Chromatin condensation and acrosome development during spermiogenesis of Ensis ensis (Mollusca,Bivalvia)

We describe chromatin condensation and acrosome development during spermiogenesis of Ensis ensis. The overall shape of the mature spermatozoon corresponds to the primitive type. The nucleus is oval and on its superior pole there is an elongated acrosome; the middle piece contains four mitochondria around the centriolar complex. The condensation of the nuclei seems to occur in three steps: first the diameter of chromatin fibers increases slightly from 17 to 20 nm; second, in midspermatids fiber pairs coalesce; and third, the coalescence continues by addition of other fibers until the nuclei become highly compacted. Chromatin changes are related with nuclear protein composition. Small proacrosomal vesicles show two regions of different electron density. At a later stage they fuse to give a single, spherical vesicle in round spermatids, which migrates to the upper pole and transforms into a tapered acrosome (18 μm long) with a central channel filled with finely fibrous material. © 1994 Wiley-Liss, Inc.     

Intestinal Ultrastructure of Nerita picea (Mollusca: Gastropoda), an Intertidal Marine Snail of Hawaii

The neritid snail Nerita picea is a marine prosobranch mollusc which resides high in the intertidal zone on the Hawaiian Islands. Since other studies have shown considerable variations in molluscan gut histology and the relatively few recent ultrastructural reports have revealed novel cellular structures in the molluscan gastrointestinal tract, this investigation was directed toward ultrastructural clarification of the neritid intestine. Seven principal cell types constituted the intestinal architecture, including absorptive cells, zymogen cells, neural and endocrine cells, myocytes, pigment and gland cells. The intestinal epithelium was composed mainly of tall ciliated (9 plus 2 complement of microtubules) columnar absorptive cells which also possessed microvilli, extensive deposits of non-membrane-bound lipid-like droplets, and large reservoirs of glycogen-like granules. Less frequent, columnar zymogen cells contained numerous large zymogen secretory granules and possessed microvilli but not cilia. Small endocrine-like cells with secretory granules were observed basolaterally between some absorptive cells, resembling mammalian gut endocrine cells. Nerve fibers were prevalent in close association with the epithelial cells. A thin layer of non-striated muscle was present, as well as a serosally located gland composed of storage cells with a granular matrix and large granules.     

Anatomy and affinities of cocculinid limpets (Mollusca, Archaeogastropoda)

The anatomy of seven cocculinid species, representing four genera, is described. Two new genera are erected, Paracocculina and Coccocrater, and a comparative account of all cocculinid species so far anatomically investigated is given. Definitions of six genera, mainly based on anatomical data, are presented. Variation at the species level is also discussed. The closest relatives of the Cocculinidae are found in the Bathysciadiidae, sharing a distinct gill type and similar coelomic conditions. Therefore, both families are united as the Cocculinoidea. The Lepetelloidea (Lepetellidae, Pseudococculinidae, Osteopeltidae, Bathyphytophilidae (?), Cocculinellidae, Addisoniidae and Choristellidae) are more distantly related and are classified, together with the Cocculinoidea, within the suborder Cocculiniformia. The Cocculiniformia are regarded as one of the earliest offshoots at the archaeogastropod level of organization, being secondarily modified in several respects.     

Anatomy and affinities of pseudococculinid limpets (Mollusca, Archaeogastropoda)

