. To identify potential evolutionary patterns, we compared data from a total of 48 specimens of P. fallax with 22 specimens of Orconectes limosus. Visualizations (2D and 3D) were carried out using a combination of classical and modern morphological techniques. Our data were compared to the existing literature. Like all Decapoda, both P. fallax and O. limosus have a hemolymph vascular system, consisting of a globular heart with seven off-branching arteries. We were able to visualize in detail the heart of crayfish for the first time, i.e., the myocard with its clusters of muscles running through the lumen of the heart, the valves and flaps of ostia and arteries. Furthermore, the branching patterns of the seven artery systems were analyzed. Anatomical structures identified to be consistent in all specimens of both species were combined as ground pattern of hemolymph vascular system features for Astacida.

The snapping shrimp dactyl plunger: a thermomechanical damage-tolerant sandwich composite.

The dactyl plunger of Alpheus sp. was found to be a layered composite, with mineral-rich outer and inner layers and a chitin-rich middle layer of high porosity. The chitin-rich middle layer is itself composed of several porous chitin laminae. Modelling heat conduction through the plunger cross-section revealed that the chitin-rich layer is able to insulate heat and retard its progress through the material. Heat accumulates in the plunger after a series of successive snaps and as such, its thermally resistant design can be considered most useful under the conditions of successive snapping. The plunger has a concurrent mechanical damage-tolerant design with biogenic mineral layers, viscous (chitin-mineral) interfaces, energy-dissipating porous chitin, and sidewalls composed of ordered, layered aragonite. The snapping shrimp plunger has a design that may protect it and internal soft tissues from thermomechanical damage during plunger-socket compression prior to cavitation bubble release.

Development and functional morphology of the mouthparts and foregut in larvae and post-larvae of Macrobrachium jelskii (Decapoda: Palaemonidae).

The morphology of the mouthparts and foregut of the larvae and post-larvae of Macrobrachium jelskii was investigated to determine their functional roles in feeding, in order to understand the larval feeding behaviour and the changes that occur during its development. The mouthparts and foregut of the zoea I and II are morphologically similar, rudimentary and non-functional in feeding. Only in the final larval stage, zoea III, do the external mouthparts and foregut become structurally more complex and thus likely to play a potential role in feeding. Two behavioral trials (point of no return, point of reserve saturation) evaluated the resistance to starvation in zoea I, II, and III. The results indicate that they have sufficient nutritional reserves to permit them to complete metamorphosis without feeding. Overall, our results suggest that the zoea I and II of Macrobrachium jelskii engage in obligate lecithotrophy and zoea III in facultative lecithotrophy.

Comparative ultrastructure of spermatozoa of the redclaw Cherax quadricarinatus and the yabby Cherax destructor (Decapoda, Parastacidae).

Ultrastructure of spermatozoa of redclaw Cherax quadricarinatus and yabby Cherax destructor were described and compared. The acrosome complex and nucleus are located at the anterior and posterior region of the spermatozoon, respectively. The acrosome is a complex vesicle divided into two parts: the main body of the acrosome appears as a dense cup-shaped structure in longitudinal sagittal view, with the subacrosome zone occupying the central area of the vesicle. The acrosome is larger in C. quadricarinatus (width 2.37±0.27µm, length 1.31±0.23µm) than in C. destructor (width 1.80±0.27µm, length 1.01±0.15µm). There was no significant difference in L:W ratios of the studied species. The subacrosome zone in both species consists of two areas of different electron density. The nucleus is substantially decondensed and irregular in shape, with elaborate extended processes. The examined species exhibited a well-conserved structure of crayfish spermatozoon, similar to those of Cherax cainii and Cherax albidus. Small acrosome size, the absence of radial arms, and an extracellular capsule seem to be the morphological features that mostly distinguish Cherax from the Astacidae and Cambaridae.

Optical coherence tomography as a tool for characterization of complex biological surfaces.

