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Orientador: Maria Célia Portella / Banca: João Martins Pizauro Junior / Banca: Rodrigo Takata / Banca: Rosangela Kyoko Jomori Bonichelli / Banca: Fernando José Zara / Resumo: O conhecimento sobre a organogênese das larvas de peixes é uma ferramenta útil para estabelecer suas capacidades funcionais sistêmicas e suas necessidades fisiológicas a fim de garantir o bem-estar ideal e o crescimento sob condições de aquicultura A manipulação de protocolos de alimentação na larvicultura pode influenciar a capacidade das funções digestivas, levando os animais a um maior ou menor desenvolvimento. Desta forma, o objetivo deste estudo foi verificar o crescimento e o desenvolvimento morfofisiológico do trato digestório de larvas de pacu, Piaractus mesopotamicus, cultivadas com diferentes dietas alimentares e protocolos de transição do alimento vivo para o formulado, por meio de análises de desempenho zootécnico, histológicas, histoquímicas e enzimológicas (tripsina, amilase, lipase, leucina-aminopeptidase, maltase e proteases ácidas), contribuindo assim, para a compreensão do desenvolvimento do trato digestório das larvas de pacu e da sua capacidade digestiva / Abstract: The knowledge about organogenesis of fish larvae is a useful tool to establish their functional capabilities and systemic physiological needs in order to ensure welfare and optimal growth under aquaculture conditions. Feed in hatchery affects digestive functions inducing faster or slower development. Thus, this study aimed to evaluate growth and morphophysiological development of the gastrointestinal tract in pacu Piaractus mesopotamicus larvae, rearing under live food and formulated diet with different weaning protocols, through the analysis of production performance, histology, histochemistry and enzymology (trypsin, amylase, lipase, leucine-aminopeptidase, maltase and acid proteases). Therefore, this research contributes to the better understanding of the development of the gastrointestinal tract in pacu larvae and its digestive capacity / Doutor

Resumo: A reprodução de peixes é um dos processos mais importantes para a piscicultura, sendo a reprodução artificial um dos manejos de criação mais estressantes para os animais, devido a todo o manejo envolvido. Esse estresse desencadeia a produção de cortisol, que pode provocar problemas na reprodução e nas larvas. Dessa forma, um maior conhecimento do estresse na reprodução é necessário, para se estabelecer rotinas e modelos de manejo mais adequados. Assim, o presente estudo teve como objetivo, verificar o efeito do cortisol administrado por meio de injeção intraperitoneal na reprodução de fêmeas de matrinxã (Brycon amazonicus), no desenvolvimento embrionário e no desempenho inicial das larvas. Para isso, foram realizados 2 experimentos, nos meses de novembro e dezembro de 2007 e 2008. Utilizou-se no total, 24 fêmeas e 18 machos de matrinxãs, onde as fêmeas, no momento da indução hormonal com EPC para a reprodução, recebiam uma injeção intraperitoneal de solução de cortisol, de acordo com os tratamentos (4 fêmeas por tratamento), 2007: E1 (Controle) e E2 (10 mg/kg cortisol) e em 2008: E3 (Controle), E4 (0,1 mg/kg cortisol), E5 (1 mg/kg cortisol) e E6 (5 mg/kg cortisol). Após a reprodução, foram verificadas as taxas de fertilização e sobrevivência pré-eclosão e coletados sangue, ovos e larvas dessas fêmeas para as analises de microscopia e biométricas. Os tratamentos com cortisol apresentaram uma menor taxa de fertilização e menor sobrevivência pré-eclosão, além de alguns tratamentos apresentarem tamanho reduzido das larvas na eclosão e ao longo das coletas. Na microscopia, podemos observar que ovos de tratamentos com cortisol tiveram uma aceleração nas divisões celulares, no desenvolvimento embrionário das dosagens mais baixas e maior desenvolvimento do sistema digestório nas larvas com até 24h de vida, porém com muitas deformidades... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The reproduction of fish is one of the most important for fish farming and the artificial breeding is one of the most handling stressful, because of all the management involved. This stress triggers the production of cortisol, which can cause problems in reproduction and larvae. Thus, a greater understanding of stress on reproduction is necessary to establish routines and management models more suitable. The present study aimed to evaluate the effect of cortisol administered via intraperitoneal injection on reproduction in female fish (Brycon amazonicus), in embryonic development and initial performance of the larvae. For this, 2 experiments were conducted in the months of November and December of 2007 and 2008. We used a total of 24 females and 18 males matrinxãs. Females in moment of hormonal induction EPC for reproduction received an intraperitoneal injection of cortisol solution, according to the treatments (4 females per treatment), 2007: E1 (Control) and E2 (10 mg / kg cortisol) and in 2008: E3 (Control), E4 (0.1 mg / kg cortisol), E5 (1 mg / kg cortisol) and E6 (5 mg / kg cortisol). After reproduction, were observed fertilization rates and survival pre-hatching and collected blood, eggs and larvae of these females for microscopic analysis and biometrics. Treatments