Dissertations / Theses on the topic 'Gambusia holbrooki'

Mosquitofish (Gambusia holbrooki) are native to the southeastern United States but invasive elsewhere, and are dominant predators in many ecosystems that they inhabit. Information on dispersal behavior will help better understand and predict mosquitofish metapopulation dynamics and invasions. I experimentally tested dispersal behavior of individual mosquitofish under a range of laboratory conditions relevant to field situations. Preliminary experiments showed that gender, lighting conditions, hunger and acclimation time did not significantly affect net dispersal rate. Power analysis based on this preliminary experiment determined that 6 replicate fish were sufficient for each subsequent experiment; I used 24 fish, and each fish was tested one time. Three factors that potentially could affect net swimming rate were tested: habitat of origin (permanent vs. temporary waters), water depth (3-24 mm), and the interaction between water depth and leaf litter type (upland and wetland). Fish from a temporary pond dispersed significantly faster than fish from a permanent pond, and fish dispersed significantly faster in deeper water than in shallower water. However, leaf litter significantly inhibited fish dispersal at all depths tested. Based on these experiments, G. holbrooki disperse more readily through relatively open and deeper (several centimeters) pathways between habitats such as roadside ditches, drainage canals and trails in flooded conditions. My results are useful for understanding mosquitofish dispersal behavior based on the abiotic and biotic factors examined in this experiment. I predict that mosquitofish can spread from a point of introduction at about 800 m per day, given and unobstructed path of only > 6 mm depth.
M.S.
Department of Biology
Arts and Sciences
Biology

This thesis aims to contribute to the understanding of the invasive success of two invasive fishes (Gambusia affinis and G. holbrooki). We reviewed four Internet databases and the literature to clarify their introduction history and geographical distribution, establish their introduction routes, and analyse predictors of their invasive success. A meta-analysis of the ecological impacts of mosquitofishes shows that the overall impact is: i) similar for the two species; ii) clear and strong for fish, macroinvertebrates, amphibians, and some zooplanktonic groups and more variable for taxa at lower trophic levels and for ecosystem features; iii) general for the number of aggressive acts received and decreases in density and biomass but more variable for other response variables; and iv) highly heterogeneous and context-dependent on a number of features. We estimated the mean critical swimming speed (Ucrit) of G. holbrooki as 14.11 cm s-1, which is lower than for many other fish of similar size and confirms that this species is limnophilic and its invasive success might be partially explained by hydrologic alteration. However, we demonstrate that Ucrit and maximal metabolic rate vary markedly with fish size and sex, with males having much higher values for the same weight, and thus probably being more resistant to strong water flows.
Aquesta tesi té com a objectiu contribuir a millorar la comprensió de l'èxit invasor d'aquests dos peixos (Gambusia affinis and G. holbrooki). Es van revisar quatre bases de dades d'Internet i la bibliografia per aclarir la seva història d’introducció i distribució geogràfica, establir les rutes d'introducció i analitzar els predictors del seu èxit invasor. Una meta-anàlisi de l'impactes ecològics de les gambúsies mostra principalment que l'impacte global és: i) similar per a les dues espècies; ii) clar i fort per a peixos, macroinvertebrats, amfibis, i alguns grups de zooplàncton i més variable per als tàxons de nivells tròfics més baixos i a nivell d’ecosistema; iii) general per al nombre d'agressions rebudes i la disminució de la densitat i la biomassa, però més variable per altres variables; i iv) altament heterogeni i dependent del context per una sèrie de característiques. Hem estimat la velocitat crítica de natació mitjana (Ucrit) de G. holbrooki com 14.11 cm s-1, que és inferior a la de molts altres peixos de mida similar i confirma que aquesta espècie és limnòfila i el seu èxit invasor parcialment s'explica per l'alteració hidrològica. No obstant això, vam demostrar que Ucrit i la taxa metabòlica màxima varien notablement amb la mida i el sexe dels peixos, amb valors molt més alts als mascles que a femelles del mateix pes, els quals deuen ser per tant menys vulnerables a cabals forts.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
O interesse crescente dos estudos ecotoxicológicos nas várias classes farmacoterapêuticas tem sido o reflexo da frequente deteção destes compostos nos ecossistemas aquáticos. Esta exposição pode resultar em alterações adaptativas dos indivíduos expostos, como modificações a nível tecidular ou ativação de mecanismos antioxidantes, podendo estas ser monitorizadas de modo a determinar o impacto destes compostos nos organismos e respetivos ecossistemas. Dentro destes compostos destacam-se os antibióticos como sendo um grupo largamente utilizado na medicina humana e veterinária, resultando na sua frequente deteção nos compartimentos aquático. A tetraciclina é o antibiótico com a segunda maior taxa de deteção a nível ambiental, sendo utilizado largamente para o tratamento de patologias em humanos e animais. Na medicina veterinária, o uso dos antibióticos é ainda alargado ao tratamento profilático de infeções e promotor de crescimento. Com o interesse de determinar os potenciais efeitos ecotoxicológicos da tetraciclina em peixes, efetuou-se uma exposição aguda (96 h) de Gambusia holbrooki a concentrações crescentes deste composto (5, 50 e 500 ng/l) e pesquisou-se eventuais modificações tecidulares das brânquias e do fígado, bem como alterações da atividade das enzimas antioxidantes e a existência de danos lipoperoxidativos. O estudo da atividade enzimática permitiu averiguar quanto à presença de uma resposta de stress oxidativo (atividades das enzimas glutationa-S-transferases no fígado e nas brânquias, e a catalase no fígado), e neurotoxicidade (atividade da acetilcolinesterase no tecido nervoso). A quantificação de TBARS no músculo permitiu determinar a ocorrência de danos lipoperoxidativos. Os resultados obtidos sugerem uma relação causa-efeito entre a concentração de tetraciclina usada e as alterações histológicas nas brânquias e a atividade enzimática alterada, nomeadamente nas enzimas catalase no fígado e glutationa-S-transferases nas brânquias, sugerindo assim que este composto tem uma atividade pro-oxidante. Assim, nas brânquias as células poderão já ter entrado em stress oxidativo, o que levou a alterações histopatológicas detetáveis. No entanto, ao nível do fígado, e nas concentrações utilizadas no presente estudo, não foram observados danos histológicos significativos. The increasing number of ecotoxicological studies performed with several drugs is a natural consequence of the increasingly frequent detection of these compounds in the aquatic ecosystems. This exposure can result in adaptive changes of individuals, such as tissue alterations or activation of antioxidant mechanisms, which can be monitored to determine the impact of these compounds on the aquatic organisms and ecosystems. Antibiotics stand out as a group widely used in human and veterinary clinic, resulting in its detection in the aquatic environment. Tetracycline is the antibiotic with the second highest level of detection in the environment. It is widely used for human and veterinary treatment of infections, and as a prophylactic treatment of infections and growth promoter on animals. To determine the potential ecotoxicological effects of tetracycline on fish, Gambusia holbrooki individuals were exposed during 96 h to increasing concentrations of this compound (5, 50 and 500 ng/l) and histological alterations in gills and liver, changes in antioxidant enzyme activity and lipoperoxidative damage were recorded. The enzymatic study allowed assessing the presence of an oxidative stress response (glutathione S-transferases in the liver and gills, and catalase in the liver) and neurotoxicity (head acetylcholinesterase). The quantification of TBARS in muscle also allowed the assessment of lipoperoxidative damage. The results indicate a cause-effect relationship between the concentration of tetracycline used and the detected histological and enzymatic alterations in the liver and gills, suggesting that this compound has a pro-oxidative activity.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
Atualmente, existem diversos grupos de agentes biocidas que podem ser utilizados em diferentes áreas, como por exemplo higiene, desinfeção, ação antiparasitária e anti-incrustantes, verificando-se assim um maior recurso à sua utilização pelo homem. Os compostos biocidas apresentam diversos usos, incorporando sobretudo tintas anti-incrustantes. O piritionato de zinco (bis (1 hidroxi -2 (1H)-piridetionato-o,s) - (T-4) zinco) classifica-se como um biocida metálico, e possui ação bactericida, algicida e fungicida. Tem sido formulado em tintas anti-incrustantes, que protegem as superfícies que se encontram expostas a um ambiente aquático, evitando assim a formação de biofilmes. Devido à sua ação biológica tem sido também utilizado para fins cosméticos, como em champôs para o tratamento da caspa e seborreia. As alterações fisiológicas em organismos aquáticos decorrentes da presença de compostos poluentes nos diversos compartimentos ambientais são hoje em dia tema de pesquisa, em face das alterações eventualmente causadas que podem ter variadas consequências deletérias ao nível dos indivíduos e dos ecossistemas. Os dados existentes recolhidos em estudos de monitorização destes compostos demonstram a presença sistemática dos mesmos em compartimentos aquáticos. No presente trabalho avaliou-se a potencial ecotoxicidade do piritionato de zinco em termos de stress oxidativo (catalase, GSTs e TBARS), neurotoxicidade (acetilcolinesterase) e danos histopatológicos no fígado e nas brânquias do peixe dulçaquícola Gambusia holbrooki, após uma exposição aguda (96 h) ao composto. O piritionato de zinco esteve na base do aparecimento de alterações histopatológicas, quer nas brânquias quer no fígado, dos organismos expostos. No entanto não se obteve nenhum resultado significativamente diferente na determinação dos índices patológicos. Relativamente aos biomarcadores testados, apenas a atividade das GSTs poderá indicar a ocorrência de efeitos pro-oxidantes, visto que para as outras enzimas utilizadas para avaliação de stress oxidativo não se obtiveram resultados significativamente diferentes do grupo controlo. At the present time, diverse biocidal agents can be used in distinct areas, such as hygiene, disinfection, antiparasitic activity, and antifouling effect, being increasingly used by humans. Biocidal compounds can be employed in a large set of applications, such as antifouling paints. Zinc pyrithione ritionato de (bis (1 hidroxi -2 (1H)-piridetionato-o,s) - (T-4) zinco) is a metallic biocide, with bactericide, algicide and fungicide activity. It has been formulated in antifouling paints, that protect surfaces exposed to the aquatic environment from the instalation of a biofilm. It has also been used in cosmetics, such as anti-dandruff shampoos. Physiological alterations in aquatic organisms exposed to pollutants in different environmental compartments have been theme of research, to diagnose deleterious effects at the individual and ecosystems levels. Already reported data show the systematic presence of these substances in the aquatic compartment. The present work intended to assess the potential ecotoxicity elicited by zinc pyrithione in terms of oxidative stress (activity of catalase, GSTs and TBARS levels), neurotoxicity (acetylcholinesteras activity), and histopathological damage in liver and gills of the freshwater fish Gambusia holbrooki, after an acute (96h) exposure to the compounds. Zinc pyrithione was capable of causing histopathological alterations, both in liver and gills of exposed organisms. However, pathological indexes were not altered. In terms of oxidative stress biomarkers, only the activity of GSTs may indicate the ocurrence of pro-oxidative effects, since the other enzymes were not responsive.

