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No description available.

Arthropod parasites and micropredators, such as ticks and mosquitoes, influence terrestrial ecosystems and harm their hosts directly and indirectly by vectoring micro-organisms. Whether micropredators similarly affect marine ecosystems and hosts is not well understood. In coral reef fish communities, the most abundant micropredators are isopods, in particular, gnathiids. Our understanding of how isopods affect fishes has been restricted by a lack of information regarding basic isopod biology including; patterns of abundance, parasite identity, host specificity and host pathology. Also it is unknown if small juvenile fish are parasitised by isopods, and if this affects their survival. Sequentially, the aims of my PhD thesis were to understand the ecology of several lesser known parasites in sufficient detail to perform manipulative experiments that could test the effects of micropredation on small juvenile reef fish. All field studies took place at Lizard Island, Great Barrier Reef My first study aimed to describe temporal and spatial patterns of isopod abundance by measuring emergence rates. Gnathiid isopod juveniles emerge from the benthos into the water column to find hosts or change locations. Although diel patterns have been demonstrated, the relationship between substrate and emergence on coral reefs is not clear. I measured emergence rates of parasitic isopods (Gnathiidea and Flabellifera) in 6 “habitats” at 2 very different sites at Lizard Island. Isopods were collected from the periphery and centres of 3 sizes of reef, and from the substrate between these reefs (sand or rubble). At both sites, the most abundant gnathiid species (Gnathia falcipenis and Gnathia sp C), was exclusive to that site. Although strong diel patterns in emergence were observed, gnathiid abundance could not be predicted by habitat. However, gnathiids were larger and more often fed over reef borders than in the centres of reefs. To explain these patterns, I suggested that first stage larvae had the largest influence on total abundance and were patchily distributed in accordance with adults from which they had recently hatched. As later stage larvae also depend on fish, more successful (fed and older larvae) are found on the edges of reefs where appropriate hosts may be more abundant. Gnathiids were over-dispersed in all habitats investigated, including apparently homogeneous beds of coral rubble and sand. This indicated that gnathiid distribution may be better predicted by very fine scale differences in substrate, or that larvae are simply gregarious, and that abundance may be difficult to predict on the basis of substrate. Without reliable differences in parasite abundance among habitats at Lizard Island, subsequent studies would rely on manipulating parasite abundance via excluding wild parasites in the field (which proved very difficult) or by controlling abundance in laboratory simulations. I then investigated host specificity in the 2 most abundant gnathiid species from the previous study. Discrete species distributions between the two sites suggested that the 2 parasites may have had different diets. Host-specificity data for gnathiid isopods are scarce because the parasitic juvenile stages are difficult to identify and host-parasite contact is often brief. Engorged third stage gnathiids were photographed and permitted to moult into adults to allow identification. I compared mtDNA sequences from their blood meals to host sequences available on GenBank. The host frequency distributions used by each species were significantly different; only four host families were shared. I concluded that G. falcipenis and Gnathia sp C operate as preferential feeders. Importantly, this work showed that G. falcipenis was indeed a natural parasite of several species of damselfish (Pomacentridae) that could be collected as young recruits and used in manipulative experiments. I then used G. falcipenis as a model parasite to investigate the effects of isopods on recently recruited Dischistodus perspicillatus and small juvenile Acanthochromis polyacanthus damselfishes. Working with honours student Ms Rose Penfold, we determined that A. polyacanthus was infected by gnathiids in the wild at sizes as small as 4.2 mm SL. Laboratory infections revealed that larger A. polyacanthus were much better at eating gnathiids, a behaviour which prevented infection, and that smaller fish were more likely to die post-infection. Infection prevalence was > 3 % during the day, but we could not sample fish for nocturnal infection prevalence. A caging experiment suggested that most micropredation on damselfish took place at night. For D. perspicillatus, I found that exposure to 2 parasites each evening for 7 nights after settlement halved the growth of these fish. Lastly, numerous free-swimming cymothoid isopods were found associated with larval fish in light traps. Cymothoids have a multi-morphic life cycle composed of micropredatory stages that eventually become females, which are permanently attached to fishes. Because cymothoid taxonomy is based around female morphology, natatory-stage cymothoids can not be identified. I sequenced mtDNA from natatory and adult female life history stages and matched 4 of 11 natatory cymothoid morphotypes. Molecular data were also used to produce a phylogeny exploring the evolution of different forms of host attachment within the Cymothoidae. This phylogeny suggests that external attachment, formerly thought to be plesiomorphic, is a derived condition and has evolved several times independently. I suggest that attachment to the buccal cavity or gills is a primitive form of attachment. This research has provided information on emergence patterns and hostspecificity which is necessary for the future study of isopod vector biology. It also provides a platform for future taxonomic and phylogenetic studies on cymothoids. I demonstrate that gnathiids infect juvenile coral reef fish and suggest that they may influence survival both directly and indirectly by reducing growth and predisposing infected fish to size-selective mortality. Thus, interactions between isopod micropredators and recruiting fishes may determine the survivorship of individual fish and influence the subsequent community structure.