with cortisol had a lower fertilization rate and lower survival pre-hatching, and some treatments make small size of larvae at hatching and during the experimental period. In microscopy, we observed that treatment of eggs with cortisol had an acceleration of cell division in embryonic development of lower dosages and further development of the digestive system in larvae up to 24 hours of life, but with many deformities in body structures and head, reducing the lifetime of these larvae. It was concluded that cortisol causes deleterious effects on reproduction of female matrinxãs and development of their offspring... (Complete abstract click electronic access below) / Orientadora: Elisabeth Criscuolo Urbinati / Coorientador: Sérgio Fonseca Zaiden / Banca: Antonio Fernando Gervásio Leonardo / Banca: Sérgio Ricardo Batlouni / Mestre

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The citrus industry is subject to a number of serious insect pests. Of these, the most important is a sedentary group known as the Armoured Scale Insects (Diaspididae). Ebeling (1950) states that they are of greater economic importance to the industry than all the other pests put together. A great deal has been published on various aspects of the biology, ecology and control of the Diaspididae, much of the work appearing in the books of Quayle (1938), Ebeling (1950) and Bodenheimer (1951). It is very noticeable, however, that the study of the active larvae, or "crawlers", of these insects has been relatively neglected. This lack of work on crawlers is surprising, since they are the only distributive stage in the life cycle of the species. The adult male is also free-living during its brief life span, but takes no part in the actual distribution of the species. It is the position of the female Scale Insect which is important in determining the further spread of the species, since this is the point from which subsequent crawlers will start out on their wanderings. The stage in the life cycle responsible for the position of the female is, of course, the crawler. The crawler is also responsible for the parallel distribution of the male Scales, so that the female can be fertilised. The role of the adult male is confined to the maintenance of the gene flow. From an economic point of view it seems desirable that a complete study be made of the biology of this distributive stage of these important pests.

Phthorimaea operculella, commonly known as the Potato Tuber Moth, is an economically important agricultural pest worldwide. The baculovirus, Phthorimaea operculella granulovirus (PhoGV) has been considered as a means of control alternative to chemical control because of its host specificity and harmless impact on other organisms and ecosystems. An isolate of PhoGV obtained from a South African PTM population would be beneficial in the production of a biopesticide, which is not yet available. An efficient and cost-effective rearing method would be advantageous for potential commercial production. Commercial table and seed potato plantations and storage facilities located in Patensie, Bathurst, Howick and Ivanhoe were surveyed for PTM infestations. Patensie was the only site where milky discoloured larvae were found, a potential symptom of PhoGV infection. TEM analysis revealed no virus in these samples. Since no virus was found in the field-collected samples, PTM insects were collected to initiate rearing in the laboratory. PTM was raised by three different methods in the laboratory. A cost/benefit analysis, survival rate, fertility and sex ratio were recorded for each rearing method. Rearing method one was deemed unsuccessful for efficient commercial rearing, as survival percentage and fertility were low. Rearing methods two and three had high survival rates and high fertility, and were efficient and less labour intensive than rearing method one. Rearing method three was the most productive technique, but for commercial production rearing method two was considered the most manageable and efficient. The sex ratio was 1:1 for all three cultures. The cost analysis revealed that rearing methods two and three were less expensive than rearing method one because less labour was required to monitor insects. The success of rearing PTM for 19 months will enable these cultures to be up-scaled to a large production facility for mass rearing. Virus was not found in the field surveys or in laboratory cultures, therefore chemical, temperature, humidity and carbon dioxide stressors were used in an attempt to initiate a baculoviral infection. Symptoms were exhibited in larvae subjected to chemical, temperature and humidity treatments, but these were confirmed by TEM analysis not to be a result of PhoGV infection. The success of rearing PTM in the laboratory suggests that the method could be used in the commercial rearing of the insects in a large mass-rearing facility. The data obtained from induction protocols have allowed for better understanding for future induction for PhoGV and other baculoviruses in other insect species. The failure to isolate a South African PhoGV strain for developing a biopesticide against PTM has motivated further studies in obtaining a baculovirus in order for South Africa to develop a commercial product against this pest.