The anatomy of 12 pseudococculinid species, representing the two subfamilies and 10 (of 11 known) genera, is described in detail. Three new genera are erected, Yaquinabyssia, Copulabyssia and Amphiplica. Subfamilial and generic systematics are reconsidered and additionally based on anatomical characters. Characters defining subfamilies and genera (Pseudoeoeculininae: Pseudococculina, Notocrater, Tentaoculus, Mcsopelex, Bandabyssia, Kurilabyssia; Caymanabyssiinae: Caymanabyssia, Colotrachelus, Yuquinabyssia, Copulabyssia, Amphiplica) are the modifications in shell and radula, the number and arrangement of gill-leaflets, the size of the pedal gland, the degree of eye-reduction and the modifications of the seminal groove and of the copulatory organ. Separation of the Pseudococculinidae Hiekman, 1983 from the Cocculinidae Dall, 1882 at the superfamilial level is confirmed by great differences in their anatomy. Whereas the Cocculinidae are closely related to the Bathysciadiidae (Cocculinoidea). the Lepctcllidae, Pyropeltidae, Pseudococculinidae,? Bathyphytophilidae, Osteopeltidae, Cocculinellidae, Addisoniidae and Choristellidae form a clearly separated supcrfamily (Lepetelloidea). Both superfamilies compose the Cocculiniformia. a primitive but highly specialized arehaeogastropod suborder.     

Anatomy and systematics of bathyphytophilid limpets (Mollusca, Archaeogastropoda) from the northeastern Pacific

Bathyphytophilus diegensis sp. n. is described on basis of shell and radula characters. The radula of another species of Bathyphytophilus is illustrated, but the species is not described since the shell is unknown. Both species feed on detached blades of the surfgrass Phyllospadix carried by turbidity currents into continental slope depths in the San Diego Trough. The anatomy of B. diegensis was investigated by means of semithin serial sectioning and graphic reconstruction. ‘The shell is limpet-like; the protoconch resembles that of pseudococculinids and other lepetclloids. The radula is a distinctive, highly modified rhipidoglossate type with close similarities to the lepetellid radula. The anatomy falls well into the lepetelloid bauplan and is in general similar to that of Pseudococculinidae and Pyropcltidae. Apomorphic features are the presence of gill-leaflets at both sides of the pallial roof (shared with certain pseudococculinids), the lack of jaws, and in particular many enigmatic pouches (bacterial chambers?) which open into the postcrior oesophagus. Autapomorphic characters of shell, radula and anatomy confirm the placement of Bathyphytophilus (with Aenigmabonus) in a distinct family, Bathyphytophilidac Moskalev, 1978. As revealed by a cladistic study, the Bathyphytophilidae should be classified within the Lepetelloidea close to the Lepetellidae, Pyropeltidae, and Pseudococculinidae.     

The relative importance of geography and ecology in species diversification: evidence from a tropical marine intertidal snail (Nerita)

Aim In this study, I examined the relative contributions of geography and ecology to species diversification within the genus Nerita, a prominent clade of marine snails that is widely distributed across the tropics and intertidal habitats. Specifically, I tested whether geographical patterns of speciation correspond primarily to allopatric or sympatric models, and whether habitat transitions have played a major role in species diversification. Location Indo‐West Pacific, eastern Pacific, Atlantic, tropical marine intertidal. Methods I used a previously reconstructed molecular phylogeny of Nerita as a framework to assess the relative importance of geographical and ecological factors in species diversification. To evaluate whether recently diverged clades exhibit patterns consistent with allopatric or sympatric speciation, I mapped the geo‐graphical distribution of each species onto the species‐level phylogeny, and examined the relationship between range overlap and time since divergence using age–range correlation analyses. To determine the relative contribution of habitat transitions to divergence, I traced shifts in intertidal substrate affinity and vertical zonation across the phylogeny using parsimony, and implemented randomization tests to evaluate the resulting patterns of ecological change. Results Within the majority of Nerita clades examined, age–range correlation analysis yielded a low intercept and a positive slope, similar to that expected under allopatric speciation. Approximately 75% of sister species pairs have maintained allopatric distributions; whereas more distantly related sister taxa often exhibited complete or nearly complete geographical overlap. In contrast, only 19% of sister species occupy distinct habitats. For both substrate and zonation, habitat transitions failed to concentrate towards either the tips or the root of the phylogeny. Instead, habitat shifts have occurred throughout the history of Nerita, with a general transition from the lower and mid‐littoral towards the upper and supra‐littoral zones, and multiple independent shifts from hard (rock) to softer substrates (mangrove, mud and sand). Main conclusions Both geography and ecology appear to have influenced diversification in Nerita, but to different extents. Geography seems to play a principal role, with allopatric speciation driving the majority of Nerita divergences. Habitat transitions appear insignificant in shaping the early and recent history of speciation, and promoting successive diversification in Nerita; however, shifts may have been important for respective divergences (i.e. those that correspond to the transitions) and enhancing diversity throughout the clade.     

