Master of Science

Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 54-60). Description: xi, 60 leaves : ill. ; 29 cm.

The Microsporidia are obligate intracellular parasites that have jettisoned oxidation phosphorylative capabilities during their early evolutionary history and so rely on ATP import from their host and glycolysis for their energy needs. Some species form tight associations with the host’s mitochondria and this is thought to facilitate ATP sequestration by the developing intracellular microsporidian. The human parasite, Enterocytozoon bieneusi has however lost glycolytic capabilities and may rely entirely on ATP import from its host for energy. E. bieneusi belongs to the Enterocytozoonidae microsporidian family and recent rDNA-based phylogenetic studies have suggested it has close evolutionary ties with Enterospora canceri, a crab-infecting intranuclear parasite. Such a close evolutionary relationship implied that glycolysis might also be absent in the intranuclear parasite raising questions as to how this parasite obtains energy from its unusual niche that is physically walled off from the host mitochondria, the main source of ATP in the host cell. In this study, draft genomes of four species of the Enterocytozoonidae namely, Ent. canceri, E. hepatopenaei, Hepatospora eriocheir and Hepatospora eriocheir canceri and one non-Enterocytozoonidae species, Thelohania sp. were assembled and annotated (The genome assembly of Hepatospora eriocheir was provided by Dr. Bryony Williams). Phylogenomics performed with this and publicly available genomic data confirmed the close evolutionary ties between Ent. canceri and E. bieneusi. Comparative genomic analyses also revealed that glycolysis is indeed lost in all members of the Enterocytozoonidae family sequenced in this study, hinting to the relaxation of evolutionary pressures to maintain this pathway at the base of this microsporidian family. Despite this absence, the hexokinase gene was retained in all aglycolytic genomes analysed, and that of Ent. canceri was fused to a PTPA gene. Functional assays and yeast complementation assays suggest that this chimera is able to recognise glucose as a substrate but the heterologously expressed homolog of H. eriocheir cannot. Finally, phylogenomics have been used here to demonstrate that despite the morphological differences between three Hepatospora-like organisms parasitizing different crab hosts, they are the same species. This finding adds more weight to current evidence suggesting that morphology is not an ideal marker for taxonomical classification in the Microsporidia.

H.rotundifrons A. Milne Edwards, 1867, the big-handed crab, belongs to the Belliidae and is the only crab representative of this family in New Zealand. It lives in the intertidal zone. The megalopae undergo 13-16 moults to become fully mature at a size of 11-13mm CWo These mature crabs live up to a maximum of 19 years and attain a size of 23-25mm CW (24-26 instars). The laboratory growth curve of H.rotundifrons is asymptotic and there is no terminal ecdysis. Growth is similar between the sexes up to maturity but decreases more in males after puberty thus exhibiting an "anomalous" pattern. Average weight gain after ecdysis was found to be same for both sexes and independent of size (approx. 18%). Environmental and physical factors which normally affect growth have no profound effect on H.rotundifrons. However, moult frequency was found to be increased by removal of eyestalks or when crabs were maintained in constant darkness. Starvation and limb removal considerably reduced the moult increment. Alpha ecdysone concentration increased to a peak 40 days before ecdys is and returned to base 1eve1s at moulting. Moulting is seasonal having a high peak in mid-summer and a smaller peak in April-May. A field study of dispersal showed that short-range movements (5.06m), within the intertidal zone, are common for H.rotundifrons (n=212). Many crabs left their refuge between low tides and their movements were independent of one another. Neither home range nor long dispersal movement (>21m) have been observed in H.rotundifrons. Diet of H.rotundifrons comprised algae (60.7%), crustaceans (19.6%), sand & unidentified materials (11.7%), sponges & worms (5.7%), and molluscs (2.3%). Although brown algae were always the single dominant food item, diet composition varied seasonally. The majority of the population at Kaikoura (602(M):858(F)) consisted of pre-pubertal males and females and fewer post-pubertal males were found than post-pubertal females, a reflection of slower male growth and suggesting higher male mortality.