Population dynamics of many intertidal organisms are highly influenced by the abundance and distribution of planktonic larvae in the water column and their arrival on the shore. The brown mussel, Perna perna was used to investigate two of the primary processes that affect population size and dynamics, larval availability and settlement, on the south coast of South Africa. Perna perna is a dominant species on rocky shores of the southern and eastern coasts of South Africa. It creates three-dimensional beds that provide habitats for many other species and hence promotes biodiversity. Larval availability and settlement were examined at different spatial and temporal scales using a nested experimental design. To detect possible relationships between larval availability and settlement, the studies were simultaneous. Two sites, 4km apart, were chosen to investigate mussel settlement patterns. Within each site, three locations (300m from each other) were selected. At each location, five artificial settler collectors were placed at approximately 20cm intervals. Collectors were replaced at a range of time intervals, from daily to seasonal, for 16 months. Each intertidal location was paired with an offshore station, 500m from the shore, where larval availability was measured. At each offshore station, three vertical hauls were collected twice a month using a plankton net. Plankton sampling lasted for 14 months and was designed to examine variability on three temporal scales: seasonal, lunar and daily. The results showed no correlation between the distribution of larvae in the water and settlers on the shore. While larvae were abundant in the water at the start of sampling, they became very rare throughout the rest of the study at both sites and all locations. In contrast, distinct peaks of settler abundance were observed during the seasonal settlement study. In addition to the expected, strong temporal variation that emerged from both studies at all time scales, spatial patterns of variability were also observed. While no spatial effect was detected for the larvae in the water column, there was distinct spatial variation in settlement at the location level: some locations always showed higher settlement than others. These results suggest that, on scales of hundreds of meters to kilometers, larval availability and settlement are very unpredictable in time and that differential delivery of larvae occurs from nearshore waters to the shore. Although the effect of the state of the moon (new or full) was not significant in either study, more settlers seemed to arrive on the shore during new moon. Wind direction did not correlate significantly with settlement. However, the dropping of offshore winds and the prevalence of onshore winds, which are characteristic of summer, may be linked to the start of settlement. Nevertheless, further investigations on tidal or lunar cycles and on the influence of wind on surface currents are required to clarify the effects of moon and wind on settlement.