Sub-populations inhabiting the length of a stream may be subject to a number of substantially different spatial and temporal environmental influences. To be able to monitor the effects of localised disturbance throughout a catchment is of prime import, not only to the study of evolutionary processes, but also to the assessment of human intervention in catchment dynamics. To understand the ultimate effects of localised disturbance, either natural or man-maAe, on the evolution of sub-divided populations, it is essential to determine the patterns of genetic differentiation, the levels of genetic exchange between sub-populations, and the potential for natural selection or genetic drift associated with a particular habitat type or disturbance. The research outlined in this thesis, aimed at determining how disturbance over part of a species range interacts with gene flow to alter the evolutionary processes in each deme of a sub-divided population. These aims involved assessing the relative conthbütion of gene flow, natural selection and genetic drift, to the genetic structure of Mosquitofish Gambusia holbrook! populations at particular times during their yearly breeding cycle. Spatial and temporal variation in Pgi allozyme frequencies were examined relative to distance, flow velocity, and bathers to dispersal, for Gambgsia hoibrooki populations inhabiting an intennitant stream in South-eastern Queensland. Isolation by distance explained only a small amount of the genetic variation between sites. Similarly, levels of differentiation could not be explained by a simple relationship between distance and flow velocity common to both catchments examined. Barriers to dispersal allowed movement only in the downstream direction during peak flooding. Different alleles occurred in high frequencies in the headwaters of each catchment. The dominance of a single allele in headwater populations is attributed to the lack of upstream movement, and the ephemeral nature of these populations. This same allele was maintained as the most common within each catchment by downstream emigration. Inter-catchment differences were therefore attributed to the random fixation of different alleles in the headwaters of each catchment, and the dispersal of these alleles downstream during peak flood events. Significant allele frequency differences between populations in one catchment occurred despite immigration. Differentiation fluctuated over a yearly cycle, and was dependent on the level of immigration entering the lower site from alternative upstream sources. Immigration from source populations was in turn related to flow velocities in the tributaries which converged above this site. All alleles were maintained in the lower reaches of both catchments despite the input of a single allele from headwater populations. Given the mechanism by which frequency differences between sites in the lower western catchment were maintained, and severe restrictions only on upstream movement, it is hypothesised that the less common alleles are preserved in the lower reaches of each catchment, by immigration originating in tributaries where alternative alleles have drifted to relatively high frequencies. Natural selection associated with saltwater inundations and the preferential movement of Ga,nbusia size classes created and maintained genetic differences between populations in the lower western catchment, not only while these populations were isolated, but also during periods of low flow. Preferential movement also enabled selection effects to be carried into adjacent populations, causing them to differentiate from populations not receiving immigrants. Selection effects were not consistent, ocurring in some years and not others. When selection took place allele frequency differences could be maintained at the site of selection for up to a year. The influence of selection at an adjacent site receiving immigrants only maintained differences between this and other sites for a period of about one month. No selection effect could compete with gene flow during high flow velocities each year. The maintenance of genetic diversity in lower reach populations was not dependent on selection associated with habitat heterogeneity in this region of the creek, but on the input of different alleles from tributaries converging upstream. Selection would only have a significant effect on the maintenance of diversity following high flow, if it had produced allele frequency differences in the upper reaches of each catchment. The restriction of upstream movement, and the small effective population sizes associated with the ephemoral nature of headwater populations, suggests genetic drift was more important in determining the final allele frequencies, and maintaining diversity in this system.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Australian School of Environmental Studies
Science, Environment, Engineering and Technology
Full Text

The eastern mosquitofish (Gambusia holbrooki) is one of the most widely distributed and invasive inland fish. It is known that it has not been established in northern Europe or upstream of rivers, where the temperature is too low for survival and reproduction. This thesis examines how temperature affects the competitive ability and life history traits of mosquitofish. We sampled mosquitofish along latitudinal gradients and along rivers and we found that mosquitofish in more favorable environments (i.e. low latitudes and downstream river reaches) had a higher reproductive effort and lower body condition but these patterns of variation were weak and dependent on fish age. We also carried out an experiment which showed that as the temperature rises mosquitofish increases its competitive ability for resources and its aggressive behavior towards Aphanius iberus, an endangered Iberian endemism. Our results imply that climate change will favor the invasive success of mosquitofish and will increase its impact on native fish
La gambúsia (Gambusia holbrooki) és un dels peixos continentals més àmpliament distribuït i més invasors. Se sap que no s’ha pogut establir al nord d’Europa ni a trams alts de rius, on la temperatura és massa baixa per a la supervivència i la reproducció. Aquesta tesi analitza com afecta la temperatura a la capacitat competitiva i a les característiques del cicle vital de la gambúsia. Es van mostrejar gradients latitudinals i al llarg de rius i es va trobar que la gambúsia que vivia a ambients més favorables (és a dir, latituds menors i zones baixes dels rius), presentava un major esforç reproductiu i una menor condició corporal però que aquests patrons de variació eren febles i depenien de l'edat dels peixos. També es va portar a terme un experiment en què es va demostrar que a mesura que puja la temperatura la gambúsia incrementa la seva capacitat de competència pels recursos i el comportament agressiu contra el fartet (Aphanius iberus), endemisme ibèric en perill d'extinció. Els nostres resultats impliquen que el canvi climàtic afavorirà l'èxit invasor de la gambúsia i augmentarà el seu impacte en els peixos natius

Actualment una de les principals amenaces a la biodiversitat és la introducció d'espècies. Revisant 26 variables de les 69 espècies de peixos continental de la Península Ibèrica concloem que la filogènia, variabilitat i els usos de l'home són necessaris per entendre millor les diferències entres les espècies natives i invasores.
Entre les especies més afectades per la introducció de peixos es troben els ciprinodontiformes endèmics del Mediterrani. Aportem les primers dades sobre l'ús d'hàbitats ocasionalment inundats i la selecció de preses del fartet (Aphanius iberus), observant un canvi ontogenètic, clarament relacionat amb el microhàbitat.
També demostrem que la salinitat influeix en l'èxit invasor de la gamúsia, afectant la seva densitat i biologia reproductiva. Per altra banda, demostrem experimentalment que amb l'increment de salinitat la gambúsia disminueix la seva agressivitat i captura menys preses, reduint la seva eficàcia competitiva respecte dels ciprinodonts natius.
One of the main current threats to biodiversity is the introduction of invasive species. By reviewing 26 life-history and ecological variables of the 69 inland fish species of the Iberian Peninsula we conclude that phylogeny, variability and human use are needed to a better understanding of the differences between native and invasive species.
Among the most threatened species by invasive fish are the Mediterranean endemic cyprinodontiform fish. We report the first data on the use of occasionally-inundated habitats ad prey electivity by the Spanish toothcarp (Aphanius iberus). We observed an ontogenetic diet shift clearly linked to a microhabitat change.
We also demonstrate that salinity limits the invasive success of mosquitofish (Gambusia holbrooki), affecting density and life history traits. ON the ohter hand, we experimentally demonstrate that mosquitofish decreases its aggresive behavior and capture less prey, reducing its competitive efficiency with salinity increases regarding to native ciprinodonts.

La introducció d’espècies invasores, juntament amb altres pertorbacions com l’augment dels nutrients, amenacen la biodiversitat nativa globalment. Les invasions biològiques d’espècies exòtiques es troben íntimament lligades a l’activitat humana, des de l’etapa inicial d’introducció en el nou hàbitat fins a l’establiment i l’expansió. La cerca dels factors ecològics i socioeconòmics que fan que una espècie introduïda esdevingui invasora i provoqui un impacte és l’objectiu fonamental de la ciència d’invasions biològiques. Aquesta tesi s’emmarca en el context d’una invasió d’un petit peix, la gambúsia (Gambusia holbrooki) en ecosistemes subjectes a degradació ambiental a causa d’una excessiva fertilització amb nitrats. Mitjançant estudis experimentals en laboratori i en mesocosmos, però també amb estudis de camp, s’explora la hipòtesi de si l’eutrofització té una influència significativa sobre l’èxit invasor de G. holbrooki. Concretament, el primer objectiu és comprendre els mecanismes ecològics resultat de la introducció d’aquests peixos i la contaminació per nitrats i com aquests alteren la comunitat biòtica nativa i certs processos ecològics com la producció primària i la descomposició de matèria orgànica. També es vol avaluar la toxicitat directa dels nitrats sobre la salut de mascles, femelles i juvenils de G. holbrooki mitjançant l’ús de diferents biomarcadors. L’augment de la concentració de nutrients, però també la qualitat de l’habitat físic, l’abundància de depredadors i la prevalença de paràsits, podrien influir en els trets d’història de vida i l’èxit invasor de G. holbrooki. Si la seva condició física estigués influenciada per qualsevol d’aquests factors, els gestors del medi natural podrien dur a terme un control de les poblacions invasores de G. holbrooki mitjançant un enfocament que tingués en compte el conjunt de l'ecosistema, afavorint certes condicions ambientals que reduïssin el seu èxit invasor. Tanmateix, fos per eliminació directa o mitjançant aquest enfocament ecosistèmic, la reducció de l’abundància de G. holbrooki hauria de ser compensada amb la recuperació d’altres espècies natives, com ara el fartet (Aphanius iberus) i proporcionar una eficiència similar en el servei ecosistèmic del control biològic de mosquits, tant en aigües netes com eutròfiques. Els resultats indicaren una interacció insignificant entre els impactes exercits tant per la introducció del peix com per la contaminació per nitrats en l’ecosistema receptor, sent per tant els seus efectes de tipus additiu. La pèrdua de biomassa de peixos observada en concentracions altes de nitrats s'atribuiria a efectes indirectes, com ara una disminució de l'abundància de preses, i no pas per una alteració directa en la fisiologia dels peixos. Aquesta hipòtesi es confirmà en els tests de laboratori, on només els mascles i juvenils patiren alteracions lleus en l’alimentació i l’emmagatzematge de reserves. Aquesta baixa susceptibilitat de G. holbrooki als nitrats, especialment en femelles, confirma la seva alta tolerància a un ampli gradient de condicions ambientals descrita anteriorment. A més, la baixa prevalença de paràsits i l'alta variabilitat en els trets d’història de vida, aquesta última atribuïda principalment a la pròpia densitat de G. holbrooki per sobre d’altres factors ambientals, ofereix poques esperances als gestors per tal de controlar aquesta espècie invasora, a part de la completa eradicació en localitats concretes. Atesa la similar eficiència, però amb una major lentitud, de les espècies autòctones, inclús en absència de peixos, per reduir les abundàncies de mosquits en aigües eutròfiques i netes, presenta una prova evident que la introducció de G. holbrooki representa una amenaça innecessària per al servei ecosistèmic natural. La superioritat competitiva de G. holbrooki sobre les espècies natives, juntament amb la dificultat d’eradicar-la una vegada establerta, fa que siguin prioritaris la prevenció de noves introduccions de G. holbrooki per al control biològic de mosquits, però també la restauració de les poblacions de competidors natius.
Invasive species, together with other perturbations like nutrient enrichment, threaten native biodiversity worldwide. This thesis is framed within the context of the invasion of a small fish, the eastern mosquitofish Gambusia holbrooki (Girard, 1859) in ecosystems subject to environmental degradation due to excessive fertilisation with nitrates. By means of experimental and field observational studies, the hypothesis of whether eutrophication mediates the invasive success of G. holbrooki is explored. Specifically, the initial aim is to understand the ecological mechanisms exerted by fish introduction and nitrate pollution in altering the native communities, the ecological processes and their interaction. Nutrient enrichment causing eutrophication, but also physical habitat quality, abundance of predators and parasites prevalence could influence the life history and the invasive success of G. holbrooki. Should the invader fitness be mediated by any factor, managers could carry out an ecosystem approach by favouring such desired conditions against the invader success. Either by direct elimination or through an ecosystemic approach, the loss of G. holbrooki populations should be substituted by other native species, such as Aphanius iberus and provide a similar mosquito control efficiency in clean and eutrophic waters. Results indicate a negligible interaction between the impacts exerted by both fish introduction and nitrate pollution on the recipient ecosystem. The loss of fish biomass in high nitrate concentration would be attributed to indirect effects such as a decline in prey abundance, rather than a direct alteration on fish physiology. This low susceptibility of G. holbrooki to nitrates, especially in females, confirms its high tolerance to a wide gradient of environmental conditions reported by previous authors. Moreover, the low prevalence of parasites and the high variability of life history traits, mainly attributed to G. holbrooki density in the pond over other environmental factors leave managers with low chances of controlling the invader apart from its eradication. Given the similar but slower efficiency of the native community both in eutrophic and clean waters in reducing mosquito abundances and the difficulty of eradicating G. holbrooki once established, favouring the presence of native competitors and preventing further introductions of G. holbrooki for biological control seems a priority.

