The mucus of Sabella spallanzanii (Annelida, Polychaeta): Its involvement in chemical defence and fertilization success

In several species mucus typically forms a slippery coating that performs many specialised roles including the defence against infection and protection for eggs. In the present study we focused on the mucus of the polychaete Sabella spallanzanii in order to ascertain its involvement in chemical defence and fertilization success. The observations were carried out over an annual cycle according to different phases of the reproductive cycle. Mucus of S. spallanzanii showed a natural lysozyme-like activity with a maximum at pH 6.0 and I = 0.175. This activity was present in all the sampling times except for January (T₄) which corresponds to the reproductive period of this species. The electrophoretic analysis revealed at least ten major protein bands, with molecular weights ranging from 16 to 90 kDa, and six minor components, with molecular weights ranging from 14 to 116 kDa. This pattern was reported all over the year except for the reproductive period when not only the lysozyme-like activity decreased but the number of protein bands was also reduced. During this period microscopical observations of the mucus indicated that only fertilised eggs enveloped in the mucus were released out of the tubes where the worms live. This supports previous hypothesis that fertilization does not occur in the water column but it takes place in the tube just after egg release (in situ fertilization). This fertilization strategy, not involving egg dispersal in the water column, ensures a high success of fertilization. The mucus of S. spallanzanii therefore represents the ideal mean where eggs are released and fertilized as well as a compartment involved in defending the worms from bacterial attack serving as medium into which the antibacterial substances are exuded. The defensive role of lysozyme is important taking into account that this polychaete lives in eutrophic environments such as harbours where bacteria, including pathogens to man and marine organism, are abundant.     

New insights into the larval development of Macandrevia cranium (Müller, 1776) (Brachiopoda: Rhynchonelliformea)

The terebratulid Macandrevia cranium (Müller, 1776) is a representative of articulate brachiopods. However, little is known about its embryology and larval development. In order to obtain reproducible results we used a strict protocol of artificial fertilization under controlled temperature conditions as a basis for our morphological study. Sampling of embryos or developing larvae at frequent intervals led to the most comprehensive collection of preserved developmental stages, ranging from early zygotes to late three-lobed stage larvae. SEM studies of all these stages showed that the development of M. cranium is similar to that of other terebratulid brachiopods. This includes the presence of four bundles of larval setae in three-lobed stage larvae. Our results contradict earlier observations on the development of M. cranium and show that this species exhibits more typical features of articulate brachiopod development than previously thought.     

External calcium concentration and fertilization in the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz) (Echinodermata)

The role of external calcium ions in the fertilization of the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz), was studied. When eggs were inseminated with jelly-treated sperm in artificial sea waters containing various concentrations of calcium, the percentage of fertilization decreased with decreasing concentrations of calcium. A small amount of external calcium ions was also found to be essential for fertilization by spermatozoa with reacted acrosomes. The binding capacity of jelly-treated spermatozoa to eggs was, however, not adversely affected by a calcium deficiency. These results suggest that calcium ions are required not only for the initiation of the acrosome reaction, but also for successful levels of fertilization following sperm-binding to eggs even after the acrosome reaction has occurred.     

Competition between two invasive Hydrocharitaceae (Hydrilla verticillata (L.f.) (Royle) and Egeria densa (Planch)) as influenced by sediment fertility and season

Competition between two invasive plants of similar growth form, Hydrilla verticillata (L.f.) (Royle) and Egeria densa (Planch), was studied in response to season and sediment fertility. These two invasive species were grown in outdoor concrete tanks in monocultures and mixtures. Five fertilization rates were tested for monocultures and two for mixtures where six combinations of planting densities were used in two seasons (spring and fall). Monitoring of plant biomass was made at the end of each of these 2-month-experiments. In contrast to E. densa, clear seasonal patterns in biomass production and in reproductive allocations of H. verticillata were evident. Competitive pressure for both species was lower during the fall experiment. Biomass production increased with fertilization for H. verticillata in monocultures and changes either in allocative ratios or in tuber production patterns were shown in response to nutrient availability. However, E. densa growth was not affected by fertilization. In most cases, H. verticillata was a better competitor than E. densa except when sediment was pure sand. Competition occurred mainly for nutrient uptake rather than for light harvesting. These results suggest that despite the similar ecology, H. verticillata may outcompete E. densa in many situations, probably due to its higher plasticity.     