This project is to search for Very High Energy (VHE) (10¹¹ eV to 10¹⁴ eV) gamma rays from the Crab nebula and pulsar using the atmospheric Cherenkov imaging technique. The technique uses an array of 37 photomultiplier tubes to record the images of the Cherenkov light pulses generated by energetic particles in the air showers initiated by VHE gamma rays or charged cosmic rays. Gamma-ray-like events are selected from numerous cosmic-ray events based on the predicted properties of the image, such as the size, shape, and orientation with respect to the axis of the detector. A steady weak flux of VHE gamma rays from the Crab is detected at high statistical significance (9 sigma), which is not usually achieved in VHE gamma-ray astronomy. No strong evidence of pulsed emission is found when the same data is folded at the Crab pulsar's radio ephemeris. The angular resolution of the technique cannot separate the emission coming from the nebula from that from the pulsar. Although it is generally believed that the unpulsed emission is coming from the nebula, there may be an unpulsed component coming at only a couple of light cylinder radii away from the pulsar too. Using the outer gap model of pulsar, the spectrum is derived and is found to be compatible with the observations.

Hypoxia or dissolved oxygen concentrations < 2 mg/L is a problem in estuaries worldwide. In Lake Pontchartrain, a 250 km2 de-faunated zone exists as a result of salinity stratification and episodic hypoxia. Mature common rangia clams (Rangia cuneata) are not found within this zone. Blue crabs (Callinectes sapidus) are important estuarine predators and may move in and out of the hypoxic zone to feed on hypoxia-stressed rangia clams. To test the effects of hypoxia on predation, rangia clams were exposed experimentally to severe hypoxic conditions (< 0.75 mg/l) for 72-hours and then presented to blue crabs. One hypoxic and one normoxic clam were added to each aquarium containing a blue crab for each trial, and crab feeding choices were observed and recorded. I found prey choice varied among crabs, but in general, the experimental data demonstrates that crabs chose to feed on hypoxia-stressed clams over clams kept under normoxic conditions.

"Lipid-based hormones known as the juvenile hormones (JH) are ubiquitous among the arthropods, but their presence, functions, and sites of production in the horseshoe crab, Limulus polyphemus, remain unknown. Large size and lack of secondary sex characteristics in adult female horseshoe crabs may indicate continuous growth and molting throughout life, which is the outcome of high JH levels in insects and crustaceans. Here a study was undertaken to detect and localize lipid-based hormones in horseshoe crab hemolymph and tissue. Capillary electrophoresis and RP-HPLC analyses indicate the presence of a JH-like compound in subadult horseshoe crab hemolymph. The compound is present only in much lower amounts in the hemolymph of adult male and adult female horseshoe crabs. Identification of this compound was based on its similar retention time to standard JH, co-migration with added JH, and cross-reactivity with a polyclonal antibody to JH III. In addition, immunohistochemistry was used to localize the production site of this compound. Analysis of neural tissue, the assumed site of production, yielded no reactivity with labeled anti-JH III antiserum. In larval animals, however, reactivity was noted in yolk contained within the digestive tract. Since the larvae are lecithotrophic and feeding only on their yolk reserves, JH in the gut may be maternal, deposited in the egg before laying. Based on these results, we conclude that horseshoe crabs produce a lipid-based, JH-like hormone, with functional similarity to JH III in insects (i.e., maintenance of the juvenile form during growth and molting.) This paper is the first substantiation of such a hormone in horseshoe crabs. Our findings suggest that JH will be found in other chelicerates as well."