Monografia apresentada à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de licenciada em Ciências Farmacêuticas.
A presença de compostos com capacidade neuroactiva nos ambientes aquáticos tem sido uma das principais preocupações dos ecotoxicologistas que avaliam os potenciais efeitos de fármacos nos organismos aquáticos. As alterações fisiológicas decorrentes da presença de resíduos de medicamentos de utilização humana nos diversos compartimentos ambientais são hoje em dia tema de pesquisa, em face das alterações eventualmente causadas nos organismos não alvo. Os dados existentes actualmente, recolhidos em estudos de monitorização destes agentes em compartimentos aquáticos e que visam avaliar o seu impacto, demonstram a presença sistemática de compostos deste tipo nas mais variadas matrizes; no entanto, existe uma manifesta falta de dados científicos que demonstrem, ou não, que estes compostos podem ser potencialmente nocivos do ponto de vista ambiental. O composto sobre o qual incidiu o presente estudo, o diazepam, é um composto de utilização como anticonvulsivante, tranquilizante e relaxante muscular, e existem já evidências que decorrem de estudos já publicados, de que pode estar envolvido em cenários de stress oxidativo. Sendo um composto que actua sobre uma via de comunicação fisiológica altamente conservada (inibição da neurotransmissão por aumento da condutância ao cloreto ao nível das células neuronais), este composto pode potencialmente comprometer um vasto leque de funções neuronais em muitas espécies. Assim, e visto actuar directamente ao nível do sistema nervoso central dos organismos, foi o nosso objectivo estudar alterações enzimáticas e histológicas na espécie Gambusia holbrooki, após exposições agudas (96 horas) a diversas concentrações deste agente (0,0025; 0,005; 0,02; 0,02; 0,04μg/l). Os efeitos decorrentes da exposição ao referido composto foram avaliados ao nível da actividade enzimática (catalase e glutationa S-transferases) e de alterações histológicas observadas no fígado e brânquias dos organismos expostos. O diazepam causou evidentemente alterações regressivas histológicas nas brânquias e no fígado dos animais expostos às três concentrações mais altas. A actividade da catalase mostrou também estar aumentada, indicando stress oxidativo. Já o caso da glutationa S-transferase demonstrou não ser um biomarcador viável para a avaliação do risco ambiental, pelo menos em exposições agudas.

Trabalho apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Licenciada em Ciências Farmacêuticas
A libertação de compostos antropogénicos no meio ambiente tem merecido especial atenção, uma vez que alguns exibem conhecida actividade biológica. Entre os compostos antropogénicos encontrados no meio ambiente, os fármacos são muito frequentemente detectados, e podem ser responsáveis por alterações fisiológicas em organismos expostos. As alterações fisiológicas decorrentes da presença de resíduos de medicamentos nos diversos compartimentos ambientais são hoje em dia tema de pesquisa, em face das alterações eventualmente causadas nos organismos não alvo. Os dados existentes recolhidos em estudos de monitorização com o intuito de avaliar o seu impacto demonstram a presença sistemática destes compostos em compartimentos aquáticos nas mais variadas matrizes ambientais. A fenitoína é um composto farmacoterapêutico utilizado como anticonvulsivante e anti-epilético, habitualmente presente em efluentes domésticos e industriais, como consequência da sua baixa eliminação nas estações de tratamento de águas residuais. Neste trabalho avaliou-se as alterações enzimáticas (actividade das isoenzimas glutationa-S-transferases) e histológicas observadas no fígado e nas brânquias de Gambusia holbrooki, após exposições agudas (96 horas) a diversas concentrações deste agente (50; 25; 12,5; 6,25; 3,125 μg/l). A fenitoína esteve relacionada com o aparecimento de lesões histopatológicas tanto nas brânquias como no fígado dos animais expostos. No que concerne à avaliação enzimática, foi possível observar uma diminuição da actividade das isoenzimas GSTs no fígado, o que não aconteceu nas brânquias, tecido no qual não se verificaram alterações significativas relativamente ao grupo controlo.

Le choix du partenaire sexuel est l'une des décisions les plus importantes auxquelles sont confrontés les animaux au cours de leur vie. Une manière pour un individu de choisir un partenaire sexuel est de copier le choix de ses congénères. Bien que décrit dans différentes espèces de vertébrés et chez la Drosophila, ce processus d'imitation du choix du partenaire reste malgré tout peu compris. L'objectif de ma thèse a été d'étudier la capacité des femelles à choisir leur partenaire sexuel par imitation dans deux espèces de poisson, le poisson zèbre (Danio rerio) et la Gambusie de l'Est (Gambusia holbrooki), et d'étudier si cette capacité des femelles à imiter est corrélée à leur personnalité, plus particulièrement à leur caractère sur l'axe timide-audacieux. Les femelles poisson zèbre et G. holbrooki ont une préférence naturelle pour les grands mâles. Afin d'évaluer leur capacité à imiter, nous avons testé si, comme décrit dans d'autres espèces de poissons, les femelles augmentent leur préférence pour le petit male après avoir observé le grand male seul et le petit mâle en interaction avec une autre femelle. Nous n'avons pas pu mettre en évidence une tendance à imiter le choix de ses congénères chez des femelles poisson zèbre, suggérant que ce modèle n'est pas adapté pour étudier les mécanismes cognitifs sous-tenant le processus d'imitation du choix du partenaire (Article 1, Chapitre 2). Afin d'évaluer leur caractère exploratoire-audacieux, nous avons testé le comportement de ces mêmes femelles poisson zèbre dans un labyrinthe toutes les deux semaines, cinq fois de suite. Bien que nous ayons trouvé une répartition claire des individus le long de l'axe timide-audacieux dans le premier test, nous n'avons cependant pas pu détecter une répétabilité des comportements individuels à travers les tests suivants. Ceci était sans doute du au fait qu'après le premier test de personalité dans le labyrinthe, les femelles avaient appris que ce labyrinthe ne contenait aucun attracteur (alimentation ou congénère), ce qui les conduisaient à bouger de moins en moins dans le labyrinthe. Ainsi, bien que les traits de personnalité doivent persister au fil du temps, nos données suggèrent que la répétabilité des mesures dans un même test pourrait être faussée par l'apprentissage et l'habituation (Article 2, Chapitre 3). Finalement, nos données sur les femelles Gambusie de l'Est suggèrent que la capacité à imiter le choix d'un partenaire sexuel est liée à la personnalité des femelles: comme anticipé, seules les femelles timides, et non les plus audacieuses, montrent une réelle capacité à imiter le choix de leurs congénères femelles. Ce comportement des femelles timides est maximum lorsque la différence de taille entre les mâles est comprise entre 10% et 25%. Enfin, l'imitation du choix du partenaire semble également être affectée par la pression atmosphérique au moment de l'expérience, les femelles étant plus susceptibles d'imiter leurs congénères lorsque la pression de l'air augmente, en prévision de condition climatique favorable. Si nous avions ignoré ces effets confondants (personnalité, différence relative de taille des mâles attracteurs et pression atmosphérique), nous n'aurions pas détecté le comportement d'imitation du choix du partenaire sexuel chez la gambusie. Nous concluons qu'il est important de prendre en compte de tels effets confondants dans l'étude du comportement d'imitation du choix du partenaire social chez les autres espèces (Article 3, Chapitre 4)
Le choix du partenaire sexuel est l'une des décisions les plus importantes auxquelles sont confrontés les animaux au cours de leur vie. Une manière pour un individu de choisir un partenaire sexuel est de copier le choix de ses congénères. Bien que décrit dans différentes espèces de vertébrés et chez la Drosophila, ce processus d'imitation du choix du partenaire reste malgré tout peu compris. L'objectif de ma thèse a été d'étudier la capacité des femelles à choisir leur partenaire sexuel par imitation dans deux espèces de poisson, le poisson zèbre (Danio rerio) et la Gambusie de l'Est (Gambusia holbrooki), et d'étudier si cette capacité des femelles à imiter est corrélée à leur personnalité, plus particulièrement à leur caractère sur l'axe timide-audacieux. Les femelles poisson zèbre et G. holbrooki ont une préférence naturelle pour les grands mâles. Afin d'évaluer leur capacité à imiter, nous avons testé si, comme décrit dans d'autres espèces de poissons, les femelles augmentent leur préférence pour le petit male après avoir observé le grand male seul et le petit mâle en interaction avec une autre femelle. Nous n'avons pas pu mettre en évidence une tendance à imiter le choix de ses congénères chez des femelles poisson zèbre, suggérant que ce modèle n'est pas adapté pour étudier les mécanismes cognitifs sous-tenant le processus d'imitation du choix du partenaire (Article 1, Chapitre 2). Afin d'évaluer leur caractère exploratoire-audacieux, nous avons testé le comportement de ces mêmes femelles poisson zèbre dans un labyrinthe toutes les deux semaines, cinq fois de suite. Bien que nous ayons trouvé une répartition claire des individus le long de l'axe timide-audacieux dans le premier test, nous n'avons cependant pas pu détecter une répétabilité des comportements individuels à travers les tests suivants. Ceci était sans doute du au fait qu'après le premier test de personalité dans le labyrinthe, les femelles avaient appris que ce labyrinthe ne contenait aucun attracteur (alimentation ou congénère), ce qui les conduisaient à bouger de moins en moins dans le labyrinthe. Ainsi, bien que les traits de personnalité doivent persister au fil du temps, nos données suggèrent que la répétabilité des mesures dans un même test pourrait être faussée par l'apprentissage et l'habituation (Article 2, Chapitre 3). Finalement, nos données sur les femelles Gambusie de l'Est suggèrent que la capacité à imiter le choix d'un partenaire sexuel est liée à la personnalité des femelles: comme anticipé, seules les femelles timides, et non les plus audacieuses, montrent une réelle capacité à imiter le choix de leurs congénères femelles. Ce comportement des femelles timides est maximum lorsque la différence de taille entre les mâles est comprise entre 10% et 25%. Enfin, l'imitation du choix du partenaire semble également être affectée par la pression atmosphérique au moment de l'expérience, les femelles étant plus susceptibles d'imiter leurs congénères lorsque la pression de l'air augmente, en prévision de condition climatique favorable. Si nous avions ignoré ces effets confondants (personnalité, différence relative de taille des mâles attracteurs et pression atmosphérique), nous n'aurions pas détecté le comportement d'imitation du choix du partenaire sexuel chez la gambusie. Nous concluons qu'il est important de prendre en compte de tels effets confondants dans l'étude du comportement d'imitation du choix du partenaire social chez les autres espèces (Article 3, Chapitre 4)

