Thesis (Ph. D.)--University of North Texas, Aug., 2009. / Title from title page display. Includes bibliographical references.

As selection pressures exist for not only adults, but for every life history stage, it is important to understand how environmental factors shape developing animals. Despite the significance placed on aquatic hypoxia as a driving force in the evolution of air breathing, this is the first known study to examine the effects of hypoxia on cardio-respiratory ontogeny of an air breathing fish. Blue gouramis are obligatory air breathing fish that possess a labyrinth-like structure that serves as the air breathing organ. Gouramis were reared for up to 90 d in normoxia or hypoxia, and morphological and physiological development was observed. Hypoxic larvae had increased lamellar and labyrinth organ surface areas. Bradycardia and increased gill ventilation rates were observed when larvae from either rearing group were briefly exposed to hypoxia. Hypoxic larvae also showed a reduced heart rate and gill ventilation rate in the absence of a hypoxic stimulus, possibly indicative of a more comprehensive, long-term respiratory plasticity. The similarity of routine oxygen consumption between rearing groups suggests that metabolic demand did not change for hypoxic larvae, but that they were more efficient at oxygen acquisition. This is further supported by increased resistance time of hypoxic gouramis to extreme hypoxia. The onset of air breathing was between 20 and 25 d post-fertilization, and was not affected by either rearing or exposure environment. It may be that this behavior is associated with the inability of smaller larvae to successfully overcome water surface tension, rather than with the necessity of aerial respiration at this stage. Hypoxia is commonly experienced by most air breathing fishes, and studies of hypoxia-induced developmental effects may provide critical insights into the evolution of air breathing. The studies presented here provide novel data on the plasticity of cardio-respiratory development of an air breathing fish reared in hypoxia, and can serve as a solid foundation for future studies.

The blue gourami, Trichogaster trichopterus, is a small, freshwater tropical fish, native to Southeast Asia. Parental behavior is normally shown only by males after spawning. Females and non-parental males eat small numbers of test eggs which they are given, while parental males retrieve them to their nests. The goal of this thesis was to investigate the factors responsible for the sudden change in behavior from egg-eating to parental care which takes place at the time of spawning. In the initial section, the patterns of parental behavior are described, and quantitative data on the development and maintenance of parental behavior in male fish spawning for the first time are presented. The experimental studies reveal that stimuli from the eggs are an important factor in the induction of parental behavior. Many naive males developed parental behavior within 0.5-2.5 min when given eggs spawned by other fish. This response was dependent upon the number of eggs presented: a large proportion of fish became parental when given 2000-3000 eggs, a smaller proportion did so when given 500-1000 eggs, and none did so when given 100 eggs. However, even 100 eggs evidently affected parental responsiveness because some fish eventually developed complete parental behavior when repeatedly given 100 eggs. These results indicate that eggs can induce parental behavior, not that they do so in the natural spawning situation. However, tests of males spawning with females whose oviducts were plugged indicated that exposure to eggs was an important component in the natural development of parental behavior. Spawning by itself did not induce fish to become parental, but it did reduce the number of eggs necessary for parental behavior to develop. Besides spawning and stimuli from the eggs, androgens also play a role in the development of parental behavior. It was shown that, after spawning, males performed more parental behavior than females, although detailed observations of females revealed that they had some capacity to perform parental behavior. The masculinization of females by means of methyl testosterone implants allowed them to develop parental behavior in response to large numbers of eggs. Whether castration of males eliminated their capacity to perform parental behavior could not be determined with certainty. The presence of young facilitates the maintenance of parental responsiveness to eggs when males with developing broods are compared with males whose broods have been removed. Apparently, physical contact with the young is necessary because males lose their parental responsiveness when their broods are placed in baskets which allow only visual and chemical contact. An important result of this study is the demonstration of the significance of stimulus strength in the induction of parental responsiveness. This factor has not often been taken into consideration in other studies of parental behavior. By varying stimulus strength, repetition of stimuli, and sexual experience, patterns of the induction of parental behavior were found in gouramis which were similar to those observed in other studies on a variety of mammals, birds, and fish. That is, parental behavior developed either almost immediately or gradually with the presentation of young to non-parental fish, or it was shown only if the young were presented during the "sensitive period" after spawning. The finding of such different patterns in a single species as a result of varying the strength of stimuli from the eggs suggested that the motivating effects of eggs and young on parental responsiveness may represent a common pattern underlying the control of parental behavior in a variety of vertebrate species. This possibility and the relationship between hormones and stimuli from the young in the control of parental behavior are discussed in the final chapter. / Science, Faculty of / Zoology, Department of / Graduate