Comparative sperm ultrastructure and spermiogenesis in basal heterobranch gastropods (Valvatoidea, Architectonicoidea, Rissoelloidea, Omalogyroidea, Pyramidelloidea) (Mollusca)

Three distinct groups of basal heterobranch gastropods (='Allogastropoda/Heterostropha') can be distinguished on the basis o f sperm and spermiogenic features: (1) Valvatoidea; (2) Architectonicoidea; (3) Rissoelloidea + Omalogyroidea + Pyramidelloidea. Sperm of pentaganglionate heterobranchs (Opisthobranchia sensu stricto + Pulmonata) conform in all respects to the pattern shown by group (3). Heterobranchia are united, and differentiated from other gastropods, by a shared suite of sperm and spermiogenic features (rounded acrosomal vesicle, nucleus usually helical, highly modified mitochondrial derivative, spermatid acrosome associated with a dense nuclear plaque, formation of the mitochondrial derivative through fusion of numerous small mitochondria along the length of the axoneme). Sperm autapomorphies of the Architectonicoidea and the Valvatoidea suggest that these two superfamilies arc distinct but terminal offshoots from ancestral heterobranchs.     

Nuclear changes and acrosome formation during spermiogenesis in Euchistus heros (Hemiptera: Pentatomidae)

Ultrastructural and cytochemical studies were carried out on nuclear changes and acrosome formation during the spermiogenesis of the phytophagous bug Euchistus heros. The development of the nucleus involves changes in the shape and in degree of chromatin condensation: initially it is dispersed and with a low-electron density, then assumes a fibrillar arrangement and finally compacts in an electron-dense material. The acrosome is formed by the Golgi complex and presents unusual morphological features during its development. The reaction product of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase activities were detected during various stages of acrosome development. In contrast, residues of alpha-N-acetylgalactosamine and basic proteins were only reported in the intermediate and late stages of the differentiation process, respectively.     

Reproductive systems and breeding condition in Nerita birmanica (Archaeogastropoda: Neritacea) from Malayan mangrove swamps

The complex reproductive systems of the mangrove-dwelling Nerita birmanica are described and related to those of other neritids. Gonad condition in both sexes and numbers of spermatophores in males and females varied slightly during a one-year survey. It is concluded that a slight tendency for larger number of "full" ovaries with big oocytes to occur in collections taken shortly before full and new moon does not represent a significant lunar cycle of gonad activity since almost all gonads bore gametes in all stages of development at all times of year. It appears merely that ovulation may be held up during relatively rainless periods after which substantial rain is followed by reduced ovaries lacking large oocytes. In brief, breeding appears to be continous with but slight variation which does not consistently follow observable environmental factors.     

Fine structure of the germinating cells during the spermiogenesis of Arion rufus (Mollusca,Pulmonate Gastropoda)