Mosquitofish is a small, voracious, highly fecund freshwater fish species originated from northeaster America, that was introduced worldwide to control mosquito populations. In this thesis we have studied the genetics of the invasion of Mediterranean streams by the mosquitofish (Gambusia holbrooki) to discover some of the aspects that drive their successfully invasive life history. Comparison of introduced populations with the American sources of invasion indicated that there was no loss of genetic diversity due to the invasion process. Local genetic diversity levels and overall population structure were maintained among generations in introduced populations. We observed adaptive response to pollutants in the Flix reservoir, Ebro River, but that human perturbations do not prevent mosquitofish dispersal along rivers. Finally, unregulated human-assisted translocations probably increase the opportunities for colonization of new environments, and therefore need to be controlled.
La gambúsia és un peix molt voraç i fecund originari de les masses d’aigua dolça del nord-est d’Amèrica, que va a ser introduïda en tot el món pel control biològic de les poblacions de mosquit. En aquesta tesis se analitzen els processos genètics relacionats amb l’èxit invasor de la gambúsia (Gambusia holbrooki) en les conques mediterrànies. Les poblacions de gambusia introduïdes catalanes mantenen els nivells de diversitat genètica presents a les poblacions Americànes que van ser les fonts dels invasors de Europa. En aquestas poblacions introduïdes, els nivells locals i patrons hidrogràfics de diversitat genètica es mantenen entre generacions. Hi a una resposta selectiva als contaminants en les poblacions de gamusia a l’embassament de Flix, riu Ebro, però aquestes pertorbacions humanes no limiten la dispersió de la gambúsia. Finalment, les translocacions d’exemplars per l’home augmenten les oportunitats de colonitzar nous ambients i per això han de ser controlats.

Les travaux présentés dans le cadre de notre travail de thèse nous ont permis d'évaluer les potentialités d'utilisation de la gambusie, poisson pœciliidœ, en écotoxicologie aquatique expérimentale dans une perspective d'analyse des effets des xénobiotiques à différents niveaux d'organisation biologique incluant la population. Les résultats obtenus nous ont permis de constater que : le 4-NP est activement métabolisé par la gambusie ; la différenciation sexuelle et la croissance des jeunes stades de gambusie sont altérées lors des expositions au 4-nonylphenol et à la 17α -ethynylœstradiol, se traduisant par une altération des processus de gamétogenèse ; l'étude conduite en mésocosmes lentiques au niveau des populations a permis de révéler d'une part, qu'il est possible de travailler expérimentalement au niveau des populations avec cette espèce et que, par ailleurs, les effets constatés au niveau des individus lors des expositions en laboratoire se traduisent, pour partie, au niveau des populations. Les informations obtenues permettent donc d'émettre l'hypothèse que la gambusie pourrait constituer un modèle biologique à retenir dans le cadre des procédures d'évaluation du risque lié à l'utilisation de xénobiotiques, d'une part du fait de la sensibilité des réponses observées à des concentrations en polluants proches de celles détectées dans l'environnement, d'autre part du fait de son utilisation possible dans différents contextes expérimentaux permettant des approches intégratives des effets des toxiques (de la cellule à la population)
The results of our study were used to evaluate the abilities of the mosquitofish, Gambusia holbrooki, in the perspective of analysis of xenobiotics effects at different level of biological organization including population. Our results showed that : 4-n-nonylphenol is biotransformed by mosquitofish ; sexual differentiation and growth of larvae were impaired during a 4-nonylphenol and 17α -ethynylestradiol exposure. These effects were persistent ; Gametogenesis of adults were impaired during a 4-nonylphenol exposure ; Mesocosms study showed that it is possible to conduct experimental study at the population level with this species and that effects observed in laboratory are partly translated at the population level. So all this results can allow us to hypothesize that mosquitofish might be an interesting model in risk assessment process concerning the xenobiotic use. Indeed, on the other hand we have showed that biological responses were observed at environmental concentrations of xenobiotics and in the other hand we have showed that it was possible to use this species in different experimental context that permit integrative approach of xenobiotics effects (from the cellular level on the population level)

Monografia apresentada à Universidade Fernando Pessoa para obtenção do grau Licenciada em Ciências Farmacêuticas
A poluição antropogénica de natureza química tem vindo a aumentar de forma considerável. A acção mais importante que os poluentes poderão exercer serão contribuir para o desenvolvimento de efeitos tóxicos, que pode decorrer da sua capacidade de interagir de forma deletéria com os sistemas biológicos. Os detergentes são compostos de origem antropogénica amplamente disseminados a nível ambiental, o que conduz à possibilidade de exercer alterações fisiológicas em organismos expostos. De forma a avaliar os riscos que os xenobióticos de origem antropogénica poderão colocar às espécies aquáticas, têm-se recorrido a biomarcadores, já que a alteração de qualquer mecanismo ou via fisiológica/metabólical/comportamental de um organismo exposto pode indicar o modo e a extensão de actuação do contaminante ambiental. No presente estudo recorreu-se ao uso da acetilcolinesterase e marcadores histológicos como biomarcadores, de forma a fazer uma avaliação ecotoxicológica dos efeitos do cloreto de benzalcónio no peixe mosquito (Gambusia holbrooki) após exposição aguda. Não foram observadas alterações significativas a nível da actividade colinesterásica nem histopatológica após exposição aguda do peixe mosquito, o que leva a questionar a utilização destas ferramentas em estudos ecotoxicológicos envolvendo detergentes.
Anthropogenic pollution of chemical nature has increased considerably. The most important action that pollutants may exert is related with their contribution to the development of toxic effects, which may derive from their ability to interact with biological systems. Detergents are anthropogenic compounds widely disseminated in the environment, a fact that leads to the possibility of exerting physiological changes in exposed organisms. To assess the risks that anthropogenic xenobiotics can pose to aquatic species, biomarkers have been commonly used. The modification of any pathway involved in physiological/metabolic/behavioural parameters may indicate the mode and extent of activity of a given environmental contaminant. The present study involved the use of acetylcholinesterase inhibition and histopathological alteration as biomarkers, to assess the ecotoxicologic effects of benzalkonium chloride in mosquitofish (Gambusia holbrooki), following acute exposure. Acetylcholinesterasic activity and histological features remained unchanged following acute exposure of the mentioned species. These observations lead us to question the use of these tools in ecotoxicologic studies for the assessment of effects caused by detergents.
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El Poecílido Gambusia holbrooki, mundialmente introducido en sistemas acuáticos epicontinentales, presenta una alta capacidad invasora y supone una seria amenaza para las comunidades nativas. En la Península Ibérica, destaca su impacto sobre los Ciprinodóntidos nativos, aunque también supone una amenaza para los sistemas salinos, principalmente en regiones áridas. Para contribuir al establecimiento de criterios científicos para el manejo de esta especie, la presente tesis doctoral aporta información básica sobre la estructura y parámetros de la biología de poblaciones habitantes de sistemas salinos del sureste peninsular. Se plantearon tres objetivos generales: (1) Ampliar el conocimiento sobre la biología y ecología de G. holbrooki en sistemas lóticos de carácter salino. (2) Estudiar la interacción trófica entre G. holbrooki y la especie nativa Aphanius iberus en un sistema salino. (3) Realizar y analizar una experiencia de control sostenido sobre una población de G. holbrooki aislada en la cabecera de un arroyo salino.
The Poeciliid Gambusia holbrooki, globally widespread in freshwater aquatic systems, is highly invasive and represents a serious threat to native communities. In the Iberian Peninsula, its impact on the native Cyprinodontids has been emphasized, although it also poses a threat to arid saline systems. To contribute to the establishment of scientific criteria in management plans of its invasive populations, the present PhD thesis aims to provide basic information about population structure and biological traits of G. holbrooki populations inhabiting saline systems in the southeastern Iberian Peninsula, with three general objectives: (1) To increase knowledge of the biology and ecology of G. holbrooki in saline lotic systems. (2) To analyse trophic interaction between G. holbrooki and the native Aphanius iberus in a saline system. (3) To conduct and examine the results of an experience of sustained control of a population of G. holbrooki isolated in the headwaters of a saline stream.

Trabalho apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
A contaminação antropogénica dos ecossistemas aquáticos por uma classe emergente de poluentes, os resíduos de fármacos de utilização generalizada, tem sido um assunto de forte debate na comunidade científica internacional. Os fármacos reúnem características que os tornam um foco de crescente preocupação do ponto de vista ambiental e de saúde pública, visto possuírem actividade farmacológica, serem resistentes aos processos de metabolização, poderem sofrer fenómenos de biomagnificação e bioacumulação, e serem ambientalmente ubíquos e libertados em elevadas quantidades para o ambiente, por vezes sem qualquer tratamento prévio. Entre os compostos antropogénicos encontrados no meio ambiente, os detergentes iónicos são muito frequentes. A presença destes agentes pode causar alterações em organismos expostos, passíveis de serem quantificadas por via de ferramentas analíticas apropriadas. Os efeitos decorrentes da exposição de fêmeas imaturas de Gambusia holbrooki (n.v. peixe mosquito) a um detergente de uso farmacêutico generalizado, o cloreto de benzalcónio, foram avaliados através do estudo de eventuais alterações histológicas, em dois órgãos-alvo em particular, as brânquias e o fígado. Vários exemplares de G. holbrooki foram expostos a diferentes concentrações subletais de cloreto de benzalcónio (0,025 mg/l; 0,1 mg/l; 0,4 mg/l) durante vários períodos de tempo (4, 14 e 28 dias). Ao nível das brânquias foram detectadas alterações histopatológicas, tais como, levantamento epitelial, fusão das lamelas secundárias, hiperplasia, aneurismas e descamação das lamelas secundárias. Ao nível hepático foram detectados sinais ligeiros de vacuolização. Contudo a análise semi-quantitativa e quantitativa dessas alterações não detectou quaisquer diferenças significativas entre os grupos controlo e os grupos de teste nas diferentes concentrações e nos diferentes tempos de exposição, sugerindo uma acção relativamente inócua do cloreto de benzalcónio nos animais expostos. Benzalkonium chloride is a detergent with effective germicide and preservative properties, commonly used in several pharmaceutical and personal care products. The histopathological effects of benzalkonum chloride on the liver and gills of mosquitofish, Gambusia holbrooki, were studied by light microscope in tissues stained with hematoxylin and eosin. The fish were exposed to sublethal concentrations (0,025 mg/l, 0.1 mg/l and 0.4 mg/l) of benzalkonium chloride for 4, 14 and 28 days. Qualitative histological examination of both organs showed a variety of minor cellular alterations (e.g. lifting up of epithelium, intraepithelial oedema, fusion of adjacent secondary lamellae and hepatocytes vacuolization), also present in the control group. Almost changes are of minimal pathological importance, suggesting that the lesions were easily reversible as exposure to benzalkonium chloride ceased. In addition, gill morphometrical indices were used to test any physiological disturbance of the respiratory surface with exposure to benzalkonium chloride. However, statistical analysis did not find any relationship between the concentration and/or duration of exposure, suggesting that benzalkonium chloride has little effect on mosquitofish.