The advent of scanning electron microscopy has facilitated our understanding of the biology in relation to surface microstructure of many invertebrates. In recent years, interest in biomimetics and bio-inspired materials has further propelled the search for novel microstructures from natural surfaces. As this search widens in diversity to nurture deeper understanding of form and function, the need often arises to examine rare specimens. Unfortunately, most methods for characterization of the microtopography of natural surfaces are sacrificial, and as such, place limiting constraints on research progress in situations where only a few rare specimens are known, such as the rich resources lodged in natural history museum collections. In this paper, we introduce the use of optical coherence tomography (OCT) as a noninvasive tool for bioimaging surface microtopography of crab shells. The technique enables the capture of microstructures down to micron level using low coherence near-infrared light source. OCT has allowed surface microtopography imaging on crab shells to be carried out rapidly and in a nondestructive manner, compared to the scanning electron microscope technique. The microtopography of four preserved crab specimens from Acanthodromia margarita, Ranina ranina, Conchoecetes intermedius and Dromia dormia imaged using OCT were similar to images obtained from scanning electron microscope, showing that OCT imaging retains the overall morphological form during the scanning process. By comparing the physical lengths of the spinal structures from images obtained from OCT and scanning electron microscope, the results showed that dimensional integrity of the images captured from OCT was also maintained.

Description of the newly-hatched juvenile of Aegla paulensis (Decapoda, Anomura, Aeglidae).

The postembryonic development of Aegla paulensis is epimorphic (= direct), in which the hatching form is a juvenile that morphologically resembles the adult. Hatching is asynchronous, taking 3-5 days for all juveniles from a single brood to hatch. This paper provides a complete description of the external morphology of the newly-hatched juvenile of A. paulensis as analyzed through light microscopy (LM) and scanning electron microscopy (SEM). One of the striking features ob- served in the newly-hatched juvenile of A. paulensis was the presence of four pairs of rudimentary pleopods, a trait never described before in early juveniles of Aegla. Additional novelties include three unique types of setae and two types of pore sensilla.

Functional morphology of mouthparts and digestive system during larval development of the cleaner shrimp Lysmata amboinensis (de Man, 1888).

Mouthpart and alimentary canal development was examined in Lysmata amboinensis larvae using scanning electron microscopy and histology. The gross morphological features of external mouthparts and internal digestive tract structures of larvae at different developmental stages indicate that ingestive and digestive capabilities are well developed from early on. With increasing age of the larvae the mouthpart appendages increased in size, the hepatopancreas in tubular density and the midgut in length. The density of setae and robustness of teeth and spines of individual structures increased. The most pronounced changes from early to late stage larvae involved formation of pores on the paragnaths and labrum, transformation of the mandibular spine-like teeth to molar cusps, development of the filter press in the proventriculus and of infoldings in the previously straight hindgut. The results suggest that early stage L. amboinensis larvae may benefit from soft, perhaps gelatinous prey, whereas later stages are better equipped to handle larger, muscular or more fibrous foods.

In vitro fertilization of the rock shrimp, Rhynchocinetes typus (Decapoda, Caridea): a review.

This review compiles all the research done on gametes and fertilization in the rock shrimp, R. typus, and describes the sequence of events from the first gamete interaction to zygote formation and the first cleavage of the embryo, with light, fluorescence confocal and electron microscopes. Early studies showed that sperm from the vas deferens have a tack-shape with a "needle-like process" or rigid spike (RS) that extends from a semi-spherical body that contains the arms with chromatin and spines. Upon contact with seawater and by action of Na +, the arms and spines extend, producing an inverted umbrella form of the spermatozoa. The first sperm-oocyte interaction occurs between protein receptors type lectins of the sperm RS and oocyte chorion sperm ligands. These ligands contain residues of α-Glu, Man (α 1-3) Man, α and ß-GlcNAc and α-GalNA terminal residues. It was found that α-Man and GlcNAc residues are the ligands that are directly related to the adhesion process and further penetration of sperm. After this first interaction, the RS enters the oocyte envelope by the action of a trypsin-like enzyme, rhynchocinecine, present in the acicular process. Later, arms and spines penetrate the oocyte cytoplasm, where the chromatin of the arms begin to migrate to the central area of the sperm, condensing in a cup-shaped structure near the connecting piece, which forms the male pronucleus.