Intensive grow-out of goldfish, Carassius auratus (L.), larvae and juveniles in closed recirculating systems requires the control of environmental conditions and feeding. This study investigated the effect of different diets, environmental condition and feeding level on growth, development and survival of goldfish larvae and juveniles. Prey preference for Artemia nauplii or decapsulated Artemia cysts as well as agonistic behaviour was recorded. The micrometer device used to measure mouth-gape was shown to produce accurate measurements which could be used to estimate the maximal particle size that can be ingested by goldfish larvae of a particular age. Goldfish completed metamorphosis earlier with an increased feeding level of Artemia cysts and by making the cysts more accessible to the fish using up-welling water movement. Morphometric plasticity in goldfish larvae was exhibited within two weeks of growth and it may be possible to induce changes in morphology by manipulating diet and rearing environment. Fish that fed on moving prey items had a significantly larger mouth-gape than those that browsed cysts from the tank bottom or dry food items. The development of mouth-gape was not affected by the feeding level of cysts. Condition factor increased with an increase in the number of cysts fed per fish per day. Goldfish larvae and juveniles grew faster and had a higher survival when fed on decapsulated Artemia cysts than on instar I Artemia nauplii or a mixed live/dry diet of Artemia nauplii and dry food. Feeding at least 155 cysts per fish per day, in tanks with upwelling water movement, gave the best growth and survival and the smallest size variation. In addition, cysts remained available to the fish for longer periods, and were easier to prepare and feed. Goldfish larvae preferred decapsulated Artemia cysts to nauplii and rejected fewer prey items as they grew older. The frequency of agonistic behaviour increased as fish grew but no cannibalism was recorded for cyst-fed fish. This study showed that decapsulated Artemia cysts are a good alternative to Artemia nauplii as a diet for larval goldfish. Good growth and high survival was achieved for cyst-fed goldfish larvae and juveniles at 23 ± 1.5°C and at an initial stocking density of 12 fish per litre. This research also contributes to an understanding of feeding behaviour and attempts to minimise under- or over-feeding of Artemia cysts in order to reduce grow-out costs due to the high value of the feed type.

Manipulation or control of the microbiological aquaculture environment has been identified as an important focus area for future hatchery development. Subsequently, alternatives to obtain control of the microbiological environment are being sought of which the field of probiotics appears highly promising. Probiotics are usually selected based on various in vitro characteristics, however, the methods used differ and are sometimes unsuccessful due to poor experimentation. The aim of this work is to contribute towards the development of a protocol for the in vitro screening of bacterial candidate probiotics for marine fish larviculture. To reduce the number of candidate probiotics to be tested in vivo, various in vitro experiments need to be conducted, each screening for a particular mode of action – antagonism towards pathogen through production of antimicrobial compounds, growth and attachment to fish intestinal mucus, and the production of other beneficial compounds such as vitamins, fatty-acids and digestive enzymes. A total of 108 bacteria species were isolated from the digestive tract of the adult common clownfish, Amphiprion percula to screen for potential probiotics to be used in clownfish larval rearing. The antagonistic compounds assay identified twelve isolates which showed antagonism towards two or more aquatic pathogens. This was followed by an in vitro test that involved growing the organisms in fish intestinal mucus and modeling their growth parameters. A ranking index (RI) was developed using the lag period (λ) and doubling time (td) of the organism, where (1over λ x td) x 100. Five candidate probionts (AP1-AP5) with varied growth parameters were used for further in vitro experiments. The attachment to mucus assay introduced a novel tool for quantifying competition for attachment sites between candidate probionts and pathogens on mucus. Candidate probiont Pseudoalteromonas AP5 reduced the attachment ability of Vibrio alginolyticus when added before the pathogen and partially out-competed the pathogen for attachment sites when added second. In vitro screening for the production of beneficial compounds tested the candidate probiotics’ ability to produce digestive enzymes - trypsin, lipase and alkaline phosphatase as well as carotenoids and vitamin C. Candidate probiont Pseudoalteromonas AP5 produced high levels of the enzymes (98.2, 34.1 and 91.3 mU product liberated.ml⁻¹, respectively) and contained carotenoids while Kocuria AP4 contained carotenoids but produced low quantities of enzymes (7.8, 0 and 59 mU product liberated.ml⁻¹, respectively). None of the candidate probiotics produced vitamin C. To eliminate potential pathogenic or toxic candidate probionts, Artemia nauplii were exposed to each candidate probiont and the percentage Artemia mortality after 24-hours was determined. Candidate probiont AP2 caused high mortality of Artemia nauplii (98.4%) and was excluded from further studies. Identification of candidate probionts AP3-AP5 was performed using 16S-rDNA molecular techniques and the bacteria were assigned the names Bacillus AP3, Kocuria AP4 and Pseudoalteromonas AP5, respectively. Two methods of larval probiont delivery were tested – attachment to Artemia, and in-water delivery. Attachment to Artemia was high for both Kocuria AP4 and Pseudoalteromonas AP5 (7.2 x10³ and 2.7x10⁴ bacteria.nauplius⁻¹, respectively) while the in-water viability experiment showed that Kocuria AP4 comprised 23.9% of the total culturable water microflora after 24 hours while Pseudoalteromonas AP5 contributed 100%. To validate the findings from the in vitro experiments, in vivo trials using clownfish larvae were performed. Of the four candidate probiotics tested, only Kocuria AP4 showed potential to increase larval survival. In vitro tests produced a better understanding of the possible mode of action and strategies of competition between bacteria, however, the number of criteria in which a candidate probiont is successful in vitro may not be the best predictor for its effectiveness in vivo. Commercial studies that reduce between-treatment variation are required to test predictions about the most suitable probiont or combinations thereof.