The production and use of manufactured metal (oxide) nanoparticles has exploded in recent years as they are exploited for their novel physical and chemical properties. Cerium oxide NPs (CeO2NPs) help increase combustion in diesel engines and their reported ability to scavenge free radicals has been exploited in therapeutic treatments. Silver NPs (AgNPs) are now used in consumer products such as socks and sticking plasters due to their antibacterial properties. Once released into the environment, their ultimate fate is predicted to be the oceans The aims of this thesis are to investigate the bioaccumulation and biological effects (oxidative stress and lipid peroxidation) of CeO2NPs and AgNPs on the mussel Mytilus edulis, and to understand the potential for trophic transfer of CeO2NPs to the crab Carcinus maenas, and subsequent induced biological effects. It was found in acute exposures that, at the suggested regulatory limit of 3mg/l, less than 5% of a CeO2NP dose will be accumulated by the digestive gland of M. edulis within 4 hours, before being depurated over at least 56 hours. There were no significant biological effects of CeO2NPs, yet larger, micron-size particles had significant anti-oxidant effects. Most effects were transitory, returning to normal levels after 24 hours. In uptake comparisons between AgNPs and Ag-nanorods (AgNRs) at 10μg/l (towards the regulatory limit of 1.9μg/l), AgNRs were accumulated in the digestive gland within 2 hours, but were depurated by 4 hours. Similarity in accumulation between AgNPs and ionic Ag – including continuous accumulation in the gills over 48 hours – suggested dissolution was mostly responsible for this. Both nanoforms instigated isolated oxidative stress responses over 4-24 hours, yet none were significantly worse than AgNO3, which instigated the greatest suite of significant oxidative stress responses. In trophic transfer experiments C. maenas accumulated CeO2NPs in the hepatopancreas at less than 1% of the fed dose. Stomach accumulation was high but transitory, with most particles removed in the faeces. Gills were also a site of accumulation and it was thought that the haemolymph provided a route of transit between the digestive organs and the respiratory organs. This novel experiment used NPs crafted from 140Ce; changes in isotopic ratios of Ce in the crab following trophic transfer could therefore be used to determine absolute increases in concentration against high, and highly variable, background concentrations. There were no significant biological effects following trophic transfer of these 140CeO2NPs. It was found that the current regulatory limits are predicted to be sufficient to protect M. edulis and C. maenas from acute exposure to CeO2NPs and AgNPs, yet chronic exposures should be investigated since the relationship between the uptake and elimination rate of NPs will determine the extent of bioaccumulation and biological effects.

Ho Hoi Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 107-118). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.v / Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.5 / Chapter 2.1 --- Introduction to phylogenetic biology --- p.5 / Chapter 2.1.1 --- Definition of phylogenetics --- p.5 / Chapter 2.1.2 --- Why employ molecular genetic markers in phylogenetics? --- p.5 / Chapter 2.2 --- DNA analysis and the contributions to phylogenetics --- p.7 / Chapter 2.2.1 --- Nuclear ribosomal DNA --- p.7 / Chapter 2.2.2 --- Animal mitochondrial DNA (mt DNA) --- p.10 / Chapter 2.3 --- Molecular phylogeny of crustaceans --- p.11 / Chapter 2.3.1 --- Phylogenetic studies of crustaceans using nuclear rRNA --- p.11 / Chapter 2.3.2 --- Population genetic and phylogenetic studies of crustaceans using mtDNA --- p.14 / Chapter 2.4 --- Taxonomy of the genus Eriocheir --- p.23 / Chapter 2.5 --- Distribution of Eriocheir s.l --- p.27 / Chapter 2.6 --- Population genetics of Eriocheir --- p.28 / Chapter 2.6.1 --- Population genetics of Eriocheir japonica --- p.28 / Chapter 2.6.2 --- Population genetics and history of invasion of Eriocheir sinensis --- p.29 / Chapter Chapter 3 --- Materials and Methods --- p.32 / Chapter 3.1 --- Specimens --- p.32 / Chapter 3.1.1 --- Specimens for phylogenetic reconstruction --- p.32 / Chapter 3.1.2 --- Specimens for population genetic study on E. sinensis --- p.33 / Chapter 3.2 --- DNA extraction --- p.33 / Chapter Chapter 3 --- Materials and Methods --- p.32 / Chapter 3.1 --- Specimens --- p.32 / Chapter 3.1.1 --- Specimens for phylogenetic reconstruction --- p.32 / Chapter 3.1.2 --- Specimens for population genetic study on E. sinensis --- p.33 / Chapter 3.2 --- DNA extraction --- p.33 / Chapter 3.3 --- Amplification of genes --- p.36 / Chapter 3.3.1 --- PCR profile --- p.38 / Chapter 3.3.1.1 --- 16S rRNA gene --- p.38 / Chapter 3.3.1.2 --- COI gene --- p.40 / Chapter 3.3.1.3 --- ITS-1 gene --- p.41 / Chapter 3.4 --- DNA sequencing --- p.41 / Chapter 3.4.1 --- Purification of extension products --- p.43 / Chapter 3.4.2 --- Electrophoresis and data collection --- p.44 / Chapter 3.4.3 --- Sequence alignment --- p.44 / Chapter 3.5 --- Phylogenetic construction --- p.45 / Chapter Chapter 4 --- Results --- p.47 / Chapter 4.1 --- Phylogenetic reconstruction of Eriocheir --- p.47 / Chapter 4.1.1 --- PCR products of mitochondrial 16S rRNA and COI gene and nuclear ribosomal ITS-1 gene --- p.47 / Chapter 4.1.2 --- Intraspecific variation --- p.49 / Chapter 4.1.3 --- Genetic variability in mitten crabs based on partial sequences of 16S rRNA gene --- p.49 / Chapter 4.1.4 --- Genetic variability in mitten crabs based on partial sequences of COI gene --- p.53 / Chapter 4.1.5 --- Genetic variability in mitten crabs based on complete sequences of ITS-1 gene --- p.62 / Chapter 4.1.6 --- Phylogenetic analysis --- p.66 / Chapter 4.1.6.1 --- Phylogenetic analysis based on 16S rDNA sequences --- p.66 / Chapter 4.1.6.2 --- Phylogenetic analysis based on COI gene sequences --- p.70 / Chapter 4.1.6.3 --- Phylogenetic analysis based on ITS-1 gene sequences --- p.73 / Chapter 4.2 --- Population genetic study of E. sinensis --- p.77 / Chapter 4.2.1 --- PCR products and intraspecific variation of mitochondrial COI gene for population genetic study of E. sinensis --- p.77 / Chapter 4.2.2 --- Genetic variability in E. sinensis based on partial sequences of COI gene --- p.79 / Chapter 4.2.3 --- Population genetic analysis based on COI gene sequences --- p.86 / Chapter Chapter 5 --- Discussion --- p.93 / Chapter 5.1 --- Phylogeny of Eriocheir --- p.93 / Chapter 5.1.1 --- Phylogenetic relationships --- p.93 / Chapter 5.1.2 --- Taxonomic implications --- p.95 / Chapter 5.1.3 --- Evolutionary history of Eriocheir --- p.97 / Chapter 5.2 --- Population study of E. sinensis --- p.101 / Chapter 5.2.1 --- Genetic variation between Chinese populations --- p.101 / Chapter 5.2.2 --- Genetic variation between native and introduced populations --- p.102 / Chapter Chapter 6 --- Conclusions --- p.104 / Literature Cited --- p.107

