Functional anatomy of the female reproductive system of the American lobster (Homarus americanus).

Light microscopy studies of the female American lobster Homarus americanus reproductive system are essentially nonexistent or outdated. Based on samples taken in the spring, summer, and autumn from the southern Gulf of St. Lawrence between 1994 and 2014, and using a combination of histological and scanning electron microscope techniques, we propose an ovarian cycle with 10 stages, identifying for the first time a recovery stage. Also, an atypical resorption stage, characterized by massive reabsorption of mature oocytes, is occasionally observed during summer months. The oviducts are composed of connective tissue (elastic and collagen fibers) with no muscle or secretory activities. Their epithelium shows a cyclic pattern and phagocytosis activities linked to spawning. Although the role of the seminal receptacle is to store and protect semen, free spermatozoa (i.e., without the spermatophoric wall and the acellular gelatinous substance that constitute the semen) were also observed in its posteriolateral grooves immediately prior to spawning, which is consistent with an external fertilization mechanism at the seminal receptacle. Unexpectedly, free spermatozoa were observed externally near two pore-like structures located on the gonopore's operculum, not at the seminal receptacle, after spawning; hence, more work is needed to fully understand the fertilization mechanism for the American lobster.

Internal anatomy and ultrastructure of the male reproductive system of the Norway lobster Nephrops norvegicus (Decapoda: Astacidea).

The Norway lobster (Nephrops norvegicus) is economically important in Europe. However, apart from the female reproductive system, very little is known about its internal anatomy. This article focuses on studying the internal anatomy and ultrastructure of the male reproductive system. This system follows the general pattern found among decapod crustaceans, with several peculiarities. Testes are composed of lobular sperm ducts in which the spermatozoa are fully constituted. The spermatozoa present three lateral arms and a long acrosome, which gives a false appearance of flagellated spermatozoa. The two testes form a double H under the heart, and the vas deferens (VD) arise from each side at the posterior edge of the double H. The main characteristic of the VD is the presence of a sphincter in the enlarged area of the distal end of the middle VD. The MVD here shows an increase in musculature of the wall as compared to the VD, which regulates the passage of the sperm cord to the distal VD (DVD) and thence to the thelycum of the female. The wall of the spermatophore is formed in the distal part of the proximal VD, which surrounds the unique sperm cord present in the VD. Isolated spermatophores are not observed in the VD. The sperm cord is pinched off during copulation by the musculature of the DVD. Then, a portion of the sperm cord is transferred from each VD to form the isolated spermatophores. The wall of the spematophores and the spermatozoa that are observed inside the thelycum have the same morphology as those observed in the VD.

Plastic contamination in the decapod crustacean Nephrops norvegicus (Linnaeus, 1758).

The aim of this study was to determine the extent Nephrops consumes plastics in the Clyde Sea and if this intake occurs through their diet. Plastic contamination was found to be high in Nephrops, 83% of the animals sampled contained plastics (predominately filaments) in their stomachs. Tightly tangled balls of plastic strands were found in 62% of the animals studied but were least prevalent in animals which had recently moulted. No significant difference in plastic load was observed between males and females. Raman spectroscopy indicated that some of the microfilaments identified from gut contents could be sourced to fishing waste. Nephrops fed fish seeded with strands of polypropylene rope were found to ingest but not to excrete the strands. The fishery for Norway lobster, Nephrops norvegicus, is the most valuable in Scotland and the high prevalence of plastics in Nephrops may have implications for the health of the stock.

Morphologic effects of in vivo acute exposure to the pesticide methoprene on the hepatopancreas of a non-target organism, Homarus americanus.

Methoprene is a pesticide widely used for mosquito control. It is an endocrine disruptor, acting as an analog of juvenile hormone. While targeting insect larvae, it also impacts non-target animals including crustaceans. Anecdotal reports suggested that methoprene has unintended effects on adult arthropods. Earlier, we documented effects in adult lobsters at the metabolic and gene expression levels. In this study we have documented morphologic corollaries to our prior observations. We examined the light and electron microscopic changes in the hepatopancreas of adult lobsters following in vivo acute exposure to methoprene. Changes by light and electron microscopy levels were evident following exposure to sub-lethal concentrations of methoprene for 24h. Tissue from exposed animals showed the formation of extensive cytoplasmic spaces (vesiculation) with disruption and loss of specific subcellular organelles. The findings provide morphologic correlates to the metabolic and genomic alterations we have observed in previous investigations.