Six rivers within the upper Mumbidgee catchment were sampled for larval and juvenile fish. The rivers represented both regulated and unregulated flow regimes and varied widely in size. There was wide variation in the larval fish communities supported by each river, both in terms of the species diversity and total abundance of fish sampled. The highly regulated reach of the Mumbidgee River sampled during this study had the highest numbers of native species and native individuals of any river sampled. In the two rivers selected for further study, the Murmmbidgee and Goodradigbee, there was a high level of inter-annual consistency in the species composition within the reaches sampled, despite considerable change in the temperature and flow regimes of both rivers. This indicates that at least some spawning of those species sampled may occur each year, regardless of environmental conditions. Estimates of the relative abundance of each species sampled changed markedly between years, and it is argued, on the basis of growth information contained in the otoliths, that differential survival of larvae and juveniles was largely responsible for this shift in relative abundance. Otolith microstructure provided information on the date of spawning and early growth patterns of all species sampled in the upper Mumumbidgee catchment. In addition to determining the age and thus 'birth-date' of an individual, the effect of a particular event or series of events has on growth, and subsequent survival, is permanently recorded in the otolith microstructure. This enables accurate back-calculation and correlation to management actions or natural events. No other research tool has this ability to retrospectively assess, on a daily basis, the impacts of management actions on condition and subsequent survival of fish larvae. Species sampled could be separated into three groups based on spawning requirements; those linked with flow, those linked with temperature and generalist species that appear to have river independent cues, such as photoperiod or moon phase. Patterns in growth rate during the early life history stages enabled quantification of the consequences of variation in environmental conditions on the survival and recruitment of various species. Growth was not always highly correlated with water temperature, in fact, for mountain galaxias, high temperatures appear to negatively affect larval condition and subsequent survival. Conversely, carp exhibited a strategy more consistent with common perceptions, with growth and survival increasing with increasing temperature. The study uncovered spawning and growth patterns that were unexpected. Age analysis of western carp gudgeon demonstrated that they had undertaken a mid-winter spawning, when the water temperature in the main channel was far lower than that at which spawning was previously recorded for this species. Redfin perch from the unregulated Goodradigbee River exhibited growth rates exceeding the published upper limits for this and other closely related species. This growth could not be correlated with either temperature or flow, indicating that there are additional factors that dominate growth rates of redfin perch in the Goodradigbee River. The proportion and abundance of native species alone is not necessarily indicative of a 'healthy' or pristine system; some native species may be positively affected by river regulation, at least as juveniles. Comparison of the current larval fish community with likely pre-European fish communities does provide an indication of change to the system. The results of this study suggest that larval fish growth rates can be strongly influenced by environmental conditions, thus providing a powerful tool for monitoring future change and the factors which cause it. This study has demonstrated the value of larval and juvenile fish age and growth information, derived from otolith microstructure techniques, for many aspects of river management. Current river management priorities for which these techniques provide unique information include the determination of environmental flow regimes and the control of undesirable exotic species such as carp.

The study of contemporary evolution and eco-evolutionary dynamics is classically defined in terms of genetic evolution, but the actual suite of processes driving contemporary trait change is likely much more complex than often credited. This dissertation considers additional mechanisms of trait change that might be important to an emerging model system for study of contemporary evolution and eco-evolutionary dynamics. Specifically, the research focuses on phenotypically plastic and demographic trait variation in Eastern and Western Mosquitofish (Gambusia affinis and G. holbrooki) facing the major ecological gradient of predation risk. Plasticity experiments employed a common-garden rearing design to manipulate fish predator cues experienced by individuals, their parents, or their grandparents and in turn quantify reaction norms in mosquitofish size, shape, and behavior. The two species of mosquitofish showed divergent plastic responses in behavior, with the relatively bolder G. holbrooki becoming even bolder in response to predator cues. In contrast, males and females within species showed parallel behavioral responses. Despite strong sexual dimorphism, both sexes and both species showed parallel patterns of plasticity toward streamlining of body shape when exposed to predators. Interestingly, mosquitofish also showed evidence of transmitting predator cues across generations, where female G. affinis become shyer and more streamlined when their parents or grandparents experienced predators. In contrast, male G. affinis showed little evidence of transgenerational plasticity and appear to rely more heavily on their own experience. Another set of field surveys and experiments with G. hoibrooki considered the potential role of sexual dimorphism and demographic variation in sex ratios as another form of trait variation with possible community and ecosystem consequences. Natural population surveys revealed female-biased sex ratios and higher primary production in the absence of predators. Mesocosm experiments suggested males and females differed in dietary preferences and that both sex ratio and density influence community responses. Although these findings support a need to expand the current eco-evolutionary synthesis to mechanisms beyond just genetic evolution, they also support some general patterns in these mechanisms and ways in which they might work with evolution to produce an even more dynamic interaction of ecology and trait change in nature.