Changes in spermatozoan ultrastructure have been studied during spermiogenesis of the slug Arion rufus (Gastropoda, Pulmonata, Stylommatophora). The ovotestis was investigated during the male stage, definite by the presence of spermatozoa. Some peculiar characteristics are shown by early spermatids: Around the nucleus, the nuclear envelope presents two thick layers located on opposite sides, the apical and basal plates, that will determine the antero-posterior axis of the spermatid. The chromatin, first dispersed throughout the nucleoplasm gives later on thick filaments which become attached over the inner surface of these plates. The chromatin filaments are then arranged parallel to the antero-posterior axis as the nucleus elongates. The position of the plates determines the antero-posterior axis of the spermatid. In the mature spermatozoa, the chromatin is more condensed and the nucleus presents an helical organization. The acrosome and flagellum are respectively attached externally to the center of the apical and basal plates. The acrosome consists of a membrane-bound vesicle and forms a column of homogeneous material. In the middle piece, the mitochondria have been transformed into a mitochondrial derivate by the way of a complicated metamorphosis. The axoneme is surrounded by three mitochondrial helices but only one of them contains glycogene granules. © 1996 Wiley-Liss, Inc.     

Control of membrane fusion during spermiogenesis and the acrosome reaction

Membrane fusion is important to reproduction because it occurs in several steps during the process of fertilization. Many events of intracellular trafficking occur during both spermiogenesis and oogenesis. The acrosome reaction, a key feature during mammalian fertilization, is a secretory event involving the specific fusion of the outer acrosomal membrane and the sperm plasma membrane overlaying the principal piece of the acrosome. Once the sperm has crossed the zona pellucida, the gametes fuse, but in the case of the sperm this process takes place through a specific membrane domain in the head, the equatorial segment. The cortical reaction, a process that prevents polyspermy, involves the exocytosis of the cortical granules to the extracellular milieu. In lower vertebrates, the formation of the zygotic nucleus involves the fusion (syngamia) of the male pronucleus with the female pronucleus. Other undiscovered membrane trafficking processes may also be relevant for the formation of the zygotic centrosome or other zygotic structures. In this review, we focus on the recent discovery of molecular machinery components involved in intracellular trafficking during mammalian spermiogenesis, notably related to acrosome biogenesis. We also extend our discussion to the molecular mechanism of membrane fusion during the acrosome reaction. The data available so far suggest that proteins participating in the intracellular trafficking events leading to the formation of the acrosome during mammalian spermiogenesis are also involved in controlling the acrosome reaction during fertilization.     

Light and electron microscopical investigations on shell pores (caeca) of fissurellid limpets (Mollusca: Archaeogastropoda)

The organic content (caecum) of the shell pores of five species of fissurellid (key-hole) limpets was investigated by means of light and electron microscopy. Fissurellid caeca are unbranched, polycellular extensions of the mantle epithelium, which penetrate the whole shell and contact the periostracum via organic fibres called 'brush'. They are built up by two cell-types, which enclose a central lumen, and are formed concurrently with the shell itself. Because of fundamental structural and developmental differences, the superficially similar shell pores of bivalves (caeca) and polyplacophorans (aesthetes) are considered as analogous rather than homologous organs. Similarities are found with brachiopod caeca, probably owing to similar functions.     

Self-fertilization and cross-fertilization in the land snail Arianta arbustorum (Mollusca, Pulmonata: Helicidae)

The simultaneously hermaphroditic land snail Arianta arbustorum (L.) (Pulmonata: Helicidae) was long believed to be self-incompatible. In the present study, however, uniparental reproduction was observed under laboratory conditions. Snails from two natural populations and from laboratory culture reared in isolation from the subadult stage produced fertile eggs. Over a period of three years after maturation, 38.5% of the unmated snails produced fertile eggs, mostly in the second and third years instead of the first year. Egg production and hatching success (the proportion of eggs that hatched) were lower in unmated snails than in mated snails. The number of hatchlings produced by unmated snails was 1–2% of that produced by mated snails. Hatchlings from unmated snails survived as well as those from mated snails under laboratory conditions. The result indicate that A. arbustorum can self-fertilize but with a great fitness reduction.     

