Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / Withering syndrome is a severe disease of abalone, Haliotis species that has been associated with mortality ranging from 99% in black, H. cracherodii Leach and 30% in red abalone, H. rufescens Swainson. The disease was first observed in California, along the west coast of North America and is an economically important disease that has led to the closure of the black abalone fishery throughout the southern California State. The causative agent of withering syndrome is a gram-negative intracellular Rickettsiales-like prokaryote designated Candidatus xenohaliotis californiensis. The geographical range of C. xenohaliotis californiensis is broad, besides red and black abalone it has also been reported in yellow, H. corrugate and blue abalone, H. fulgens all caught in Baja California, Mexico. In 2000 a Rickettsiales-like prokaryote resembling the disease-causing agent was observed in the digestive gland of the South African abalone H. midae. In this study we aimed to determine the relationship of the bacterium observed in the local abalone species, H. midae to the disease-causing agent of withering syndrome. A specific PCR and in situ hybridization test using primers and probes specific for the C. xenohaliotis californiesis 16S rDNA gene were used to screen H. midae digestive gland tissues showing classical features of the Rickettsiales-like prokaryote. Both analyses indicated that C. xenohaliotis californiensis is not present in the local abalone species. We therefore aimed the identification of the organism parasiting the local abalone species by DNA sequence analysis of the 16S rDNA gene. The 16S rDNA gene was amplified by PCR, cloned and sequenced. Phylogenetic trees, constructed by maximum parsimony analysis revealed a diverse community comprised of α - Proteobacteria, Mollicutes and Spirochaetes. In the class α - Proteobacteria a novel group of sequences showing phylogenetic affinities to the order Rickettsiales was identified as likely candidate for forming the Rickettsiales-like inclusions in the digestive gland of H. midae. Oligonucleotide probes that bind to four variable regions of the novel group were used to confirm their presence in infected H. midae digestive gland tissue by in situ hybridization. Although these probes did not recognize the inclusions formed by the Rickettsiales-like organisms, they revealed the presence of a group of free-living bacteria abundant in the host tissue. We therefore conclude that (1) C.xenohaliotis californiensis is not present in the South- African abalone, H. midae; (2) the organisms isolated from the digestive gland of H. midae are part of the normal microflora and (3) the group of sequences showing phylogenetic affinities to the order Rickettsiales is not responsible for the Rickettsiales-like inclusions in infected digestive gland tissues but represent a novel group of organisms that are abundant in the host tissue.

Terebrasabella heterouncinata is a small K-selected sabellid polychaete. It is a simultaneous hermaphrodite with a semi-continuous mode of reproduction, producing relatively few large eggs that are brooded within the parental burrow until the larvae emerge, to settle on the growing edge of the abalone shell. Despite its low fecundity, this worm has become problematic on abalone farms in South Africa. The present study was conducted to gain an understanding of the life history patterns of T. helerouncinata to determine how they contributed to the success of these worms under altered conditions. This study demonstrated that conditions prevalent on abalone farms were conducive to enhancing the reproductive success of this worm, and suggests that larger, more fecund worms may have been selected for in the decade that these worms have been present on the farms. Increased nutrient availability, and possibly the increased stability of the farm environment relative to its natural environment, has led to a 1.5-fold increase in the average size of the worms. Body size was found to be positively correlated with brood size, and this resulted in worms on farms brooding 3 to 4.5 times more offspring at a time than worms from wild abalone. The ability to increase the number of eggs produced at a time may have been limited by the fact that these worms have only two ovaries. Thus, the increase in fecundity may have been related primarily to the increase in the rate at which the eggs were laid by the worms on the farms, and the increase in the coelomic space available for the storage of these rapidly developing eggs. The ability to increase the rate at which oocytes develop may be related to the vitellogenic mechanisms employed by these worms. Vitellogenic oocytes are able to incorporate high molecular weight yolk precursors from the surrounding coelomic fluid through endocytotic activity. This may allow the oocytes to increase the rate at which they incorporate yolk material under conditions of nutrient enrichment. The increase in fecundity did not occur at the expense of offspring size and, presumably, quality. The increased reproductive output on the farms was compounded by a proportionate increase in the number of reproducing worms within the population. In addition, these worms are long-lived (worms from farmed abalone reached a maximum age of approximately 40 months) and exhibit negligible senescence. Thus, their reproductive output did not change significantly with an increase in age. Furthermore, the proportion of the reproductive worms did not decrease with an increase in age. Thus, within the age range tested, worms of all ages have the potential to make equal contributions to population growth. While diet and abalone stocking density could not be identified as having a significant effect on reproductive output and infestation rate under intensive culture conditions, it was demonstrated that in a naïve abalone population, the total intensity of infestation increased exponentially with time. This increase may be a consequence of an increase in fertilisation success. These worms continuously produce entaquasperm that are released into the water column. The sperm are collected by other individuals that then store the sperm in a single spermatheca. The ability to store sperm relieves individuals of a dependence on the synchronisation of spawning of eggs and sperm. As the population size and density increases, there could be more individuals releasing sperm into the water column, resulting in a continuous supply of sperm. The increased production of eggs would therefore not be constrained by a lack of sperm. The stored sperm are released into the brood chamber to fertilise eggs as they are laid, and this would probably increase the fertilisation success in the species. This study also provides evidence to suggest that reproduction in this worm has a seasonal component. Future studies should concentrate on measuring fertilisation success in greater detail, measuring the effect of season on reproduction, determining whether there are genetic differences between worms on farmed and wild abalone and determining whether wild worms have similar life-spans and age-related fecundity as worms on farms.