Electron-microscopical localization of gelsolin in various crustacean muscles.

Gelsolin was localized by immunoelectron microscopy in fast and slow cross-striated muscles of the lobster Homarus americanus. When ultrathin sections of the muscles were labelled with anti-gelsolin and a gold-conjugated second antibody, 90% of all gold particles in the myoplasm were detected on myofibrils, preferentially in the I-band and AI-region of the sarcomeres. Both the region of the H-zone (lacking thin filaments) and the Z-disc contained no or little gold label. Under physiological conditions, a close association of gelsolin with the thin filaments was observed for both muscle types. The preferential localization of particles in the I- and AI-region indicated that gelsolin was distributed randomly over the whole length of the thin filaments. Preincubation of muscle strips with Ringer solution containing 0.5 mM EGTA resulted in a significantly different distribution pattern; gold particles were now localized preferentially in the cell periphery close to the sarcolemma, with significantly decreased abundance in the centre of the cell. Compared with the muscle under physiological conditions, the number of gold particles over sarcomeric structures was significantly reduced. Thus, binding of gelsolin to the thin filaments is apparently reversible in vivo and depends on the presence of calcium ions. We assume a functional role for gelsolin in the actin turnover processes in invertebrate muscle systems.

Influence of microstructure on deformation anisotropy of mineralized cuticle from the lobster Homarus americanus.

The exoskeleton of the American lobster Homarus americanus is a hierarchically organized nano-composite material consisting of organic chitin-protein fibers associated with inorganic calcium carbonate. The presence of a well-developed and periodically arranged pore canal system leads to a honeycomb-like structure. The concomitant presence of the twisted plywood arrangement of the mineralized chitin-protein fibers alters the elastic properties, the deformation behavior, and fracture behavior compared to classical honeycomb structures. By performing compression tests in various directions of the cuticle we examined the anisotropic elastic-plastic deformation and fracture behavior of mineralized parts of the exoskeleton. By applying digital image correlation during compression testing, the evolution of the elastic-plastic deformation at the microscopic scale was observed with high resolution and simultaneously global stress and strain data were acquired. Shear tests were performed in order to determine the fracture energy for different shear planes and directions. The investigation of the microstructure after plastic deformation revealed the underlying deformation mechanisms of lobster endocuticle from the claws under different loading conditions. For evaluating the effect of hydration the samples were tested both in the dry and in the wet state.

Preferred crystallographic texture of alpha-chitin as a microscopic and macroscopic design principle of the exoskeleton of the lobster Homarus americanus.

The crystallographic texture of the crystalline alpha-chitin matrix in the biological composite material forming the exoskeleton of the lobster Homarus americanus has been determined using synchrotron X-ray pole figure measurements and the calculation of orientation distribution functions. The study has two objectives. The first one is to elucidate crystallographic building principles via the preferred synthesis of certain orientations in crystalline organic tissue. The second one is to study whether a general global design principle exists for the exoskeleton which uses preferred textures relative to the local coordinate system throughout the lobster cuticle. The first point, hence, pursues the question of the extent to which and why alpha-chitin reveals preferred textures in the lobster cuticle. The second point addresses the question of why and whether such preferred textures (and the resulting anisotropy) exist everywhere in the exoskeleton. Concerning the first aspect, a strong preference of a fiber texture of the orthorhombic alpha-chitin is observed which is characterized by a 020 crystal axis normal to the exoskeleton surface for the chitin matrix. The second question is tackled by studying samples from different parts of the carapace. While the first aspect takes a microscopic perspective at the basic structure of the biological composite, the second point aims at building a bridge between an understanding of the microstructure and the macroscopic nature of a larger biological construction. We observe that the texture is everywhere in the carapace optimized in such a way that the same crystallographic axis of the chitin matrix is parallel to the normal to the local tangent plane of the carapace. Notable differences in the texture are observed between hard mineralized parts on the one hand and soft membranous parts on the other. The study shows that the complex hierarchical microstructure of the arthropod cuticle can be well described by surprisingly simple crystallographic textures.