Arthropod parasites and micropredators, such as ticks and mosquitoes, influence terrestrial ecosystems and harm their hosts directly and indirectly by vectoring micro-organisms. Whether micropredators similarly affect marine ecosystems and hosts is not well understood. In coral reef fish communities, the most abundant micropredators are isopods, in particular, gnathiids. Our understanding of how isopods affect fishes has been restricted by a lack of information regarding basic isopod biology including; patterns of abundance, parasite identity, host specificity and host pathology. Also it is unknown if small juvenile fish are parasitised by isopods, and if this affects their survival. Sequentially, the aims of my PhD thesis were to understand the ecology of several lesser known parasites in sufficient detail to perform manipulative experiments that could test the effects of micropredation on small juvenile reef fish. All field studies took place at Lizard Island, Great Barrier Reef My first study aimed to describe temporal and spatial patterns of isopod abundance by measuring emergence rates. Gnathiid isopod juveniles emerge from the benthos into the water column to find hosts or change locations. Although diel patterns have been demonstrated, the relationship between substrate and emergence on coral reefs is not clear. I measured emergence rates of parasitic isopods (Gnathiidea and Flabellifera) in 6 “habitats” at 2 very different sites at Lizard Island. Isopods were collected from the periphery and centres of 3 sizes of reef, and from the substrate between these reefs (sand or rubble). At both sites, the most abundant gnathiid species (Gnathia falcipenis and Gnathia sp C), was exclusive to that site. Although strong diel patterns in emergence were observed, gnathiid abundance could not be predicted by habitat. However, gnathiids were larger and more often fed over reef borders than in the centres of reefs. To explain these patterns, I suggested that first stage larvae had the largest influence on total abundance and were patchily distributed in accordance with adults from which they had recently hatched. As later stage larvae also depend on fish, more successful (fed and older larvae) are found on the edges of reefs where appropriate hosts may be more abundant. Gnathiids were over-dispersed in all habitats investigated, including apparently homogeneous beds of coral rubble and sand. This indicated that gnathiid distribution may be better predicted by very fine scale differences in substrate, or that larvae are simply gregarious, and that abundance may be difficult to predict on the basis of substrate. Without reliable differences in parasite abundance among habitats at Lizard Island, subsequent studies would rely on manipulating parasite abundance via excluding wild parasites in the field (which proved very difficult) or by controlling abundance in laboratory simulations. I then investigated host specificity in the 2 most abundant gnathiid species from the previous study. Discrete species distributions between the two sites suggested that the 2 parasites may have had different diets. Host-specificity data for gnathiid isopods are scarce because the parasitic juvenile stages are difficult to identify and host-parasite contact is often brief. Engorged third stage gnathiids were photographed and permitted to moult into adults to allow identification. I compared mtDNA sequences from their blood meals to host sequences available on GenBank. The host frequency distributions used by each species were significantly different; only four host families were shared. I concluded that G. falcipenis and Gnathia sp C operate as preferential feeders. Importantly, this work showed that G. falcipenis was indeed a natural parasite of several species of damselfish (Pomacentridae) that could be collected as young recruits and used in manipulative experiments. I then used G. falcipenis as a model parasite to investigate the effects of isopods on recently recruited Dischistodus perspicillatus and small juvenile Acanthochromis polyacanthus damselfishes. Working with honours student Ms Rose Penfold, we determined that A. polyacanthus was infected by gnathiids in the wild at sizes as small as 4.2 mm SL. Laboratory infections revealed that larger A. polyacanthus were much better at eating gnathiids, a behaviour which prevented infection, and that smaller fish were more likely to die post-infection. Infection prevalence was > 3 % during the day, but we could not sample fish for nocturnal infection prevalence. A caging experiment suggested that most micropredation on damselfish took place at night. For D. perspicillatus, I found that exposure to 2 parasites each evening for 7 nights after settlement halved the growth of these fish. Lastly, numerous free-swimming cymothoid isopods were found associated with larval fish in light traps. Cymothoids have a multi-morphic life cycle composed of micropredatory stages that eventually become females, which are permanently attached to fishes. Because cymothoid taxonomy is based around female morphology, natatory-stage cymothoids can not be identified. I sequenced mtDNA from natatory and adult female life history stages and matched 4 of 11 natatory cymothoid morphotypes. Molecular data were also used to produce a phylogeny exploring the evolution of different forms of host attachment within the Cymothoidae. This phylogeny suggests that external attachment, formerly thought to be plesiomorphic, is a derived condition and has evolved several times independently. I suggest that attachment to the buccal cavity or gills is a primitive form of attachment. This research has provided information on emergence patterns and hostspecificity which is necessary for the future study of isopod vector biology. It also provides a platform for future taxonomic and phylogenetic studies on cymothoids. I demonstrate that gnathiids infect juvenile coral reef fish and suggest that they may influence survival both directly and indirectly by reducing growth and predisposing infected fish to size-selective mortality. Thus, interactions between isopod micropredators and recruiting fishes may determine the survivorship of individual fish and influence the subsequent community structure.