In vitro fertilization of the rock shrimp, Rhynchocinetes typus (Decapoda, Caridea): a review

This review compiles all the research done on gametes and fertilization in the rock shrimp, R. typus, and describes the sequence of events from the first gamete interaction to zygote formation and the first cleavage of the embryo, with light, fluorescence confocal and electron microscopes. Early studies showed that sperm from the vas deferens have a tack-shape with a "needle-like process" or rigid spike (RS) that extends from a semi-spherical body that contains the arms with chromatin and spines. Upon contact with seawater and by action of Na +, the arms and spines extend, producing an inverted umbrella form of the spermatozoa. The first sperm-oocyte interaction occurs between protein receptors type lectins of the sperm RS and oocyte chorion sperm ligands. These ligands contain residues of a-Glu, Man (a 1-3) Man, a and p-GlcNAc and a-GalNA terminal residues. It was found that a-Man and GlcNAc residues are the ligands that are directly related to the adhesion process and further penetration of sperm. After this first interaction, the RS enters the oocyte envelope by the action of a trypsin-like enzyme, rhynchocinecine, present in the acicular process. Later, arms and spines penetrate the oocyte cytoplasm, where the chromatin of the arms begin to migrate to the central area of the sperm, condensing in a cup-shaped structure near the connecting piece, which forms the male pronucleus.

Reciprocal changes in calcification of the gastrolith and cuticle during the molt cycle of the red claw crayfish Cherax quadricarinatus.

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l(-1)). On the other hand, pH varied greatly, from 8.7+/-0.15 in the gastrolith cavity through 7.6+/-0.2 in muscle to 6.9+/-0.5 in the stomach.

Characteristics and fate of the spermatozoa of Inachus phalangium (Decapoda, Majidae): description of novel sperm structures and evidence for an additional mechanism of sperm competition in Brachyura.

Various aspects of the reproductive anatomy of the spider crab Inachus phalangium are investigated utilizing light and electron microscopy. Spermatozoal ultrastructure reveals the presence of a glycocalyx in the peripheral region of the periopercular rim, never recorded before in crustacean sperm cells. Sperm cell morphological traits such as semi-lunar acrosome shape, centrally perforate and flat operculum, and absence of a thickened ring, are shared only with Macropodia longirostris, confirming a close phylogenetic relationship of these species and their separation from the other members of the family Majidae. Spermatozoa are transferred to females inside spermatophores of different sizes, but during ejaculate transfer, larger spermatophores might be ruptured by tooth-like structures present on the ejaculatory canal of the male first gonopod, releasing free sperm cells. Such a mechanism could represent the first evidence of a second form of sperm competition in conflict with sperm displacement, the only mechanism of sperm competition known among Brachyura, enabling paternity for both dominant and smaller, non-dominant, males. In addition, we propose several hypotheses concerning the remote and proximal causes of the existence of large seminal receptacles in females of I. phalangium. Among these, genetically diverse progeny, reduction of sexual harassment and phylogenetic retention seem the most plausible, while acquisition of nutrients from seminal fluids, demonstrated in other arthropods, and suggested by previous studies, could be discarded on the basis of the presented data.

Functional consequences of activity-dependent synaptic enhancement at a crustacean neuromuscular junction.