Ultrastructural studies and Na+,K+-ATPase immunolocalization in the antennal urinary glands of the lobster Homarus gammarus (Crustacea, Decapoda).

Unlike in crustacean freshwater species, the structure and ultrastructure of the excretory antennal gland is poorly documented in marine species. The general organization and ultrastructure of the cells and the localization of Na(+),K(+)-ATPase were examined in the antennal gland of the adult lobster Homarus gammarus. Each gland is composed of a centrally located coelomosac surrounded ventrally by a labyrinth divided into two parts (I and II) and dorsally by a voluminous bladder. There is no differentiated nephridal tubule between them. The labyrinth and bladder cells have in common a number of ultrastructural cytological features, including basal membrane infoldings associated with mitochondria, apical microvilli, and cytoplasmic extrusions, and a cytoplasm packed with numerous vacuoles, vesicles, lysosome-like bodies, and swollen mitochondria. Each type of cell also presents distinctive characters. Na(+),K(+)-ATPase was detected through immunofluorescence in the basal part of the cells of the labyrinth and in the bladder cells with an increasing immunostaining from labyrinth I to the bladder. No immunoreactivity was detected in the coelomosac. The cells of the labyrinth and of the bladder present morphological and enzymatic features of ionocytes. The antennal glands of the lobster thus possess active ion exchanges capabilities.

Ultrastructure and physiology of the hooded sensillum, a bimodal chemo-mechanosensillum of lobsters.

The antennules of decapod crustaceans are covered with thousands of chemosensilla that mediate odor discrimination and orientation behaviors. Most studies on chemoreception in decapods have focused on the prominent aesthetasc sensilla. However, previous behavioral studies on lobsters following selective sensillar ablation have revealed that input from nonaesthetasc antennular chemosensilla is sufficient for many odor-mediated behaviors. Our earlier examination of the setal types on the antennules of the Caribbean spiny lobster Panulirus argus revealed three types of nonaesthetasc chemosensilla. The most abundant and widely distributed of these is the hooded sensillum. The present study describes the detailed ultrastructure of antennular hooded sensilla and the physiological response properties of their receptor neurons. Light and scanning and transmission electron microscopy were used to examine structural characteristics, and electrophysiology was used to examine single-unit responses elicited by focal chemical and mechanical stimulation of antennular hooded sensilla. Hooded sensilla have a porous cuticle and are innervated by 9-10 chemosensory and 3 mechanosensory neurons whose dendrites project to the distal end of the sensillum. Hooded sensillar chemosensory neurons responded to waterborne chemicals, were responsive to only one of the six tested single compounds, and had different specificities. Hooded sensillar mechanosensory neurons were not spontaneously active. They had low sensitivity in that they responded to tactile but not waterborne vibrations, and they responded to sensillar deflection with phasic bursts of activity. These results support the idea that hooded sensilla are bimodal chemo-mechanosensilla and are receptors in an antennular chemosensory pathway that parallels the well-described aesthetasc chemosensory pathway.

Immunolocalization of NA(+),K(+)-ATPase in the branchial cavity during the early development of the European lobster Homarus gammarus (Crustacea, Decapoda).

