The effects of noise exposure and genetic background on auditory-evoked behaviors in larval zebrafish

Thesis (Ph.D.)--University of Washington, 2016-06

Entomological investigations in a focus of dengue transmission in Cairns, Queensland, Australia, by using the sticky ovitraps

Sticky ovitraps (patent pending) were used to sample female Aedes aegypti (L.) weekly in a focus of dengue activity in Cairns, Queensland, Australia. In February 2003, transmission of dengue virus serotype 2 began in the suburb of Parramatta Park, peaking in mid-March 2003. This suburb features many older, unscreened houses with high populations of Ae. aegypti. Highest densities (2-3.5 females per trap per week) were obtained during peak dengue transmission (January and February) before mosquito control was initiated. Beginning in late March, female Ae. aegypti collected in sticky ovitraps were tested for dengue viral RNA by using a TaqMan reverse transcription-polymerase chain reaction assay. Dengue viral RNA was detected in six pools of Ae. aegypti collected in late March. The highest minimum infection rate was 116/1000 mosquitoes. After the initiation of larval control (containers treated with S-methoprene or lambda-cyhalothrin) and adult control (interior harborage sites sprayed with lambda-cyhalothrin) in early March, trap collections dropped to

An in vitro larval motility assay to determine anthelmintic sensitivity for human hookworm and Strongyloides species

With the implementation of programs to control lymphatic filariasis and soil-transmitted helminths using broad spectrum anthelmintics, including albendazole and ivermectin, there is a need to develop an in vitro assay for detection of drug resistance. This report describes an in vitro assay for measuring the effects of ivermectin and benzimidazoles on the motility of larvae of the hookworm species Ancylostoma ceylanicum, A. caninum, and Necator americanus, and Strongyloides species including Strongyloides stercoralis, and S. ratti. A dose-response relationship was demonstrated with each of the parasite species, with distinct differences observed between the various species. In pilot field testing of the assay with N. americanus larvae recovered from human fecal samples, a dose-response relationship was observed with ivermectin. While the assay has demonstrated the ability to determine drug responsiveness, its usefulness in resistance detection will require correlation with the clinical outcome among individuals infected with parasite strains showing different drug sensitivities.

Distribution of calcium phosphate in the exoskeleton of larval Exeretonevra angustifrons Hardy (Diptera : Xylophagidae)

Distribution and organisation of the mineral, amorphous calcium phosphate (ACP), has been investigated in the exoskeleton of the xylophagid fly larva Exeretonevra angustifrons Hardy. While head capsule and anal plate are smooth with a thin epicuticle, the epicuticle of the body is thicker and shows unusual micro-architecture comprised of minute hemispherical (dome-shaped) protrusions. Electron microprobe analysis and energy dispersive spectroscopy revealed heterogeneity of mineral elements across body cuticle and a concentration of ACP in the epicuticle, especially associated with the hemispherical structures. Further imaging and analysis showed the bulk of the ACP to be present in nano-sized granules. It is hypothesised that the specific distribution of ACP may enhance cuticular hardness or durability without reducing flexibility. (c) 2004 Elsevier Ltd. All rights reserved.

Correlating gene expression with larval competence and the effect of age and parentage on metamorphosis in the tropical abalone Haliotis asinina

The non-geniculate crustose coralline alga (CCA) Mastophora pacifica can induce the metamorphosis of competent Haliotis asinina (Vetigastropoda) larvae. The ability to respond to this natural cue varies considerably with larval age, with a higher proportion of older larvae (e.g. 90 h) able to metamorphose in response to M. pacifica than younger larvae (e.g. 66 h). Here we document the variation in time to acquisition of competence within a larval age class. For example, after 18 h of exposure to M. pacifica, approximately 15 and 36% of 84 and 90-h-old H. asinina larvae had initiated metamorphosis, respectively. This age-dependent response to M. pacifica is also observed when different aged larvae are exposed to CCA for varying periods. A higher proportion of older larvae require shorter periods of exposure to CCA than younger larvae in order to initiate metamorphosis. In this experiment, as in the previous, a small proportion of young larvae were able to respond to brief periods of CCA exposure, suggesting that they had developed the same state of competency as the majority of their older counterparts. Comparisons of the proportions of larvae undergoing metamorphosis between families reveals that parentage also has a significant (P < 0.05) affect on whether an individual will initiate metamorphosis at a given age. These familial differences are more pronounced when younger, largely pre-competent larvae (i.e. 66 h old) are exposed to M. pacifica, with proportions of larvae undergoing metamorphosis differing by as much as 10 fold between families. As these data suggest that variation in the rate of development of the competent state has a genetic basis, and as a first step towards identifying the molecular basis to this variation, we have identified numerous genes that are differentially expressed later in larval development using a differential display approach. Spatial expression analysis of these genes suggests that they may be directly involved in the acquisition of competence, or may play a functional role in the postlarva following metamorphosis.