This study provides evidence that activity-dependent synaptic enhancement at a neuromuscular junction modifies the characteristics of force production of the receiving muscle during rhythmic motor neuron discharge patterns. Long-lasting augmentation of the excitatory junction potentials (EJPs) quickens and strengthens the muscle response to a given motor pattern. We used the muscle gm6 of the crab Cancer pagurus to study the functional consequences and temporal dynamics of facilitation and augmentation. This stomach muscle is driven by the rhythmic activity of the gastric mill central pattern generator in the stomatogastric nervous system. We tested the response of this muscle to rhythmic motor drive using a variety of gastric mill-like stimulations. EJPs recorded in muscle gm6 were initially small but are summated and facilitated strongly with continuous stimulation. Facilitation increased with shorter interspike intervals and possessed a time constant of decay <1 s. During gastric mill rhythms, motor neuron activity was by contrast represented by bursts of activity with intermittent pauses of several seconds. Recordings in intact animals and in the isolated nervous system showed a great variability in firing frequency and temporal distribution of motor neuron bursts. Train stimulations with various stimulus frequencies (5 Hz, 10 Hz, 20 Hz) and inter-train intervals (2 s, 4 s, 8 s, 16 s, 32 s) revealed that augmentation acted in addition to facilitation. Augmentation increased muscle EJPs during stimulations with inter-train intervals of 16 s or less. The effects of augmentation increased with shorter inter-train intervals, but were independent of stimulus frequency. Augmentation also contributed to the electrical response of the muscle during gastric mill rhythms, which were obtained in vitro and in vivo, and was also reflected by an increase of muscle force and the slope of force development during repetitive train stimulation. We conclude that the augmentation of EJPs at the neuromuscular junction tunes the muscle response to support force production during rhythmic motor patterns.

Exuviotrophic apostome ciliates from crustaceans of St. Andrew Bay, Florida, and a description of Gymnodinioides kozloffi n. sp.

Gymnodinioides kozloffi n. sp. is described from the eelgrass broken-back shrimp Hippolyte zostericola. The species is distinct from others in the apostome genus Gymnodinioides in that the trophont ciliature has a small group of kinetosomes located to the right of Kinety 9a, and Kinety 1 and 2 are divided. Other apostome morphologies are described from many decapod crustaceans from St. Andrew Bay, Florida, including Gymnodinioides inkystans, Hyalophysa chattoni, and variants of both H. chattoni and G. kozloffi. All of these apostome ciliates are exuviotrophic, found feeding on exuvial fluid within the exoskeleton of the host after ecdysis. The hosts surveyed for this study are the following: Callinectes sapidus, Eurypanopeus depressus, Hippolyte zostericola, Farfantepenaeus spp., Palaemonetes intermedius, Palaemon floridanus, Portunus spp., Tozeuma carolinense, and Sicyonia laevigata, which revealed a number of new host-apostome records.

Interspecific variations in the morphology and ultrastructure of the rhabdoms of oplophorid shrimps.

Interspecific variations in rhabdom structure between various oplophorid shrimps are described and the differences are related to the light environment at different depths within the mesopelagic zone. The ultrastructure of the distal rhabdom in these species is described for the first time. Quantitative measurements show that the proportion of the rhabdom layer occupied by the distal rhabdom varies from 3.5-25% in the dorsoventral plane of the eye of Systellaspis debilis. The distal rhabdom occupies less than 1% of the rhabdoms in the eye of Acanthephyra pelagica, where it can only be seen by using the electron microscope. It is suggested that the rhabdoms of those species that remain within the photic zone (such as S. debilis) are adapted to maximize contrast, whereas in those whose depth ranges extend into the aphotic zone (such as A. pelagica) they are adapted for maximum sensitivity.

Use of the confocal laser scanning microscope in studies on the developmental biology of marine crustaceans.

Confocal Laser Scanning Microscope techniques have been applied to study the developmental biology of marine copepods and decapod larvae. The lipophylic probes DiI and DiOC(6) were used to study both the external and internal morphology of these crustaceans, whereas the same DiOC(6) and the specific nuclear probe Hoechst 33342 were used to study embryonic development of copepods in vivo. To distinguish viable from non-viable copepod embryos, the vital dye dichlorodihydrofluorescein diacetate (H(2)DCFDA) was used. Major advantages and difficulties in the use of these non-invasive techniques in studies of the reproductive biology of marine crustaceans are discussed.