We examined the ontogeny of the osmoregulatory sites of the branchial cavity in embryonic and early postembryonic stages of the European lobster Homarus gammarus through transmission electron microscopy, immunofluorescence microscopy, and immunogold electron microscopy using a monoclonal antibody IgGalpha(5) raised against the avian alpha-subunit of the Na(+),K(+)-ATPase. In mid-late embryos, Na(+),K(+)-ATPase was located along the pleurites and within the epipodite buds. In late embryos just before hatching, the enzyme was confined to the epipodite epithelia. After hatching, slight differentiations of ionocytes occured in the epipodites of larval stages. Na(+),K(+)-ATPase was also located in the ionocytes of the epipodites of larvae exposed to seawater (35.%o) and to dilute seawater (22.1 %o). After metamorphosis, the inner-side branchiostegite epithelium appeared as an additional site of enzyme location in postlarvae held in dilute seawater. Within the ionocytes, Na(+),K(+)-ATPase was mostly located along the basolateral infoldings. These observations are discussed in relation to the physiological shift from osmoconforming larvae to slightly hyper-regulating (in dilute seawater) postmetamorphic stages. The acquisition of the ability to hyper-osmoregulate probably originates from the differentiation, on the epipodites and mainly along the branchiostegites, of ionocytes that are the site of ion pumping as evidenced by the location of Na(+),K(+)-ATPase.

Morphology and distribution of setae on the antennules of the Caribbean spiny lobster Panulirus argus reveal new types of bimodal chemo-mechanosensilla.

This study describes the morphology and distribution of setae on the lateral and medial flagella of the antennules of the spiny lobster Panulirus argus in an effort to identify antennular chemoreceptors in addition to the well-studied aesthetasc chemosensilla. Setae were examined using light and electron microscopy, and their distribution on flagellar annuli was analyzed. We identified ten setal types based on external morphology: hooded, plumose, short setuled, long simple, medium simple, short simple, aesthetasc, guard, companion, and asymmetric setae, with the last four types being unique to the "tuft" located on the distal half of the lateral flagellum. The three setal types whose ultrastructure was examined--hooded, long simple, and medium simple setae--had characteristics of bimodal (chemo-mechanoreceptive) sensilla. The antennules have four distinct annular types based on their setal complement, as shown by cluster analysis. This basic distribution of non-tuft setal types is similar for both lateral and medial flagella. Annuli in the tuft region have tuft setal types superimposed on a basic organization of non-tuft setal types. These results show that the antennules possess a diverse set of setae, that these setae have a highly ordered arrangement on the antennules, that at least four (and probably many more) of these setal types are chemosensilla, and suggest that most antennular chemosensilla are bimodally sensitive.

Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the norway lobster Nephrops norvegicus during infection by a parasitic dinoflagellate of the genus Hematodinium.

Changes in various biochemical and ultrastructural characteristics of the deep abdominal flexor (DAF) muscles were studied in Norway lobster Nephrops norvegicus (L.) from the Clyde estuary, Scotland, UK, at different stages of infection by a parasitic dinoflagellate of the genus Hematodinium. Abdominal DAF muscles from infected lobsters showed slight, significant increases in total water content, along with greatly depleted glycogen reserves and an altered free amino acid profile. However, protein concentration and composition remained unchanged. Ultrastructurally, parasitic infection of DAF muscle fibres caused alterations in sarcolemmal structure, and localized disruption of myofibrillar bundles around the periphery, but not throughout the centre of the fibres. Overall, the reduction in swimming performance previously reported for N. norvegicus during Hematodinium infection reflect an alteration in carbohydrate supply to the active muscle and some subtle disruption of muscle structure. The altered carbohydrate titre could reflect the Hematodinium parasites acting as a carbohydrate sink in the haemolymph, a disruption of normal tissue glycogenesis, or some alteration in the host's hormonal regulation. These changes could also adversely affect the taste, texture and marketability of infected meat.

Video microscopic analysis of ionophore induced acrosome reactions of lobster (Homarus americanus) sperm.