A study was conducted to determine the effects of incubation salinity and temperature . and post-hatching temperature on salinity tolerance of Pacific herring (Clupea pallasi Valenciennes) larvae. Calorimetry experiments were also conducted on herring larvae that were incubated and reared in various combinations of salinity and temperature. In the salinity tolerance test, herring eggs were artificially fertilized in three salinities (13, 21 and 29°/ooS) at 6°C. Newly fertilized eggs were incubated in the same salinities as those in which they were fertilized at two levels of incubation temperature (6 and 12°C), making six incubation groups. During the peak of the hatching period, groups of larvae from each incubation group were exposed to a series of test salinities, ranging from 20 to 55°/ooS at three levels of post-hatching temperatures (6, 9 and 12°C). Salinity tolerance was measured by determining the mortality of larvae occurring in each test salinity over a period of 72 hours. Two approaches were employed: 1) time-mortality, and 2) dosage-mortality. The former provided estimates of the effective time to 50% mortality (ET₅₀), the latter, the effective dosage (salinity) to 50% mortality (ED₅₀). The ET₅₀ or ED₅₀ values were used as bases for comparison of the responses of larvae to incubation temperature and salinity, and post-hatching temperature. The results indicate that both incubation salinity and temperature exert a significant influence on the salinity tolerance of larvae. Eggs incubated in the highest salinity (29°/ooS) and lower temperature (6°C) produced larvae most tolerant to higher post-hatching salinities. Post-hatching temperature also showed a possible influence, although the trends seen were not statistically significant. Salinity tolerance of larvae hatching from the 6°C incubation temperature appeared to be maximized at post-hatching temperatures between 6 and 9°C; whereas, those larvae from the 12°C incubation temperature showed maximum salinity tolerance at 9 to 12°C post-hatching temperatures. The response. of. larvae to salinity may be a result of an irreversible non-genetic adaptation of the embryo to salinity and temperature during incubation. In the calorimetry experiments, samples of herring larvae were combusted in a microbomb calorimeter to determine caloric values. These larvae were hatched and reared in the same six combinations of incubation salinities and temperatures as those used in the main experiment. Larvae were sampled at day 0 (peak of hatching), 3, 6 and 9. Standard lengths, dry weights, ash-free dry weights and ash content of the larval samples were also taken. There was a considerable variability in the data for calorimetry of the herring larva. In general, larvae from all treatments, at hatching, averaged 6,586 cal/g ash-free dry weight and had a mean ash value of 8.25%, obtained by direct ashing. Mean standard length of larvae at the peak of hatching (day 0) ranged from 7.67 mm (12°C, 29°/ooS) to 9.93 mm (6°C, 13°/ooS). There was a trend towards larger larvae (greater standard length) at lower temperatures in lower salinities in all ages tested. Data on dry weights show a linear decrease in larval (body and yolk) weight from day 0 to day 9. Mean dry weight of larva at the peak of hatching ranged from 0.142 mg (6°C, 13°/OOS) to 0.162 mg (12°C, 21°/ooS; 12°C, 29°/ooS). At day 0, the largest larvae (in terms of dry weight) were found at higher temperatures in higher salinities, while at day 9 (past yolk-sac stage), the largest larvae were found at lower temperatures in lower salinities. The same trend was observed in terms of ash-free dry weight. / Science, Faculty of / Zoology, Department of / Graduate