Characterization and comparative 3D modeling of CmPI-II, a novel 'non-classical' Kazal-type inhibitor from the marine snail Cenchritis muricatus (Mollusca)

The complete amino acid sequence obtained by electrospray ionization tandem mass spectrometry of the proteinase inhibitor CmPI-II isolated from Cenchritis muricatus is described. CmPI-II is a 5480-Da protein with three disulfide bridges that inhibits human neutrophil elastase (HNE) (K(i) 2.6+/-0.2 nM), trypsin (K(i) 1.1+/-0.9 nM), and other serine proteinases such as subtilisin A (K(i) 30.8+/-1.2 nM) and pancreatic elastase (K(i) 145.0+/-4.4 nM); chymotrypsin, pancreatic and plasma kallikreins, thrombin and papain are not inhibited. CmPI-II shares homology with the Kazal-type domain and may define a new group of 'non-classical' Kazal inhibitors according to its Cys(I)-Cys(V) disulfide bridge position. The 3D model of CmPI-II exhibits similar secondary structure characteristics to Kazal-type inhibitors and concurs with circular dichroism experiments. A 3D model of the CmPI-II/HNE complex provides a structural framework for the interpretation of its experimentally determined K(i) value. The model shows both similar and different contacts at the primary binding sites in comparison with the structure of turkey ovomucoid third domain (OMTKY3)/HNE used as template. Additional contacts calculated at the protease-inhibitor interface could also contribute to the association energy of the complex. This inhibitor represents an exception in terms of specificity owing to its ability to strongly inhibit elastases and trypsin.     

Purification and partial characterization of human neutrophil elastase inhibitors from the marine snail Cenchritis muricatus (Mollusca)

Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 degrees C for 30 min. From this extract, three human neutrophil elastase inhibitors (designated CmPI-I, CmPI-II and CmPI-III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI-I and CmPI-II was confirmed, while CmPI-III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI-I and CmPI-II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI-I (6 amino acids) and CmPI-II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI-II. Both inhibitors, CmPI-I and CmPI-II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (Ki) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI-I and CmPI-II are the first human neutrophil elastase inhibitors described in a mollusk.     

Spermiogenesis in Polyplacophora,with special reference to acrosome formation (Mollusca)

Summary The present study examines spermiogenesis, and in particular the formation of the acrosome, in ten species of chitons belonging to four families. This study emphasizes the formation of the acrosome but brings to light several other structures that have received little or no mention in previous studies. The process of spermiogenesis is essentially similar in each species, although Chaetopleura exhibits some significant differences. In early spermiogenesis the Golgi body secretes numerous small pro-acrosomal vesicles that gradually migrate into the apical cytoplasm. The chromatin condenses from granules into fibres which become twisted within the nucleus. A small bundle of chromatin fibres projects from the main nuclear mass into the anterior filament; this coincides with the appearance of a developing manchette of microtubules around the nucleus that originates from the two centrioles. Radiating from the distal centriole is the centriolar satellite complex, which is attached to the plasma membrane by the annulus. The distal centriole produces the flagellum posteriorly and it exits eccentrically through a ring of folded membrane that houses the annulus. Extending from the annulus on one side of the flagellum, in all but one species, is a dense fibrous body that has not been previously reported. The proximal centriole lies perpendicular to the end of the distal centriole and is attached to it by fibro-granular material. Pro-acrosomal vesicles migrate anteriorly through the cytoplasm and move into the anterior filament to one side of the expanding nucleus. Eventually these vesicles migrate all the way to the tip of the sperm, where they fuse to form one of two granules in the acrosome. In mature sperm the nucleus is bullet-shaped with a long anterior filament and contains dense chromatin with occasional lacunae. The mitochondria vary in both number and position in the mature sperm of different species. Both centrioles are housed eccentrically in a posterior indentation of the nucleus, where the membranes are modified. The elongate flagellum tapers to a long filamentous end-piece that roughly corresponds to the anterior filament and may be important in sperm locomotion for hydrodynamic reasons. An acrosome is present in all ten species and stained positively for acid phosphatase in three species that were tested.