Sperm from the American lobster (Homarus americanus) are normally nonmotile. However, during fertilization, the sperm undergo a calcium-dependent acrosome reaction that propels them forward about 18 microns. The reaction occurs in two phases, eversion and ejection, which take place too quickly to permit analysis by direct observation. The purposes of this study were to examine the structural changes occurring in sperm during the normal acrosome reaction and to determine the rate of the reaction using video microscopy. The reaction was induced in vitro by ionophore A23187 and recorded using a video system attached to a Nikon Nomarski interference microscope. Videotapes were played back frame by frame (30 frames/sec), and images of reactions from 10 sperm were analyzed. The acrosome reaction, including the eversion of the acrosomal vesicle and ejection of the subacrosomal material and nucleus, can be divided into 4 steps: (1) expansion of the apical cap followed by expansion of the remainder of the acrosomal cylinder; expansion of the cylinder begins at its apical end and proceeds toward its base, (2) eversion of the apical half of the acrosomal vesicle and initial contraction of the apical cap, (3) eversion of the basal half of the acrosomal vesicle, continued contraction of the apical cap, and ejection of the subacrosomal material and nucleus, and (4) final contraction of the apical cap and ejection of the acrosomal filament. During steps 2, 3, and 4, the mean forward movement of sperm is 12.7, 3.9, and 1.1 microns, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Transmission electron microscopy of the consumed hairs in an Australian lobster's stomach].

The present report is concerned with the transmission electron microscopic observations of the consumed hairs in an Australian lobster's stomach. The lamellar cuticles of these hairs were observed and their minimum diameters of melanin granules of cortices were measured by an imaging analyzer. The lamellar cuticles of these hairs consisted of two or three layers and there was no significant difference between them and Aberdeen Angus's hairs in mean the minimum diameters of melanin granules of cortices. Consequently, these hairs in an Australian lobster's stomach were identical to Aberdeen Angus's hairs.

Axonal microtubules of crayfish and spiny lobster nerve cords are decorated with a heat-stable protein of high molecular weight.

Axons of crayfish and spiny lobster ventral nerve cords contain large numbers of microtubules that are decorated with fine filaments. These microtubules can be stabilized in permeabilized axons using buffers that contain either polyethylene glycol or glycerol/dimethyl sulphoxide. In the former, the stabilized microtubules retain their filaments and their normal spacing; in the latter, the filaments are stripped off and the bare microtubules collapse onto one another. This observation has been used as the basis for a method of identifying some of the proteins that make up the filaments. Axons are first permeabilized and stabilized in either buffer and then treated with a microtubule-depolymerizing buffer. The axons treated first with polyethylene glycol release tubulin and significant quantities of microtubule-associated proteins (MAPs), while the axons pre-treated with glycerol release tubulin and only traces of associated proteins. One of the proteins released in largest quantity along with tubulin from polyethylene glycol-treated axons is a high molecular weight, heat-stable MAP that co-electrophoreses with MAP-2 from mammalian brain. This same protein co-purifies with tubulin that is obtained from crayfish nerve cords by two cycles of polymerization and depolymerization. It is concluded that this protein is a component of the filaments that decorate the axonal microtubules of the crayfish and spiny lobster.

Intramembrane organization of synapses in the lobster stretch receptor organ.

The intramembrane organization of axodendritic and neuromuscular synapses in the lobster stretch receptor organ was investigated by freeze-fracturing. Based on ultrastructural criteria which are known to be correlated with physiological properties, we identified three types of synapse: the inhibitory axodendritic, the inhibitory neuromuscular, and the excitatory neuromuscular synapse. Although these synapses have some features in common, each has a characteristic arrangement of intramembrane particles in both the presynaptic and postsynaptic membranes. All three have, in their presynaptic membranes, aggregates of P-face particles and associated depressions representing sites of synaptic vesicle exocytosis, features which together define active zones. However, in the inhibitory axodendritic synapse the P-face contains short ridges in this region. These ridges may occur singly or in pairs oriented in V-shaped configurations. The ridges are decorated with particles along their entire length. In the inhibitory neuromuscular synapse, no ridges are present. Clusters of particles are present, but they are scattered randomly over a large expanse of presynaptic membrane. In the excitatory neuromuscular synapse, isolated clusters of particles are associated with the P-face and are occasionally located on circular elevations of the membrane. The postsynaptic membrane also shows structural diversity in the three types of synapse. In the inhibitory axodendritic synapse, there is no apparent specialization. However, in the inhibitory neuromuscular synapse, P-face particles are arranged in double rows which are separated by particle-free strips of membrane. In the excitatory neuromuscular synapse, particles are confined to a narrow band that borders the synaptic cleft. This band is demarcated by a single intermittent strand of particles arranged in the direction of the long axis of the muscle fibre. Therefore, intramembrane specializations of both the presynaptic and postsynaptic membranes are sufficiently distinctive that three different types of synapse can be recognized.