Cannibalistic feeding of larval Trichogramma carverae parasitoids in moth eggs

Wasps of the genus Trichogramma parasitise the eggs of Lepidoptera. They may deposit one or many eggs in each host. Survival is high at low density but reaches a plateau as density increases. To reveal the mechanism by which excess larvae die we chose a lepidopteran host that has flattened, transparent eggs and used video microscopy to record novel feeding behaviours and interactions of larval Trichogramma carverae (Oatman and Pinto) at different densities. Single larvae show a rapid food ingestion phase, followed by a period of extensive saliva release. Ultimately the host egg is completely consumed. The larva then extracts excess moisture from the egg, providing a dry environment for pupation. When multiple larvae are present, the initial scramble for food results in the larvae consuming all of the egg contents early in development. All larvae survive if there is sufficient food for all to reach a threshold developmental stage. If not, physical proximity results in attack and consumption of others, continuing until the surviving larvae reach the threshold stage beyond which attacks seem to be no longer effective. The number of larvae remaining at the end of rapid ingestion dictates how many will survive to emerge as adults.

Predator-specific changes in the morphology and swimming performance of larval Rana lessonae

1. We investigated the morphological responses of larval Rana lessonae to the presence of two predators with substantially different prey-detection and capture techniques; larval dragonflies (Aeshna cyanea) and the Pumpkinseed Sunfish (Lepomis gibossus). 2. We also examined the functional implications of any predator-induced morphological variation on their swimming ability by assessing performance during the initial stages of a startle response. 3. We found the morphological responses of larval R. lessonae were dependent on the specific predator present. Tadpoles raised in the presence of dragonfly larvae preying upon conspecific tadpoles developed total tail heights 5.4% deeper and tail muscles 4.7% shallower than tadpoles raised in a non-predator environment, while tadpoles raised with sunfish possessed tails 2% shallower and tail muscles 2.5% higher than non-predator-exposed tadpoles. 4. Predator-induced morphological variation also significantly influenced swimming performance. Tadpoles raised with sunfish possessed swimming speeds 9.5 and 14.6% higher than non- and dragonfly predator groups, respectively. 5. Thus, the expression of these alternative predator-morphs leads to a functional trade-off in performance between the different environments.

Changes in morphology and physiology of an East Mediterranean sponge in different habitats

The sponge Tetilla sp. (Tetractinomorpha: Tetillidae) is a common species in the eastern Mediterranean. This sponge inhabits four different habitat types differing in wave impact and irradiance levels. Two of these habitats (a shallow cave and deep water) are characterized by relatively calm water, whereas the other two (shallow exposed site and tide pools) are in turbulent water with high energy flow. The present study examined the influence of physical (depth, illumination and water motion) and biotic factors on morphology, skeletal plasticity and reproductive traits among the four spatially separated populations. Sponges from tidal pools had significantly larger body volume than sponges from deep water and from shallow caves (ANOVA: tidal-deep P< 0.0001; tidal-shallow caves P< 0.05). Sponges from exposed habitats were significantly larger than deep-water sponges (ANOVA: P=0.01). In addition, individuals from tide pools and from the exposed habitat had a significantly higher proportion of structural silica than sponges from the calmer deep water and from the cave sites. Oxea spicules in sponges from the calm habitats were significantly shorter than in those from the tidal pools and the exposed habitats. The percentage of spicules out of a sponge's dry weight in individuals transplanted from deep (calm) to shallow (turbulent) water significantly increased by 21.9&PLUSMN; 12.9%. The new spicule percentage did not differ significantly from that of sponges originally from shallow water. Oocyte diameter differed significantly between habitats. The maximal size of mature eggs was found in deep-water sponges in June (97&PLUSMN; 5 μ m). In the shallow habitats, a smaller maximal oocyte diameter was found in the cave, in May (56.5&PLUSMN; 3 μ m). Furthermore, oocyte density in shallow-water sponges was highest in May and decreased in June (with 88.2&PLUSMN; 9 and 19.3&PLUSMN; 9 oocytes mm(-2), respectively). At the same time (June), oocyte density of deep-water sponges had just reached its maximum (155&PLUSMN; 33.7 oocytes mm(-2)). The difference in oocyte size and density between deep- and shallow-water individuals indicates an earlier gamete release in the shallow sponge population. The results suggest that plasticity in skeletal design of this sponge indicates a trade off between spicule production and investment in reproduction.