The eastern mosquitofish, Gambusia holbrooki, is a freshwater viviparous fish that originates from North America. This along with its sister species G. affinis, were introduced throughout the world (i.e., over 110 countries), including Australia as mosquito control agents. Due to their high adaptability and tolerance, they have rapidly established populations in new ecosystems and caused decline in native aquatic fauna through competition and predation. For example, in Australia, G. holbrooki has been implicated in the decline of nine native fish species (genera Galaxias, Chlamydogobius, Melanotaenia, Craterocephalus, Mogurnda, Pseudomugil, Ambassis, Scaturiginichthys and Retropinna) and 10 species of frog. Therefore, G. holbrooki is considered as one of the highly invasive species. Their impacts have become apparent, particularly in freshwater ecosystems, where invasive species constitute the single greatest threat to biodiversity. Hence, development of an effective ‘threat abatement strategy’ is vital. One of the most promising solutions for eradication, is the Trojan Y chromosome (TYC) approach. The strategy works by manipulating the sex ratio of the target population through the introduction of sex-reversed females carrying two Y chromosomes (Trojan Y) that produce only male offspring. However, for this to be successful, the chromosomal sex mechanism must be known. In addition, developing a phenotypic sex marker could facilitate the process of genetic screening of the embryos prior to and post-hormonal treatments (i.e., production of sex-reversed embryos) for rapid identification of sex reversed individuals. To investigate the relationship between genetic sex and phenotypic traits amongst various embryonic stages, a detailed developmental study is needed. However, little information was available on embryonic development in this species, with the exception of an earlier detailed staging system for Gambusia sp. Although less complete than the present study, the earlier study accurately describes stages of embryonic development, and includes useful sets of hand-drawn illustrations. More recently, another study established time-saving (i.e., without sacrificing the brood) approach to predict the developmental progress, by applying the gravid spot index of pregnant females as surrogates for five broad stages. Generally, proposed classifications are a guide but not precise for G. holbrooki developmental staging due to divergent traits and morphology. Regardless, several basic keys specific to different organ systems (e.g., circulation), detailed morphology and precise indices for each stage were virtually non-existent. The developmental studies are also essential to investigate the genotype-phenotype relationships as several sex-related events occur during the embryogenesis. However, these remain uninvestigated, particularly in G. holbrooki development. Low mitotic index is a common problem in cytogenetic studies when using direct (i.e., harvesting cells from tissue of a living animal instead of in vitro tissue culture) chromosome preparation methods. Moreover, to improve cytogenetic studies, physical stretching/relaxation of metaphase chromosomes is essential, primarily to enhance the resolution of the fine chromosomal region (i.e., precise detection). Intercalated agents such ethidium bromide (EtBr) is known as effective in altering the conformational and physical properties of DNA helix of the chromosomes. Although there are several reports confirming the key roles of EtBr that intercalates between DNA bases and prevents DNA folding and condensing in mammalian models like mice, there is no report of using these agents in any fish species including G. holbrooki. Several studies have suggested a XX/XY sex-determining system for G. holbrooki based on the melanic color pattern inheritance from father to son and its allelic linkage group associated to males. However, a sex determination mechanism has not been cytogenetically confirmed in this or any poecilids, with the exception of P. reticulata. Therefore, due to inconclusive evidence in the literature, multiple evidence, including cytogenetic are required to verify the chromosomal sex determining mechanism in G. holbrooki. In this context, the overarching objectives of this study were to discover early phenotypic markers of sex differentiation and ascertain the sex determining mechanism in G. holbrooki, specifically: 1) standardise developmental staging of G. holbrooki embryos for determining the timing and onset of sex determination and differentiation; 2) develop a phenotypic sex marker to assist distinguishing the sex of live embryos at very early developmental stages (e.g., prior to sex-reversal of embryos), and; 3) identify and confirm a sex determination mechanism (i.e. male or female heterogamety) using classical karyomorphology and comparative genomic hybridisation (CGH). Objectives were addressed by morphological studies of embryos during development from zygote to parturition (chapter 2), with emphasis on heart development (chapter 3) and cytogenetics (chapters 4 and 5). First (chapter 2), a comprehensive developmental staging of embryos that document sequential events of development from zygote to parturition (30 days post fertilization) at 25 ± 0.5 °C was defined. To overcome limitations associated with superfetation and obtaining an adequate number of embryos, two approaches were simultaneously employed; first, embryos were harvested from wild caught gravid females, with varying intensity of gravid spot, during peak reproductive season to ensure availability of sufficient embryos at multiple developmental stages. Second, to calibrate developmental timing, genetically sexed females (i.e., at parturition) were grown up to maturity (2–3 months) and then individually mated with males to record time of mating (i.e., first copulation behaviour) as proxy for fertilisation. Post-copulation females (n = 1 or 2) were sampled, and embryos harvested every 6 h for ten days (n = 35), then every 12 h for the remaining 20 days (n = 55). Live embryos were photographed. Morphological diagnostics were used for preliminary staging of embryos according to indices described for developmental staging in Gambusia sp. and zebrafish. For stage identification, a combination of key morphometric measurements (egg size, egg diameter and embryo total length), indices (i.e., otic vesicle closure, heart rates); and meristic counts (e.g., the number of caudal fin rays, and number of caudal fin ray elements) were also employed, as necessary. The development of nervous, circulation, musculoskeletal, visual, and digestive systems along with craniofacial and external body features, were also employed for staging. This was complimented by quantitative pixel analysis of embryonic photographs (i.e., three-dimensional pixel distribution analysis of images). The data were collectively used to describe and define each embryonic stage. The developmental process was defined into seven broad developmental phases (i.e., zygote, cleavage, blastula, gastrula, segmentation, pharyngula and parturition) and 40 stages numerically (stages 0–39; zygote to parturition, respectively). The superfetation, inadequacy of embryos, time/age-controlled during embryogenesis, non-transparency of the eggs and very limited developmental information amongst livebearers for detailed comparison posed some challenges. However, these were overcome by employing multiple strategies including the pixel intensity analysis to assist with identification of stages during early development. Traits found during development could be sex-related, for example, both pigmentation and skeletal traits (i.e., teeth) are sometimes linked to sex chromosome in teleost (e.g., Malawi cichlids). Therefore, the outputs and knowledge from the developmental staging allowed investigating the relationship between developmental phenotypic traits and their genetic sex. This was further validated using a genetic sex marker. Second (chapter 3), the heart rates (HRs) during embryogenesis of G. holbrooki, from onset of heartbeat to just prior to parturition was investigated. Genetic sex of the embryos was postverified using a sex-specific genetic marker. Heartbeat of embryos (n = 10 each sex/stage) at early organogenesis, mid-organogenesis, late organogenesis, early pharyngula, late pharyngula, just prior to parturition and adults (n = 10 each sex) were videographed for 60 s at 25 ± 0.5 °C. The HR and its frequencies were determined using a non-invasive light-cardiography (LCG) method previously described for adult zebrafish. LCG profiles for embryos corresponded to contraction of the ventricle (increased average brightness) conversely, relaxation (decreased average brightness) within the prescribed region of interests (ROI). LCG profiles for adults constitute an opposite signal intensity compared to embryos. The peaks for each LCG oscillation were found using Gauss model and peak analyser function (Origin Pro 2020). The time duration (s) between ventricular systolic (contraction) and atrial diastolic (dilation) phases, constituted a complete cardiac cycle. The time delay between the atrioventricular (A-V delay) peak values of the extracted synchronous chronologies within the same cardiac cycles were measured using time history values for ventricle and atrium peaks. A-V delay was defined as the time duration (s) between the onset of atrium peak and the onset of subsequent ventricular peak, which represents the resting phase of the heart. The average resting time per minute for all developmental stages was calculated by multiplying the total A-V delay/sec by 60. For quantification of ventricle size in adults, both ventricular surface area (mm2 ) and volume of the ventricle (mm3 ) were calculated using ImageJ. Ventricle volume measurements were normalised by condition factor (a) derived separately for each sex (male = 0.01; female = 0.02) using allometric growth equation, W = aL3 . Results reveal that heart rates and resting time significantly increase (p < 0.05) with progressive embryonic developmental stage. The total cardiac resting time per minute was approximately three times greater in the advanced embryos (just prior to parturition) compared to those at early organogenesis, when the heart first begins to beat. Initially, up to mid-organogenesis stage, the heart rates of both male and female embryos were comparable (p > 0.05). However, at late organogenesis, both ventricular and arterial frequencies of female embryos were significantly higher (p < 0.05) than those of their male sibs at the corresponding developmental stages and remained so at all later developmental stages evaluated. Consistent with the sex-specific size dimorphism in adults of this species, the size of the normalised heart (i.e., ventricle) of females were significantly larger than those of the males (p < 0.05). In addition, a clear difference in average HR of the two sexes in adults was immediately evident with females having higher HRs (p < 0.05). In addition, significant (p < 0.05) differences in ventricular diastolic state was evident between adult females and males. Collectively the results suggest that the cardiac sex-dimorphism manifests as early as mid-organogenesis and persists through adulthood in this species. This appears to be the first study to demonstrate early onset of cardiac sex-dimorphism in any teleost species. These findings also suggest that the cardiac measurements can be employed to non-invasively and rapidly sex the developing embryos, well in advance of their phenotypic sex is discernible. Although the mechanism(s) underpinning the observed sex-specific cardiac functions are as yet unknown, the results highlight the amenability of G. holbrooki as a powerful vertebrate model to study these and sex-specific consequences of human cardiovascular diseases as well as to monitor anthropogenic and climatic impacts on heart physiology. Third (chapter 4), to facilitate cytogenetic studies in this species the use of baker’s yeast to optimise frequency of mitotic chromosomes and ethidium bromide to overcome the problems associated with chromosomal condensation were experimentally determined in four different tissues (cephalic kidney, liver, spleen, and gill). Mitotic stimulation with activated baker’s yeast, Saccharomyces cerevisiae (SC) was determined over 1–4 days. For synchronisation and elongation of mitotic chromosomes, individuals (both sexes) were injected (IP) with ethidium bromide (2 or 4 µg/ml) and colcemid 1 (µg/ml), and sacrificed at three timepoints (1, 4 and 8 h) post-injections. Results showed that, cells obtained from gill had the highest number of metaphases at 3 days post-SC exposure, with no significant (p > 0.05) differences between sexes. Nonetheless, sex specific differences in the mitotic index were evidenced in spleen, kidney, and liver, suggesting sex specific difference in immune-response to yeast injections. Elongated metaphase chromosomes of female and male were obtained following injection of 4 µg/ml and 2 µg/ml of ethidium bromide, respectively, for an hour. Moreover, the average mitotic chromosome length of females was significantly (p < 0.05) longer than males at 4 µg/ml ethidium bromide treatment at all the three timepoints (1, 4 and 8 h). Such difference in chromosomal elongation between sexes, could be attributed to the sex-specific episomal (e.g., DNA methylation) and histone modifications or variations in length of linker DNA sequences between the consecutive nucleosomes. This substantially facilitated downstream cytogenetic experiments in this study by making available (1) adequate number of arrested cells at metaphase for intensive cytogenetic studies e.g., comparative genomic hybridisation (CGH) and (2) elongated metaphase chromosome allowed investigating very fine heterochromatin structure (i.e., sex-specific fluorescent signals) differences between sexes. To identify and verify the sex determination mechanism (Chapter 5), the metaphase spreads of both males and females were prepared using a conventional air-drying method. Chromosomes were counterstained using 1 mg/ml 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 0.2 µg/ml final concentration) and mounted with Vectashield medium (without DAPI). Mitotic chromosomes were imaged at 100× magnification using a Leica epifluorescence microscope equipped with single band pass emission filters. Metaphase chromosome (n = 25/sex) arms were measured and ideograms for male and female fish generated using IdeoKar. Total genomic DNA was nick translated incorporating SpectrumGreen dUTP (Vysis) and Spectrum-Red dUTP (Vysis). Briefly, slides were denatured for 3 min at 70 °C in 70 % formamide, 2 X SSC, dehydrated through an ethanol series, air-dried, and kept at 37 °C until probe hybridisation. For each slide (made using one drop of fixed-cell solution), 250–500 ng of SpectrumGreen-labelled female and SpectrumRed-labelled male DNA was co-precipitated with (or without) 5–10 µg of boiled genomic DNA from the homogametic sex (as competitor), and 5–10 µg Salmon sperm (as carrier). Since the homogametic sex was not known, reciprocal experiments were performed using alternatively male and female DNA as competitor. As various fluorescent dyes could exhibit differential signal intensities, reciprocal experiments were done incorporating either of dyes (red/green) into total genomic DNA (male or female) as necessary. The co-precipitated probe DNA was resuspended in 35–50 µl hybridisation buffer, first denatured and then hybridised on chromosomes for 3 days at 37 °C. Post washings, slides were mounted with antifade medium Vectashield (with DAPI) and imaged. Phylogenetic analysis was done by multiple sequence alignments of mitochondrial cytochrome b (COI) gene segment (1140 bp) derived from eight Poeciliid species including G. holbrooki (Tasmania and Florida populations) using neighborjoining method (Timura-nei model) with 1000 replicates. Results from karyotyping and ideograms analysis showed no morphological (i.e., size) differences in any of the chromosome pairs in both sexes. However, fluorescent in situ hybridisation (FISH) results showed heterochromatinisation of all the centromeres (CG-positive and AT-negative) and few pericentric and distal regions of the metaphase chromosomes in both sexes. Two small size autosomes in both sexes were intensively heterochromatinised, indicative of DNA repeats accumulation. These two smallest autosomes had hybridisation patterns resembling those of ancestral sex chromosome pair, perhaps originating from whole genome duplication. In addition, a pair of DAPI-positive (i.e., AT-rich) microchromosomes were observed in 20% of the male metaphases and not in females. However, CGH results showed that a single microchromosome also exists in the female metaphases, showing high CG-rich heterochromatinisation (similar to male) but DAPI-negative. Interestingly, CGH in the male metaphase revealed a large male-specific signal (i.e., indicating preferential binding of the male-derived probe) on an interstitial arm of a large chromosome and also two conspicuous male-specific signals superimposed on two weak female-specific signals in two different similar sized chromosomes of the female mitotic complements. These patterns are consistent with the hypothesis of an XX/XY sex determining mechanism but require further verification. Notably, the male specific signal was in the proximal location of the chromosome implying that the accumulation of these repeats may have initiated the Y chromosome differentiation, as well as played a critical role towards the evolution and differentiation of sex chromosomes in this species. Direct estimation of the genetic divergence timing of the Y chromosome in the G. holbrooki was showed to range between 6– 10 million years ago. Therefore, G. holbrooki represents characteristics of nascent XY sex chromosomes, where the Y appears at an early stage of differentiation. In conclusion, this study for the first time described a detailed developmental staging guide in this species, identified sex-specific heart rate during the development which can be employed as a precise and non-invasive sexing marker and provided cytogenetic evidence suggesting the species is likely male heterogametic (XX/XY). Further characterisation of the putative sex chromosomes through isolation of sex-specific sequences and their mapping may be necessary to validate the identity and role of these and the microchromosomes, in this species.