An ultrastructural study of the spermatozoa ofEulalia sp. (Phyllodocidae),Lepidonotus sp. (Polynoidae),Lumbrineris sp. (Lumbrineridae) andOwenia fusiformis (Oweniidae)

The ultrastructure of the mature spermatozoa of four polychaetes is described:Eulalia sp. (Phyllodocidae),Lepidonotus sp. (Polynoidae),Lumbrineris sp. (Lumbrineridae) andOwenia fusiformis (Oweniidae). All the sperm show features typical of externally fertilizing sperm in having a rounded nucleus, a short unmodified midpiece, and a simple flagellum with a 9+2 axoneme.Owenia fusiformis andLepidonotus sp. have a nuclear cone extending into the subacrosomal space that may act to present the inner acrosomal membrane to the egg during fertilization. The acrosome ofLumbrineris sp. is flattened and crenulated. The sperm ofEulalia sp. is unusual in having the four mitochondria of the midpiece ensheathed by a membrane. Comparisons are made with other polychaete sperm, and the use of sperm ultrastructure as a taxonomic tool within the Polychaeta is discussed.     

Larval development of nemerteans of the genus Quasitetrastemma (Nemertea: Monostilifera)

Data on the larval development of Quasitetrastemma stimpsoni and Q. nigrifrons are presented. In both species, fertilization is external; the development passes through a free-swimming larval stage, the “hidden larva.” The larva has three pairs of eyes. After settling, the eyes of the second pair fuse with eyes of the first pair or are completely reduced. The basis and stylets are formed in 7–8 days after fertilization. Larvae of Q. stimpsoni settle on day 9–10 after fertilization; and Q. nigrifrons, on day 7–8.     

The suctorial organ of the Solifugae (Arachnida,Solifugae)

The ability of members of the arachnid order Solifugae to climb smooth, vertical surfaces and the organs involved in this behavior are investigated. Macroscopic, microscopic, and scanning electron microscopic observations are made of a palpal organ called the suctorial organ. Observations of the behavior but not the microstructure have been made in the past. Histological examination illustrates the internal gross anatomy of this structure and scanning electron microscopy demonstrates the fine structure in adults of four genera: Eremobates (Eremobatidae), Eremochelis (Eremobatidae), Eremorhax (Eremobatidae), Ammotrechula (Ammotrechidae), as well as an unidentified late stage immature and third stage instar. The suctorial organ is most likely primarily used for prey capture in the wild.     

Reproduction and development of the limpet Limalepeta lima (Dall, 1918) (Gastropoda: Lepetidae) from Peter the Great Bay, Sea of Japan

Reproduction and larval development of the limpet Limalepeta lima (Dall, 1918) were studied under laboratory conditions for the first time. L. lima breeds in early July; it exhibits external fertilization and the pelagic lecithotrophic type of development. Larvae, juvenile, and adult specimens have different shell sculptures. Development from fertilization to settling and metamorphosis continues for 4–5 days at a water temperature of 16°C.     

Understanding fertilization through intracytoplasmic sperm injection (ICSI)

Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.     

Mechanism of Fertilization: A Modern View

Despite numerous studies on mammalian fertilization, the mechanisms of fertilization—including the timing of acrosome reaction—remain largely unknown; more accurately described, the classical theory built upon years of layered experimental data is being challenged by recent conflicting evidence provided by gene-manipulated animals. Although in vitro fertilization remains our central research tool, the classical theory’s decline reminds us of the importance of in vivo observations. Here, I describe the essential roles of gene-manipulated animals in elucidating the mechanism of fertilization and the pitfalls of in vitro fertilization studies trapping many researchers.     