Herbicides implicated as the cause of severe mangrove dieback in the Mackay region, NE Australia: consequences for marine plant habitats of the GBR World Heritage Area

Herbicides, particularly diuron, were correlated with severe and widespread dieback of the dominant mangrove, Avicennia marina (Forsk.) Vieth. var. eucalyptifolia (Val.) N.C. Duke (Avicenniaceae), its reduced canopy condition, and declines in seedling health within three neighbouring estuaries in the Mackay region of NE Australia. This unusual species-specific dieback, first observed in the early 1990s, had gotten notably worse by 2002 to affect > 30km(2) of mangroves in at least five adjacent estuaries in the region. Over the past century, agricultural production has responded well to the demands of increasing population with improvements in farm efficiency assisted by significant increases in the use of agricultural chemicals. However, with regular and episodic river flow events, these chemicals have sometimes found their way into estuarine and nearshore water and sediments where their effects on marine habitats have been largely unquantified. Investigations over the last three years in the Mackay region provide compelling evidence of diuron, and possibly other agricultural herbicides, as the most likely cause of the severe and widespread mangrove dieback. The likely consequences of such dieback included declines in coastal water quality with increased turbidity, nutrients and sediment deposition, as well as further dispersal of the toxic chemicals. The implications of such findings are immense since they describe not only the serious deterioration of protected and beneficial mangrove habitat but also the potential for significant direct and indirect effects on other highly-valued estuarine and marine habitats in the region, including seagrass beds and coral reefs of the Great Barrier Reef lagoon. This article reviews all key findings and observations to date and describes the essential correlative and causative evidence. (c) 2004 Elsevier Ltd. All rights reserved.

Field evaluation of anthelmintic drug sensitivity using in vitro egg hatch and larval motility assays with Necator americanus recovered from human clinical isolates

A field-applicable assay for testing anthelmintic sensitivity is required to monitor for anthelmintic resistance. We undertook a study to evaluate the ability of three in vitro assay systems to define drug sensitivity of clinical isolates of the human hookworm parasite Necator americanus recovered from children resident in a village in Madang Province, Papua New Guinea. The assays entailed observation of drug effects on egg hatch (EHA), larval development (LDA), and motility of infective stage larvae (LMA). The egg hatch assay proved the best method for assessing the response to benzimidazole anthelmintics, while the larval motility assay was suitable for assessing the response to ivermectin. The performance of the larval development assay was unsatisfactory on account of interference caused by contaminating bacteria. A simple protocol was developed whereby stool samples were subdivided and used for immediate egg recovery, as well as for faecal culture, in order to provide eggs and infective larvae, respectively, for use in the egg hatch assay and larval motility assay systems. While the assays proved effective in quantifying drug sensitivity in larvae of the drug-susceptible hookworms examined in this study, their ability to indicate drug resistance in larval or adult hookworms remains to be determined. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Immunocytochemistry and metamorphic fate of the larval nervous system of Triphyllozoon mucronatum (Ectoprocta : Gymnolaemata : Cheilostomata)