M.Sc. (Zoology) / Aphanomyces invadans is an oomycete associated with epizootic ulcerative syndrome (EUS). It affects more than a 100 freshwater and estuarine species of fish and is a serious threat to aquaculture and natural aquatic ecosystems. Currently, cases of EUS have been reported across Asia, Australia, North America and more recently Southern Africa. Outbreaks occur mostly during periods of sudden temperature change, such as heavy rainfall or change of seasons. These conditions favour sporulation of A. invadans, and low temperatures have been shown to delay the inflammatory response of fish to oomycete infection. Diagnosis of A. invadans is usually based on clinical signs and confirmed by demonstrating the presence of mycotic granulomas in histological section. Further diagnosis of EUS is made by isolation of A. invadans from internal tissues. Demonstrating typical asexual characteristics by inducing sporulation allows identification of the oomycete to the genus level. After inducing sporulation, the zoospores can be isolated for use in clinical infection of fish through subcutaneous injection or bath challenge systems. Standard molecular techniques such as polymerase chain reaction (PCR) have also been development for the fast and reliable diagnosis of the disease. The aim of this study was to perform different infection trials on EUS in two susceptible fish species, three-spot gourami, Trichogaster trichopterus, and rainbow trout, Oncorhynchus mykiss. Through initial trials, three-spot gourami was established as a suitable positive control species. Subsequently, hyphae were successfully re-isolated from infected fish and demonstrated as A. invadans, which was used in a further inoculation trial. Rainbow trout were challenged with A. invadans through intramuscular inoculation revealing varying degrees of susceptibility at different water temperatures. Detection of fungal hyphae and mycotic granulomas in tissue sections was achieved through histopathological examination, including the use of birefringence and fluorescents. Confirmation of A. invadans DNA in the various infection trials was done through PCR analyses. A histological grading system is proposed which will allow simplification of large scale qualitative microscopic analyses and identification of histological trends within a data set when analysing suspected cases of EUS. It is recommended that similar infection trials be applied to endemic species in Southern Africa to investigate their susceptibility to EUS.

This study determined the effect of hypoxia on air-breathing onset and physiological and morphological characters in larvae of the air breathing fishes Trichopodus trichopterus and Betta splendens. Larvae were exposed intermittently (12/12 h daily) to 20, 17, and 14 kPa of PO2 from 1 to 40 days post-fertilization. Survival, onset of air breathing, wet body mass, O2, Pcrit were measured every 5 dpf. Hypoxia advanced by 4 days, and delayed by 9 days, the onset of air breathing in Betta and Trichopodus, respectively. Hypoxia increased larval body length, wet mass, and labyrinth organ respiratory surface of Betta, but did not affect these factors in Trichopodus. Hypoxic exposure increased O2 by 50-100% at each day throughout larval development in Betta, but had no effect on larval Trichopodus. Hypoxia decreased Pcrit in Betta by 37%, but increased Pcrit in Trichopodus by 70%. Larval Betta reared in hypoxia showed a modified heart rate:opercular rate ratio (3:1 to 2:1), but these changes did not occur in Trichopodus. Compared to Betta, the blood of Trichopodus had a higher P50 and much smaller Bohr and Root effects. These interspecific differences are likely due to ecophysiological differences: Betta is a non- obligatory air-breather after 36 dpf with a slow lifestyle reflected in its low metabolism, while Trichopodus is an obligatory air-breather past 32 dpf with an athletic fast lifestyle and accompanying high metabolism.