Aquatic ecosystems are threatened by factors related to climate change, leading to changes in the physical and chemical water properties. Between these changes, and as a consequence of global warming and extreme weather events, coastal systems are facing sudden fluctuations in salinity. Aquatic ecosystems are also threatened by organic and inorganic compounds that increase water pollution. Metformin is an antidiabetic drug commonly used by patients of diabetes type 2. It is also used as a treatment for polycystic ovary syndrome and as an anti-cancer drug. Metformin is not metabolized by the human body and has high watery mobility. Because wastewater treatment plants have low efficacy in removing these compounds, they may reach in the aquatic environment. The aim of this study was to evaluate the influence of different salinity levels (17, 24 means control, 31) on the effects of metformin (0-Control, 1.5, 15, 150 μ/L) in Gambusia holbrooki after acute exposure (96h). A chronic exposure was also performed to assess the effects of metformin (0-Control, 0.5, 1, 5, 10 μ/L) in G. holbrooki at salinity 17 for 28 days. For both assays, several biomarkers were quantified in the exposure organisms to assess: antioxidant capacity (superoxide dismutase-SOD, catalase-CAT, glutathione peroxidase-GPx, glutathione reductase-GRed) and biotransformation capacity (glutathione S-transferases-GSTs), cellular damage (levels of lipid peroxidation-TBARS), content of energy reserves (head and body protein levels- PROT, glycogen content- GLY) and neurotoxic effects (acetylcholinesterase-AChE). The results obtain from acute exposure showed interactions between salinity and metformin in SOD activity, body PROT levels and GLY content in G. holbrooki. The data obtain showed that an increase in salinity can modulate the response of the G. holbrooki to metformin. The results obtain from the chronic assay, showed that exposure of G. holbrooki to metformin led to a significant decrease in SOD activity at almost all concentrations tested. In addition, GPx and GSTs activity increased significantly at the concentration of metformin 10 and 5 μ/L, respectively. Therefore, metformin can lead to oxidative stress in G. holbrooki. Overall, the GLY content in G. holbrooki increased after exposure to metformin concentrations. No significant effects were observed in the remaining biomarkers measured. Although this work does not show a full oxidative stress scenario in G. holbrooki after exposure to metformin under different salinities, other studies have already shown that metformin alone can lead to oxidative damage in aquatic species. Therefore, additional ecotoxicological studies should be performed to find out if different metformin concentrations combined with salinity increase might impact non-target species.
Os ecossistemas aquáticos são ameaçados por vários fatores relacionados com as alterações climáticas, levando a modificações nas propriedades físicas e químicas da água. Entre essas alterações, e como consequência do aquecimento global e de eventos climáticos extemos, os ecossistemas costeiros estão a enfrentar flutuações repentinas na salinidade. Os ecossistemas aquáticos são também ameaçados por compostos orgânicos e inorgânicos que aumentam a poluição da água. A metformina é um medicamento antidiabético usualmente utilizado por pacientes com diabetes tipo 2. É também utilizada no tratamento da síndrome do ovário poliquístico e como um medicamento anticancerígeno. A metformina não é metabolizada pelo corpo humano e tem uma alta mobilidade aquosa. Devido à baixa eficiência na remoção destes compostos pelas estações de tratamentos de águas residuais, a metformina pode chegar aos ambientes aquáticos. O objetivo deste estudo é avaliar a influência dos diferentes níveis de salinidade (17, 24- controlo, 31) nos efeitos da metformina (0- Controlo, 1.5, 15, 150 μ/L) em Gambusia holbrooki após uma exposição aguda (96h). Uma exposição crónica também foi realizada para aferir os efeitos da metformina (0- Controlo, 0.5, 1, 5, 10 μ/L) em G. holbrooki na salinidade 17 durante 28 dias. Para ambos os ensaios, vários biomarcadores foram quantificados nos organismos expostos para aferir: a capacidade antioxidante (superóxido dismutase- SOD, catalase- CAT, glutationa peroxidase- GPx, glutationa redutase- GRed) e capacidade de biotransformação (glutationa S-transferases- GSTs), dano celular (níveis de peroxidação lipídica- TBARs), conteúdo de reservas energéticas (níveis de proteína do corpo e da cabeça- PROT; conteúdo de glicogénio- GLY) e efeitos neurotóxicos (acetilcolinesterase- AChE). Os resultados obtidos da exposição aguda mostram interações entre a salinidade e a metformina na atividade da SOD, níveis de PROT do corpo e conteúdo de GLY em G.holbrooki. Os dados extraídos mostram que um aumento da salinidade pode alterar a resposta de G.holbrooki à metformina. Os resultados obtidos da exposição crónica, mostraram que a exposição de G.holbrooki à metformina levou a uma diminuição significativa da atividade da SOD em quase todas as concentrações testadas. Além disso, a atividade da GPX e das GSTs aumentou, significativamente, nas concentrações de metformina de 10 e de 5 μ/L, respetivamente. Assim sendo, a metformina pode levar ao stress oxidativo em G.holbrooki. No geral, o conteúdo de GLY em G.holbrooki aumentou após a exposição ás concentrações de metformina. Não foram observados efeitos significativos nos restantes biomarcadores medidos. Apesar deste trabalho não mostrar um cenário completo de stress oxidativo em G.holbrooki, após a exposição à metformina em diferentes salinidades, outros estudos já revelaram que a metformina pode levar a danos oxidativos em espécies aquáticas. Assim, estudos ecotoxicológicos adicionais devem ser realizados para descobrir se diferentes concentrações de metformina combinadas com um aumento da salinidade podem ter impactos em espécies não alvo.
Mestrado em Biologia Marinha Aplicada

The eastern mosquitofish Gambusia holbrooki has been listed in Australia as a noxious invasive species that requires control and eradication solutions on a large spatial scale. The Trojan Sex Chromosome (TSC) strategy has been proposed in recent years as a new genetic solution to control invasive aquatic species. The TSC approach involves the release of Trojan chromosome carriers, individuals produced via hormone sex reversal into the wild to skew the sex ratio of the population. As a first step towards applying the TSC strategy in G. holbrooki, this study systematically investigated and documented the relationship of the gravid spot with gestation, clutch size and timing of parturition – knowledge that are essential for efficient administration of hormones in this species. Importantly, by utilizing this new knowledge, the efficacy of two feminizing hormones (Diethylstilbestrol-DES and Estradiol-E2) was tested and the reproductive fitness of treated fish assessed. The study identified the gravid spot of females as an excellent marker to predict embryonic developmental progress and reproductive output in G. holbrooki by means of quantifying its visual attributes (intensity and size). An equation to predict clutch size using the relationship between gravid spot intensity and size together with fish length was ascertained [CS = 1.835 − (0.85 X SS) + (0.196 X SI) + (3.543 X FL), where SS and SI are gravid spot size and intensity respectively, and FL is fish length]. The reliability of these findings was confirmed when it facilitated design and accurate observation on G. holbrooki gestation period and parturition behaviour. The gestation was significantly longer (F=364.58; df=1,48; P˃0.05) when reared at 23°C (39±1.91 days) compared to 25 °C (28.6±1.94 days). However, temperature did not have significant impact (P>0.05) on clutch size or diel timing of parturition which occurs predominantly in the morning (0900-1100h). The first-ever description on the posture of G. holbrooki fry (progenies) during parturition was also reported where the tail of the fry emerged first with a few exceptions of head-first, twin and premature births. All this information especially the utility of the gravid spot as an external marker of embryonic development was used to structure the sex reversal experiments. Sex reversal experiments administering both DES and E2 through food targeted two life stages separately: (i) embryonic stage through gravid females and (ii) newborn juveniles. The concentrations of DES tested at both life stages ranged between 20 to 100 mg/kg feed. Two control groups were set for each experiment: (C1) normal feed (no chemical exposure) and (C2) feed mixed with 70% ethanol (vehicle control). In the first experiment, DES treatment did not affect the duration of gestation period in parturating females but as the treatment continued, DES was found responsible for gonadal atrophy in all the treated females after 30 days of exposure. The mean survival rate (MSR) of juveniles was significantly lower (F=16.79; df: 6, 15; P<0.05) compared to the controls (C1: 74.76±15.37%; C2: 71.26±18.5%) where the highest MSR was only 30.15±6.05% in fish treated at the concentration of 40 mg/kg feed for the duration of 11-23 days. Similarly, the MSR of juveniles fed with DES supplemented diet (second experiment) was also significantly lower (F=3.216; df: 6, 28; P<0.05) than controls (C1: 78.42 ±22.0%; C2: 73.8 ±20.22%) with the highest MSR shown by fish treated with DES at 20 mg/kg feed for 30 days (45.6 ±17.93%). Unexpectedly, masculinization was observed in all treated fish (in both experiments 1 and 2) where all the juveniles were phenotypically (morphologically) male based on the elongation of the anal fin. Observation of treated fish at 365 days after parturition (DAP) old revealed that all individuals possessed an under-developed gonopodium compared to normal males, suggesting a paradoxical masculinising and an incomplete sex-reversal effect on genetic females and males respectively in all the treated groups. This condition also seems to have affected the reproductive viability of the fish since none managed to breed with unexposed fish. A paradoxical effect even at relatively low doses of treatment suggests that DES is not a suitable feminising agent for G. holbrooki sex reversal. Molecular and cellular experimentation is warranted for further understanding of the mechanism underpinning this rare observation. Similar to DES treatment, E2 was administered at concentration ranging between 50-400 mg/kg of feed in two life stages —embryos and juveniles. Two control groups were also set as those in the DES experiment. The MSR of controls were found to be slightly higher (F=4.38; df: 6, 27; P<0.05) compared to the treatment groups (C1: 79.96±20.327%; C2: 77.09±10.32%). Treatment of E2 at 200-400 mg/kg feed between 12-21 days successfully produced a 100% female population in the embryo administered group, with the highest MSR of 59.33±12.54% shown by those treated at 200 mg/kg feed. The gestation period of the treated females was not altered by the exposure to E2. In the second group (juvenile administration), the MSR of controls were also significantly higher (F=7.27; df: 5, 24; P<0.05) compared to the treatment groups (C1: 71.73±22.86%; C2: 70.02±18.26%). A 100% feminization was achieved at all administered doses with juveniles treated with an E2 concentration of 50 mg/kg feed displaying the highest MSR at 66.38±12.34%. The survival rates observed in this study are substantially low compared to other E2 treated livebearers such as guppies and black mollies. High stocking densities which lead to aggression by dominant females is proposed as one of the reasons for the low survival rates in E2 treated G. holbrooki. Reproductive fitness assessment was conducted on two groups of G. holbrooki that showed the best performance (highest MSR and feminization percentage) in the sex reversal experiment namely: (i) fish that were treated with E2 at 200 mg/kg of feed (first experimental group); (ii) those exposed to E2 concentration of 50 mg/kg of feed (second experimental group). In parallel a control group (unexposed fish) was also assessed. All juveniles were reared to maturity and bred with normal males. In general the assessment shows that E2 treatments at optimum dose did not compromise the reproductive fitness of the treated fish. There was no significant difference (P>0.05) in terms of ability to breed, gestation period, clutch size and the MSR of progenies produced between treated fish and controls. These observations demonstrate that the reproductive fitness of E2 treated fish is on par with controls. Nevertheless, the number of progeny produced by females in all three groups was low (1-5 fish) warranting continued longterm observations of subsequent clutches. In conclusion, feminization of G. holbrooki was successfully achieved by using E2 as a feminizing agent. The study established a protocol to successfully feminize this species at optimum dose of E2 through oral administration either during the embryonic stage via female brood (200 mg/kg) or to newborn juveniles (50 mg/kg). The protocols and information generated in this study provide a basis for further refinement of hormone treatment and for developing the TSC strategy to control and eradicate this noxious pest. Other studies on fish reproductive biology and ecology especially in livebearing species as well as research on ecotoxicology and pest fish management will benefit from the outcomes presented in this thesis.