Autogamy in the American strain of witchweed,striga asiatica (scrophulariaceae)

American plants ofStriga asiatica (witchweed), a serious pathogen of corn and other grains that was accidentally introduced to the Carolinas in the 1950’s, exhibit a form of autogamy in which fertilization occurs before the corolla opens. Pollen is shed on the stigma while the flowers are still in bud. Pollen grains germinate immediately and by the time the corolla is open fertilization has occurred. Examination of a native population of the same species in Nigeria revealed no autogamy.     

Water salinity effects on embryogenesis of the lesser sandeel,Ammodytes tobianus (Linnaeus, 1758)

Effects of water salinity (3.0–9.0 PSU) on the duration and outcome of embryogenesis in the lesser sandeel (Ammodytes tobianus (L.) as well as on survival, size, and malformation rate of the newly hatched larvae were followed, based on observations involving equipment consisting of a microscope and still and digital cameras. The images obtained and measurements of egg and larval dimensions were analyzed using the NIS elements Br. computer software package. The lowest salinity tested (3.0 PSU), similar to that in the inshore Baltic Sea waters native for the species studied, produced a lower fertilization rate and resulted in extended embryogenesis, reduced embryo survival, changes in the hatch size, and increased larval malformation rate. The high-end salinity (7.5–9.0 PSU), higher than that prevailing in the species’s spawning grounds in the Baltic Sea, resulted — similarly to the low-end salinity — in disturbed development and growth of the embryos.     

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit,Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

Background/Aims

The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.

Methodology and Principal Findings

Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2^nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization.

Conclusions

This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.     

A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization

A trypsin-like proteinase was localized within a single subcellular compartment of unfertilized Strongylocentrotus purpuratus eggs, the cortical granules. Homogenates of eggs were fractionated by rate-zonal centrifugation. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1, 3-glucanase) in the sucrose density gradient. A bimodal distribution pattern was obtained for aryl esterase activity (substrate: β-naphthyl acetate), with one peak in the microsomal and the other in the cortical granule fractions. The cortical granule enzyme was characterized as a trypsin-like proteinase, since it also hydrolyzed another typical tryptic substrate α-N-benzoyl-l-arginine ethyl ester and was completely inactivated by soybean trypsin inhibitor (SBTI). The aryl esterase activity in the microsomal fractions was not inhibited by SBTI, while 50% of the total aryl esterase activity in the original egg homogenate was inactivated by SBTI. The identity of the enzyme(s) responsible for the aryl esterase activity associated with the microsomal particles is unknown at present.The cortical granule proteinase functions in the elevation of the fertilization membrane and establishment of the block to polyspermy at fertilization. Arbacia punctulata eggs inseminated in the presence of trypsin inhibitors, SBTI or tosyl lysine chloromethyl ketone (TLCK), failed to elevate normal fertilization membranes and became heavily polyspermic.On the basis of these results and observations made by other investigators with a wide variety of biological systems, it is proposed that trypsin-like proteinases function in the discharge of secretory granules from all types of cells.     

Chromosome studies on the Bambusoideae (Gramineae)

Meiotic chromosome numbers and observations were made in eleven species belonging to nine different genera of Bambusoideae. The numbers include counts in eight species that have never been investigated cytologically:Arundinaria aff.amabilis (n=24), Chusquea bambusoides (n=ca. 20),Cryptochloa dressleri (n=11), Olyra ciliatifolia (n=11), Sucrea monophylla (n=11), Raddia brasiliensis (n=11), Strephium distichophyllum (n=11), andPariana aurita (n=22). The generaStrephium andSucrea have not been studied previously with respect to their chromosomes. The significance of these results are discussed in relation to the over 140 published counts in the subfamily, which indicates a preponderance of polyploidy in the woody members, or bamboos, but not in their herbaceous allies. Aneuploidy is rare in the former but frequent in the latter. It is postulated that the herbaceous bambusoids, which flower every year and produce recombinants regularly and in short-time cycles, could have tolerated successive reductions of the linkage groups and their genetic flexibility. These changes would not have been favored in the bamboos, which have more genetic constancy due to the long lapse (often 20–120 years) between flowering generations.     