The Dungeness crab fishery is the most economically important on the West Coast; however, it has experienced dramatic fluctuations in annual catch. Previous research has shown the annual catch of megalopae is correlated with the commercial catch. The catch of megalopae is correlated with the phase of the Pacific Decadal Oscillation (PDO), the day of the year of the spring transition and the amount of upwelling following the spring transition. Further, the daily catch of megalopae is correlated with the internal tide. We developed individual based models of Dungeness crab dispersal, which we validated with results from a light trap. We demonstrated that the retention of larvae in the California Current is enhanced during negative phase PDOs. Further, we suggest that larvae migrate to or almost to the bottom each day. Specifically, megalopae exhibit a twilight vertical migration off of the continental shelf and remain in the neuston on the continental shelf. This concentrates megalopae at the continental shelf break. We also observed megalopae in situ and demonstrated that they swim in the neuston with the surface current at speeds of ~ 10 cm s-1. Using these results and data from a mooring, we demonstrated that this behavior would increase the distance internal waves would transport larvae. We analyzed mooring data and suggest that catch of megalopae is greater when the thermocline is deep and weak and there is less horizontal shear. We hypothesize this allows internal waves to remain coherent longer on the continental shelf. We show that the spring transition coincides with a shallowing of the thermocline, which would ultimately lead to the development of internal waves of depression rather than elevation. We hypothesize that the change in surface flow, based on whether the wave is one of elevation or depression, explains why most megalopae are caught following the spring transition. In general, these findings help us better understand the dispersal of Dungeness crabs. We suggest the dispersal patterns support Michael Sinclair’s member vagrant hypothesis. Further, we suggest these findings apply to many of the continental shelf species in the California Current. This dissertation includes both published and unpublished co-authored materials.