The dynamics of exoskeletal-epidermal structure during molt in juvenile lobster by electron microscopy and electron spectroscopic imaging.

The exoskeletal-epidermal complex of juvenile lobsters at various stages throughout the molt cycle was examined by conventional electron microscopy, freeze-etch replicas, and electron spectroscopic imaging. This latter technique which enables the direct localization of atomic elements superimposed over morphological fine structure has been applied to this tissue complex to determine the spatial distributions and interrelationships of calcium, phosphorus, and sulphur. Chitin microfibril assembly is visualized in thin sections as occurring at the surface of apical membrane plaques which in freeze-etch replicas invariably possess a rich distribution of intramembrane particles on both P and E faces. In early stages of mineralization the exo- and endocuticular zones of the exoskeleton possess a dense Ca-containing lamellar repeat. These bands are unrelated to the helicoidal arrangement of chitin microfibrils. At later stages of development mineral deposits occur within the exocuticle and advance through to the endocuticle. These deposits align with chitin microfibrils and exhibit a helicoidal pattern. Morphological and chemical alterations associated with mineralization and demineralization of the exoskeleton are discussed.

Intercellular junctions in the hepatopancreas of the lobster Nephrops norvegicus.

The hepatopancreas of the lobster has recently been found to be a rich source of material from which to isolate arthopod gap junctions biochemically (Finbow et al., 1983a; 1984). It has therefore been studied here to assess the features of these intercellular junctions and any others that may be present, in vivo. The tissue consists of columnar epithelial cells which possess apical microvilli and basal infoldings. In thin sections the lateral borders of these cells are characterized by desmosomes and smooth septate junctions as well as by gap junctions. The desmosomes exhibit no apparent freeze fracture profile but the septate junctions display parallel rows of ridges or aligned intramembranous particles (IMPs) with complementary grooves on the other membrane half; these IMPs shift in their preferential fracturing plane depending on whether the tissue has first been fixed, always remaining on the EF if unfixed. The IMPs or connexons, of which the gap junctions are composed, fracture onto the E face, leaving complementary pits on the P face, regardless of whether the tissue is fixed or not. At the base of the pancreatic cells, the lateral borders are thrown into interdigitating folds which display endocytotic profiles and possible internalization of junction-bearing membranes. This phenomenon, which is readily visualized both after tracer incubation and in replicas, may represent junctional degradation relating to membrane turnover.

Fine structure of comparable synapses in a mature and larval lobster muscle.

Neuromuscular synapses from the single excitor axon to the proximal accessory flexor muscle (PAFM) was studied by serial section electron microscopy in a 1st stage larval (less than 0.1 g) and a large adult (6.8 kg) lobster. The adult innervation of a lateral and a medial fiber, physiologically identified as low- and high-output respectively, was similar in the number and mean size of synapses but had significantly larger pre-synaptic dense bars for the high-output synapses. This correlation between quantal transmitter output and pre-synaptic dense bars and the appearance of exocytotic profiles along the dense bars strongly implicates the bars as active sites of transmitter release. Moreover the mature innervation is differentiated on the basis that the percentage of dense bar area to synaptic area is 9% for the low-output type compared to 22% for its high-output counterpart. In the larval PAFM the excitatory axon has not proliferated many branches and the innervation is therefore localized to groups of fibers in the lateral, medial and central regions of the muscle rather than to individual fibers. The lateral and medial sites of innervation representing putative low- and high-output types respectively (because of their location) do not differ in the size and number of pre-synaptic dense bars thereby suggesting a similarity in quantal synaptic transmission. However, the percentage of dense bar area to synaptic area is 40% for the lateral site compared to 67% for the medial site. Since this is a trend mimicking the mature innervation it shows an early stage in the differentiation of low- and high-output synapses. Furthermore the main axon provides half of the total innervation in the larval PAFM but none in the adult thereby demonstrating a restructuring of multiterminal innervation.