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In situ measurements of the fertilization success of broadcast spawning marine invertebrates have been conducted mainly on shallow subtidal echinoderm and coral species. In this study, field fertilization success was measured for two infaunal, intertidal polychaetes Arenicola marina and Nereis virens. Both species are epidemic spawners, with populations of A. marina spawning over a few days in autumn while N. virens spawns in early spring. The fertilization success of female Arenicola marina is highly variable, ranging from 0 to 100%, and is determined by male spawning density. It is hypothesised that fertilization success over the entire spawning period is the result of an accumulation of fertilizations each day in the spawning period. Fertilization success was measured indirectly in Nereis virens by transplanting oocytes into the field. At least two spawning periods occurred for the population studied here. Fertilization success was uniformly high during the first period, and in the second fertilization success was highest among those eggs located high in the water column as opposed to those at substratum level. The fertilization strategies of these polychaetes are discussed in the light of these results. Comparative laboratory experiments were performed on factors that affect fertilization success in Arenicola marina. Nereis virens, Asterias rubens and Echinus esculentus, including sperm:egg ratio, sperm-egg contact time and sperm concentration. The extent to which each of these factors affects fertilization success varies with species, and this is explained by gamete attributes and mathematical models. Gamete longevity significantly affects fertilization success, and eggs and sperm of Arenicola marina are extraordinarily long lived compared to those of the other species studied here and elsewhere. Eggs are viable for 5 days after spawning, while dilute sperm remains capable of fertilizing eggs for more than 48 hours. The laboratory data are discussed in terms of the fertilization strategies of each of the species.

This thesis presents ultrastructural and biochemical information on meiotic reinitiation during oocyte maturation in the polychaetes, Arenicola marina, A. defodiens and Nereis virens. The ultrastructural changes during meiotic maturation was characterised in the oocytes of Arenicola marina and Nereis virens using transmission electron microscopy in addition to germinal vesicle breakdown, release of the prophase I block was signified by major cortical changes in both species. The ultrastructure of fertilization in A. marina was independent of whether the oocytes were matured in vivo and spawned or matured in vitro by CMF. Oocyte maturation in Arenicola marina is controlled by a hormonal cascade that is initiated by the prostomial maturation hormone, PMH, and followed by the coelomic maturation factor, CMF (Watson and Bentley, 1997). Results presented here demonstrated that PMH has a molecular mass greater than 10 kDa, yet how this molecule triggers CMF activity remains unknown. M-phase promoting factor (MPF) consists of two subunits, cdkl and cyclin B, and is responsible for the control of mitosis and meiosis. The cytoplasmic "second messenger" that transduces the hormone signal to the activation of MPF in the oocyte cytoplasm was investigated in the two Arenicola species and is discussed. MPF regulation was investigated in Arenicola marina and Nereis virens oocytes. MPF activation was driven by the dephosphorylation of cdkl and phosphorylation of cyclin B. The results indicate that as with all other higher eukaryotes, the precursor of MPF in A. marina oocytes was maintained inactive by the phosphorylation of threonine 14 and tyrosine 15 (or equivalent residues) on the cdkl subunit. In contrast to other organisms, however, only a fraction of the cdkl present was complexed to cyclin B and utilised during meiotic reinitiation. All the cdkl in N. virens oocytes was joined with cyclin B but results suggest that the inactive complex contained tyrosine-only phosphorylated cdk1.