The development of gymnolaemate Ectoprocta includes a larval stage of either the coronate or the cyphonautes type. Herein, we provide the first description of the larval neural anatomy of a coronate larva using immunocytochemical methods. We used antibodies against the neurotransmitters serotonin and FMRFamide and followed the fate of immunoreactive cells through metamorphosis. The larval serotonergic nervous system of Triphyllozoon mucronatum consists of an apical commissure, one pair of lateral axons, a coronate nerve net, an internal nerve mesh, and one pair of axons innervating the frontal organ. FMRFamide is only found in the larval commissure and in the lateral axons. The entire serotonergic and FMRFamidergic nervous system is lost during metamorphosis and the adult neural structures form independent of the larval ones. In the postlarval zooid, both neurotransmitters are detected in the cerebral commissure, in cell bodies located at the base of the lophophore, and in neurites connecting these somata to the cerebral commissure. These findings differ significantly from that observed in other lophotrochozoans, where certain larval neural features are either incorporated in the adult nervous system and/or have inductive functions during its ontogeny. The occurrence of a larval commissure and the lack of a serotonergic or FMRFamidergic apical organ in T. mucronatum are unique among lophotrochozoan larvae, which usually have a distinct apical organ containing serotonergic cells. Our data show that the larval neuroanatomy and the processes that underlie the reorganization of larval organ systems during metamorphosis may vary much more among lophotrochozoan taxa than previously thought.

Evaluation of Melanotaenia duboulayi (Atheriniformes : Melanotaeniidae), Hypseleotris galii (Perciformes : Eleotridae), and larvicide Vectolex (R) WG (Bacillus sphaericus) for integrated control of Culex annulirostris

Australian freshwater fish species Melanotaenia duboulayi and Hypseleotris galii were selected for a small plot field evaluation of an integrated pest management strategy using native fish and VectoLex® WG (Bacillus sphaericus) for the control of Culex annulirostris Skuse, the principal freshwater vector of arbovirus Ross River virus in Australia. When tested alone, the level of control afforded by M. duboulayi and H. galii was highly dependent on the prerelease density of mosquito larvae; and even when stocking rates as high as 10 g per pond (>30 kg/ha) were used, larval abundance was too high to attain adequate control from fish alone. In contrast, treatment with VectoLex WG at 500 g/ha resulted in 100% mortality of Cx. annulirostris immatures, but no residual activity was evident. The delayed reduction of Cx. annulirostris immatures in ponds stocked with fish alone, and the recolonization by Cx. annulirostris in ponds after treatment with B. sphaericus, did not occur when both treatments were combined.

Larval dispersal reveals regional sources and sinks in the Great Barrier Reef

We analysed simulated connectivity patterns for reef fish larvae in the Cairns section of the Great Barrier Reef, and identified 2 key subregions that exhibit regional scale source–sink dynamics. The source and sink were separated latitudinally by a boundary at 16.1°S, with the source subregion lying to the north. Larval transport between the 2 subregions was predominantly unidirectional, from north to south. Only a few local populations, described here as ‘gateway reefs’, were able to transport larvae from the sink subregion to the source subregion and thus maintain the connectedness of the metapopulation. The northern subregion was able to persist without external larval supply, but when conditions were recruitment limited, the southern subregion depended on larval supply from the north to persist. The relative autonomy of the northern subregion, and its importance in sustaining the southern subregion, will influence the effectiveness of conservation efforts.

Microbial diversity within the water column of a larval rearing system for the ornate rock lobster (Panulirus ornatus)

The ornate tropical rock lobster, Panulirus ornatus has substantial potential as an aquaculture species though disease outbreaks during the animal's extended larval lifecycle are major constraints for success. In order to effectively address such disease-related issues, an improved understanding of the composition and dynamics of the microbial communities in the larval rearing tanks is required. This study used flow cytometry and molecular microbial techniques (clone libraries and denaturing gradient gel electrophoresis (DGGE)) to quantify and characterise the microbial community of the water column in the early stages (developmental stage I-II) of a P. ornatus larval rearing system. DGGE analysis of a 5000 L larval rearing trial demonstrated a dynamic microbial community with distinct changes in the community structure after initial stocking (day I to day 2) and from day 4 to day 5, after which the structure was relatively stable. Flow cytometry analysis of water samples taken over the duration of the trial demonstrated a major increase in bacterial load leading up to and peaking on the first day of the initial larval moult (day 7), before markedly decreasing prior to when > 50% of larvae moulted (day 9). A clone library of a day 10 water sample taken following a mass larval mortality event reflected high microbial diversity confirmed by statistical analysis indices. Sequences retrieved from both clone library and DGGE analyses were dominated by gamma- and alpha-Proteobacteria affiliated organisms with additional sequences affiliated with beta- and epsilon-Proteobacteria, Bacteroidetes, Cytophagales and Chlamydiales groups. Vibrio affiliated species were commonly retrieved in the clone library, though absent from DGGE analysis.