Consistent Paternity Skew through Ontogeny in Peron's Tree Frog (Litoria peronii)

Background

A large number of studies in postcopulatory sexual selection use paternity success as a proxy for fertilization success. However, selective mortality during embryonic development can lead to skews in paternity in situations of polyandry and sperm competition. Thus, when assessment of paternity fails to incorporate mortality skews during early ontogeny, this may interfere with correct interpretation of results and subsequent evolutionary inference. In a previous series of in vitro sperm competition experiments with amphibians (Litoria peronii), we showed skewed paternity patterns towards males more genetically similar to the female.

Methodology/Principal Findings

Here we use in vitro fertilizations and sperm competition trials to test if this pattern of paternity of fully developed tadpoles reflects patterns of paternity at fertilization and if paternity skews changes during embryonic development. We show that there is no selective mortality through ontogeny and that patterns of paternity of hatched tadpoles reflects success of competing males in sperm competition at fertilization.

Conclusions/Significance

While this study shows that previous inferences of fertilization success from paternity data are valid for this species, rigorous testing of these assumptions is required to ensure that differential embryonic mortality does not confound estimations of true fertilization success.     

Interactions between parents and infants,and the development of independence in the common marmoset (Callithrix jacchus)

Detailed observations were made of the parent-infant interactions of five adult pairs and 31 infant common marmosets (Callithrix jachus), housed in captive family groups. Records of parental measures were taken up to 4 months old and comparisons made between infants in similar social situations. These comparisons investigate sex differences, birth-order variations, those between singleton and twin infants, parental variation and the proportion of time that older siblings carry infants. Correlations between the measures reveal that both parents are responsible for promoting and regulating increasing infant independence. In the discussion, comparisons are made with other parent-infant studies and problems of the adaptiveness of the Callitrichid social system considered.     

Histopathology of Aedes aegypti (Diptera: Culicidae) larvae parasitized by Reesimermis nielseni (Nematoda: Mermithidae)

Histological observations were made of Aedes aegypti larvae parasitized for 2, 4, and 6 days by Reesimermis nielseni. Little difference was detected between the tissues of uninfected and nematode-parasitized larvae 4 days after infection, at which time most hosts were in the early fourth instar and their fat bodies were well developed containing abundant storage materials. Nematodes grew most rapidly between day 4 and day 6 of parasitism, depleting host metabolites, reducing the fat body and other host storage tissues while accumulating storage material in their trophosomes. Development of host imaginal discs was inhibited during this period. The severity of the nematodes upon host tissues depended upon intensity of infection. Dry weight measurements of nematode and host supported histological observations that the nematode developed most rapidly 4–6 days post-infection, thus causing most serious effects upon the host at that time.     

Developmental cytology of the midgut in the flesh-fly, Sarcophaga bullata (Parker)

The cytological comparisons of the midgut in Sarcophaga bullata (Parker) between the second instar, the third instar larvae and the adult are made. The adult midgut differs from that of the larvae in the following ways: (1) the peritrophic membrane is thicker than in the larvae and has become multi-layered; (2) epithelial cells are smaller; (3) branched microvilli are present throughout the entire midgut instead of being present only in the posterior region as in the larval midgut; (4) nuclear pores are less frequent; (5) lysosome-type structures occur less frequently; (6) the basal membrane is thicker; (7) the z-bands in the surrounding muscle fibers are more distinct in adults. The possible function and the significance of these structures related to previous observations in Sarcophaga and other Diptera are discussed.