This study investigated the reproduction, larval growth and metamorphosis of the nudibranchs Onchidoris bilamellata (L.) and Goniodoris nodosa (Montagu) under controlled laboratory conditions. In addition, the rare occurrence of spawning events of O. bilamellata taking place in the field outwith the winter and spring reproductive period was studied. Onchidoris bilamellata and Goniodoris nodosa differ in the size and number of eggs produced per spawning event. In the present study O. bilamellata does not exhibit a clear spawning pattern, whereas G. nodosa lays increasingly smaller eggs and larvae as the season progresses. The diameter of the eggs of O. bilamellata is not correlated with the organic content per egg. The data available for G. nodosa were insufficient to analyse this relationship. The rates of larval growth and development are reported for Onchidoris bilamellata and Goniodoris nodosa. These variables do not differ significantly for O. bilamellata between cultures maintained under various light regimes, ranging from continuous darkness to continuous illumination, and are comparable to those of G. nodosa. The shell growth pattern exhibited by larvae of G. nodosa is sigmoid, similar to that of other opisthobranchs. Spawn masses laid during the winter and spring months were collected fresh from the field and compared to spawn masses collected from the field in July and September, outwith the typical spawning period of Onchidoris bilamellata in the British Isles. The shell size of the July and September hatchlings was significantly smaller than that of the winter and spring hatchlings. Furthermore, the larvae hatched from spawn masses laid in September cultured in the laboratory exhibited high mortality rates and the overwhelming majority did not survive through metamorphosis. The significance of the spawning activity of O. bilamellata past the typical spawning period of this species is discussed. The induction of metamorphosis of Onchidoris bilamellata was investigated using seawater containing elevated concentrations of potassium ion, and the results indicate that the optimal concentration inductive of metamorphosis is 19 mM K+ ASW, Metamorphosis experiments were also performed with the natural prey of O. bilamellata, the acorn barnacle Semibalanus balanoides (L.). The results suggest that pediveligers can be induced to metamorphose at a distance from the inductive substrata. Attempts were made to identify the natural inductive cue of Goniodoris nodosa, but this was not successful.

Previous studies on Arenicola marina suggest that oocyte maturation is induced by a hormone, from the prostomium, acting directly on the oocyte (Meijer and Durchon, 1977). Results presented here, from studies on British populations of Arenicola marina, show that in this species, oocyte maturation is controlled by two hormonal steps; a prostomial maturation hormone followed by a maturation inducing substance in the coelomic fluid, the Coelomic Maturation Factor (CMF). A reliable in vitro assay for oocyte maturation in A. marina has been adopted distinguishing immature from mature oocytes enabling CMF to be investigated; CMF has a molecular mass greater than 10 kDa, is thermolabile and inactivated by trypsin suggesting a proteinaceous nature. Production of CMF was also investigated; peak production occurs at approximately 2 hours after the injection of homogenised female prostomia. Oocytes require, on average, a minimum time of 20 minutes incubation in CMF to mature and preliminary results suggest calcium may not be necessary for maturation. Immunocytochemical techniques were used to characterise microtubule structures during oocyte maturation and post-fertilization development in Arenicola marina. Different regimes capable of bringing about maturation do not affect the morphology of the meiotic spindle. However, post-fertilization development is slower in oocytes matured in vitro when compared to mature oocytes obtained from naturally spawning females and females injected with homogenised prostomia which then spawn. Changes in microtubule structures in A. defodiens and Nereis virens during post-fertilization have also been partially characterised and these are compared and contrasted with A. marina. The development of a microinjection system will enable intracellular calcium and its role in maturation and fertilization within oocytes to be examined in these polychaetes.

Populations of Arenicola marina from different localities have been reported to show variation in certain characteristics. There are morphological differences such as colour, size and annulation patterns, and there is also variation in the duration and timing of the spawning seasons on different beaches. An extensive study of gametogenesis in individuals from three selected study sites was carried out by regular sampling throughout the year. Each site shows a different spawning pattern, either epidemic such as at the East Sands, St. Andrews, or an extended season, such as at the Eden Estuary, St. Andrews. These two sites are less than three kilometres apart and yet there are significant differences in the size of the gravid adults. The third site, Fairlie Sands on the west coast of Scotland shows a very unusual type of spawning season in that a small percentage of the population are gravid throughout most of the year. Genetic variation was assessed using the technique of starch gel electrophoresis of allozymes. There is a degree of genetic differentiation between the three sites. Allele frequencies at the GOT locus are different between east and west coast sites and a third rare allele is present at Fairlie Sands. It is suggested that gene flow mediated by larval transport may be limited and this would cause inbreeding in some subpopulations. The hormones involved in spawning have been investigated with particular reference to a putative female oocyte maturation hormone. Comparative work between Arenicola marina and A. defodiens has demonstrated the presence of an oocyte maturation factor in the prostomia of both species. While this factor acts directly on A. defodiens oocytes in vitro, the presence of a cofactor or secondary product produced in vivo is required in order to reinitiate meiosis in A. marina oocytes. Preliminary studies on the nature of reproductive endocrine substances present in the prostomia of female A. marina have been carried out.