Baculoviruses are lytic insect viruses. Upon internalisation, the viral genome orchestrates a sequential expression process ultimately leading to lysis of the infected cell. Release of progeny capable of infecting other cells during the process completes the infection cycle. Studies of the infection cycle in cell culture are typically conducted by synchronous infection, i.e. near simultaneous infection of all cells, by means of high virus concentrations. The behaviour of the synchronously infected culture, such as the timing of onset of progeny release, is considered representative for the infection progression within individual cells. In reality, however, the synchronously infected culture only reflects the average behaviour of all infected cells. The infection progresses in individual cells display large variability; this is most obvious in the observation that within the same culture some cells undergo cell lysis at two days post infection while others remain viable up to four days post infection. Such variabilities or asynchronies observed in synchronously infected culture is the topic of this thesis. Using a simple phenomenological model, it is demonstrated that cell death and associated intracellular product release is adequately described assuming that the waiting time from infection to cell death follows a Gaussian distribution with a mean of 59 hours post infection (hpi) and a standard deviation of 15hpi. Unlike other deterministic models developed over the last decade (Licari and Bailey 1992; Nielsen 2000), this stochastic model does not make the biologically inconsistent assumption that cells continue to be metabolically active following loss of membrane integrity. While elegant in its simplicity, the model provides no explanation for the underlying stochasticity. Investigations into the cause of this dispersion of cell death highlighted further asynchronies in the specific recombinant protein yield, in viral DNA content, in virus budding rate, and in cell volume increase instead of clarifying the issue. A modelling framework developed by Licari & Bailey (1992) and later Hu & Bentley (2000) incorporates the number of infectious particles each individual cell receives as a possible source of the dispersions in the host cell responses. However, this was found NOT to be the cause of the observed asynchronies under non-substrate limiting conditions. The timing of cell death, cell volume increase, recombinant product yield, viral DNA content, and virus budding rate is identical in Sf9 cell cultures infected at multiplicities of infection of ~5, ~15, and ~45 infectious particles per cell. Cell cycle variation has previously been suggested as a possible cause for observed asynchronies in baculovirus infections (Brown and Faulkner, 1975). The cell cycle phase is indirectly linked to the cell volume, because a G_2-phase cell prior to division is inherently twice the cell volume of a G_1- phase cell after cell division. By the same logic, it is also apparent that a G_2-phase cell possesses twice the number of ribosomes of a G_1-phase cell and thus a doubled protein production capacity. The effect of the cell cycle or cell volume on the baculovirus infection was determined by splitting an exponentially growing Sf9 cell culture into 5 cell size dependent fractions by centrifugal elutriation. The subsequent infection of these fractions showed (1) no dependency of the timing of cell lysis and cell volume increase and (2) approximately twofold increase of a) recombinant protein yield, b) viral DNA concentration, and c) budded virus yield. The recombinant protein yield showed a strong proportionality to the initial cell volume and the total RNA concentration during the late phase of the infection. As argued in chapter 6, these proportionalities suggest that the observed differences in the responses of the cell fractions to the baculovirus infection are more likely caused by the difference in the protein production capacity than by cell cycle specific molecules. This investigation gave also reason to speculate that infected cells cannot progress beyond the G_2/M phase, and cell cycle progression continues undisturbed until ~8hpi when all cellular DNA replication appears to cease. Resuspended, infected Sf9 cells synchronised by centrifugal elutriation showed an identical cell cycle distribution as the non-infected control cultures for the first ~8hpi; G_1 and G_2/M phase cell proportions remained unchanged, whereas S phase cells progress to G_2/M phase. Subsequently, the non-infected control cells resumed normal cycling whereas all infected cells remained at the same cell cycle phase from 8 to 11hpi. The initial cell cycle arrests in G_2/M phase in both infected and non-infected cultures were caused through medium exchange.