Nephrops norvegicus is a deep water marine decapod crustacean which burrows in fine muddy substrata. It is a commercially important as a fisheries species, but knowledge of its biology, particularly concerning its endocrinology, is limited. This thesis describes the endocrinology of Nephrops norvegicus with particular reference to crustacean hyperglycaemic hormone. Histological investigations of the eyestalk of Nephrops norvegicus enabled the identification d" the X-organ sinus gland complex. The development of a microbioassay allowed the determination of increases of glycaemia following the injection of crude sinus gland extracts into a host animal. The optimal dose for induced haemolymph glycaemia was determined. HPLC separation was used to isolate and purify several neuropeptides from crude sinus gland extracts, which had varying degrees of hyperglycaemic activities. The use of ELISA (enzyme linked immunosorbent assay) showed that the peptides reacted with polyclonal rabbit antiserum raised against the CHH of the crayfish Orconectes limosus and with polyclonal guinea pig antiserum raised against the CHH of the lobster, Homarus americanus. SDS-PAGE of these peptides enabled an estimation of their molecular weights and the purity of one of the active peptides was determined using capillary electrophoresis. The effects of photoperiod and severe hypoxia on the CHH-induced hyperglycaemia of Nephrops norvegicus were also investigated. The responses of N. norvegicus appeared to differ in some respects from other decapods species. Pairs of oligonucleotide primers, based on the sequence of the lobster, Homarus americanus, complimentary to regions of the CHH sequence, were used in the polymerase chain reactions (PCR). Complimentary first strand DNA (cDNA) was synthesised from total Nephrops eyestalk RNA. 35 rounds and 40 rounds of PCR amplification produced a l00bp and 230bp double stranded DNA product respectively, which were resolved by electrophoresis on a tris-borate-EDTA gel. The product size was compared with known standards. Finally, the identification of CHH and GIH synthesis and storage in the eggs of Nephrops norvegicus at various stages of development was investigated. By the use of PCR, it was not possible to determine if synthesis of CHH was occurring in 50% developed eggs. The use of ELISA, however, demonstrated that in 90% developed eggs there was a significant increase of both CHH and GIH immunoactivity.

The spermatozoa of Arenicola marina are unlike those of most marine invertebrates, in that they become motile in the body cavity prior to spawning. This occurs in response to a Sperm Maturation Factor (SMF) which is released from the prostomium. Prior to activation, spermatozoa are held as morulae with several hundred spermatozoa connected by a common mass of cytoplasm called the cytophore. Sperm activation by SMF is characterised in vitro, in terms of the ultrastructural changes which occur as the sperm become motile, and an active role for the cytophore during sperm activation is suggested. The morphology of these spermatozoa is 'primitive', and ultrastructural observations show that they possess a discoid swelling at the distal end of the flagellum. It is suggested that this may aid in swimming efficiency. The chemical nature of SMF has been putatively identified as 8,11,14-eicosatrienoic acid from both in vitro and in vivo studies. Biochemical investigations demonstrate that sperm activation is linked to an increase in sperm respiration rate, and an elevation of intracellular pH in the order of 0.2 pH units. Levels of ATP in spermatozoa are higher than those reported in other species, and it is considered that quiescence of sperm is not mediated by the deprivation of ATP to the axoneme. It is reported that sperm activated in vitro display a motile life of less than one hour, sperm which has been spawned in vivo, however, can have a motile life of up to 48 hours. It is suggested that this extension in sperm motile life may result from capacitation-like events which occur during activation and release in vivo. A hypothesis for the synthesis of 8,11,14-eicosatrienoic acid, its release from the prostomium, its transportation and mode of action at the level of the spermatozoa is also developed.