The Poeciliid fish, Gambusia holbrooki, a native of North America, has been deliberately introduced into many countries for the purpose of mosquito control. Extensively introduced on mainland Australia since the early 1900's, the fish was absent from the island state of Tasmania until it was illegally introduced in 1992. Since that time, the fish has spread throughout the Tamar Estuary in northern Tasmania. Surveys conducted from 2004-2006 revealed that the fish have spread naturally at the modest rate of less than 2 km per year. Populations showed synchronous birth of live young occurred in mid-November each year, and mean abundance was highest in the summer months (`~60 fish``/0.5 m^2`). Gambusia fed predominantly on micro-crustaceans throughout the year, while mosquitoes and amphibians made up a very minor proportion of the diet (<2% in any season). Gambusia appeared to negatively impact tadpole communities with abundance of tadpoles significantly increasing following eradication of the fish. Population genetic analysis using microsatellite markers revealed that Australian Gambusia populations are characterized by low diversity and Tasmanian Gambusia were possibly derived from southeast Queensland. Although significant differentiation existed among most populations, Tasmanian Gambusia populations were generally genetically similar, indicating a single introduction event. The genetic pattern of relationships among Tamar populations indicated that the site, TIWR, is driving the spread of Gambusia throughout the estuary. In May 2005, an attempt was made to eradicate Gambusia from two enclosed water bodies in the Tamar region. Water was pumped from both sites prior to application of hydrated lime (`Ca(OH)_2`). Both eradications proved unsuccessful, and populations were monitored and samples taken to assess the population recovery and genetic consequences of the eradication attempts. The Riverside population recovered quickest with live fish sighted 17 days after the initial lime treatment compared to 8 months at LD2. Estimates of effective population size (`N,`) indicated that less than ten individuals survived the eradication attempt at Riverside [6 (95% CI=2-14)], and less than five at LD2 [1 (95% CI=0-3)]. Allele frequencies varied significantly at both sites following the eradication attempt, and some alleles were not detected post-eradication. However, there was no statistically significant loss of allelic diversity at either of the sites. The rapid recovery of both populations combined with the maintenance of genetic diversity and minimal changes in allele frequencies, indicate that Gambusia are particularly resistant to the negative genetic effects of bottleneck events that dramatically decrease population size. A site-based risk assessment (RA) protocol was developed from a survey of 27 sites within and around the current distribution of Gambusia in the Tamar ≥30 (all currently support large Gambusia populations). Over 85% of sites returned a risk score from 1-20, with all sites where the fish has never been encountered or failed to establish returning scores of ≤20. The RA could be utilized by managers of Gambusia in Tasmania to rapidly assess new survey sites in the Tamar region and to guide monitoring considerations in the future.

The eastern mosquitofish Gambusia holbrooki was introduced to Australia in 1928 from USA as a bio-control method for mosquitoes and has spread out over freshwater coastal and inland waters of Australia. It is an ineffective mosquito control agent but rather is responsible for depletion of several native and endangered fish and amphibians. Because of its severe negative impacts on native species, G. holbrooki is classed as an invasive species requiring control and eradication efforts. The Trojan Sex Chromosome (TSC) strategy has been suggested in recent years as a genetic control method for this noxious invasive species. The TSC theory relies on the regular release of sex-reversed fish into target wild populations to produce single sex offspring after mating with wild fish; hence biasing the sex ratios leading to eventual extinction of the target populations. As a step towards applying the TSC strategy in G. holbrooki, this study investigated the sex reversal of genetic females into phenotypic males at three different life stages: adults, neonates and 30-day old juveniles. The study observed the timing of sex differentiation in this species to inform the best time for applying hormonal treatments before systematically optimizing hormone doses and determining appropriate life stages for hormonal masculinization as well as observing sexual morphology, behavioural interactions, and reproductive fitness of neo-males. Firstly, the study identified the window when sex differentiation occurred in G. holbrooki to determine the best time for hormonal induction in this species. The embryonic stages (segmentation, pharyngula and hatching) and juveniles from 0 to 150 days after parturition (DAP) were sampled for gonadal histology. Morphometrically, the results showed that there was no significant difference in cell diameters of PGC (t-test, t = 1.71, df = 23, p = 0.1) and gonocytes (t-test, t = 0.46, df = 14, p = 0.66) between males (PGC, 13.9 ± 1.2 μm; gonocytes, 11.7 ± 1.1) and females (PGC, 14.7 ± 1.3 μm; gonocytes, 11.9 ± 1.2). However, significant differences in cell diameters between PGC, gonocytes, oogonia and spermatogonia were detected (one-way ANOVA, F (3, 85) = 113.3, p < 0.0005, ƞ\(^2\) = 0.8). There were significant decreases in size of germ cells during their development (Tukey post hoc analysis). Cell diameter decreased from PGCs (14.25 ± 1.37 μm), to gonocytes (11.81 ± 1.14 μm), to oogonia (9.16 ± 0.94 μm), to spermatogonia (8.27 ± 1.5 μm), in that order. Several migrating PCG, were clearly distinguished from the somatic cells in the somatic layer at the mid-segmentation stage. However, they have not yet coalesced into a nest. At late somitogenesis, PGC nested and formed gonadal primordia before becoming undifferentiated gonads with two distinct clusters of gonial germ cells attached to the epithelial swim bladder. In female embryos, the first signs of ovarian differentiation, e.g., the presence of mitotic oogonia and chromatin-nucleolus oocytes were found in early pharyngula stage. In males, the gonads of newly born juveniles were sexually indifferent and the morphological differentiation became apparent only about 10 DAP with the appearance of spermatogonia in clusters of more than 4 cells enclosed by Sertoli precursor cells and stromal cell aggregations. All the information related to timing of sex differentiation and gonad development was used to design the following sex reversal experiments. Sex reversal experiments administering 17α-Methyltestosterone (MT) via feed targeted three life stages separately: (i) adults, (ii) 30-day old juveniles and (iii) neonates. The concentrations of MT tested at adult stages (re-maturing and maiden females) ranged between 50 to 200 mg MT/kg feed. The treated females were fed control or respective MT incorporated feed to satiation for 50 days. Effects of the hormone on external sexual morphology (gonopodial development, internal gonad morphology), expression of the Anti-Müllerian Hormone (amh) gene and sexual behaviours of the treated re-maturing and maiden females were observed. Specifically, the treated females were anesthetized, and their anal fins photographed every seven days (from 0 to 50 days of the treatment period) for observing the effects of MT on the development of gonopodia. At the end of the 50-day treatment period, the maiden females in the 50 mg MT/kg diet group showing the most advanced gonopodial development were used to observe MT effects on sexual behaviour and the remaining females were euthanized to determine MT effects on gonad morphology and expression of the amh. The results indicated that MT at the dose of 50 mg/kg feed stimulated sexual character development, upregulated expression of amh, formation of testicular tissue and a change in the behaviour of the treated maiden females. Like adult females, MT enriched feed at concentrations ranging from 5 to 40 mg/kg diet was administered to two different life stages: 30-day old juveniles and neonates. The experiment of 30-day old juveniles included one control and four MT treatment doses (10, 20, 30, 40 mg/kg diet). At 150 DAP, there was no significant difference in survival rates of juveniles fed on MT treatment and control diets (Kruskal-Wallis H test, F = 3.05, df = 4, 20, p = 0.55). The gonadal sex ratio in the control group was not significantly different from the expected 50: 50 sex ratio (Chi-square goodness of fit, χ\(^2\) = 3.26, df = 1, p = 0.07), although the gonad sex ratios of the treatment groups of 10, 20, 30 and 40 mg/kg diet were statistically different from that of the control. The percentages of neo-males were significantly different between MT treatment groups (Kruskal-Wallis H test, χ\(^2\) = 10.44, df = 4, p = 0.034). Post hoc tests revealed significant differences in the median percentages of neo-males between control group (0 %) and 10 mg MT/kg diet group (40 %, p = 0.04), but not between any other group combinations. Therefore, the lowest MT dose (10 mg/kg diet) was the best dose rate for MT treatment on 30-day old juveniles. In the experiment of MT treatment on neonates, there were one control and three MT treatment doses (5, 10, 20 mg/kg diet). Clutches of neonates were fed respective MT enriched or control spirulina. The hormonal treatments started at 1 DAP and lasted for 30 days during which the neonates were fed twice daily to satiation. At 150 DAP, there was no significant difference in survival rates of neonates fed on MT treatment and control diets (Kruskal-Wallis H test, F = 5.7, df = 3, p = 0.13). The body morphology sex ratio in the control groups was not significantly different from the expected 50: 50 sex ratio (Chi-square goodness of fit, χ\(^2\) = 0.23, df = 1, p = 0.63). There was no significant difference in the sex ratios based on body morphology between the treatment and control groups. Intersex females were found in the 20 mg MT/kg diet group, whereas no testicular tissue was seen on the gonads of fish in the other treatments (5 and 10 mg MT/kg diet) or control groups. The percentages of neo-males out of phenotypic males were significantly different between various MT treatment groups (One-way ANOVA, F = 5.1, df = 3, 14, p = 0.014, ƞ\(^2\) = 0.52). Tukey post hoc analysis revealed that the percentages of neo-males in 5 mg MT/kg group (45.9 ± 22.2 %) and 10 mg MT/kg group (51.1 ± 26.6 %) were statistically significant to that of the control (0 %) (p = 0.28 and p = 0.14, respectively), but no other group differences were statistically significant. Therefore, the neo-males from 5, 10 and 20 mg MT/kg diet group were used to observe their gonopodial and gonad morphology. Sexual morphology of the neo-males was observed and compared thoroughly. Thirty wild males, five control males and 15 neo-males were anesthetized and their gonopodia were photographed for morphometric measurements. Nine normal males and ten neo-males were euthanized for histological observation and 3D gonopodial and testis reconstructions. The results showed that neo-males in the 20 mg MT/kg diet group possessed an under-developed gonopodium with no serrae and hooks, while neo-males in the two lowest doses (5 and 10 mg MT/kg diet) were stimulated to develop complete gonopodia. There were significant differences in the width ratio of fin rays 3 & 4 between neo-male groups, control males and wild males (Welch ANOVA, F (\({_3,}\) \(_{9.4}\)) = 14.4, p = 0.001) in which the width ratios in control males (2.54 ± 0.13) and wild males (2.76 ± 0.14) were significantly larger than those of neo-males of 5 and 10 mg MT/kg diet (2.2 ± 0.11 and 2.15 ± 0.09, respectively). The 3D simulation of the basal gonopodium was novel and shed light on the mechanism of transferring sperm packages from males to females in poeciliids. The results of histological observation and 3D reconstruction of testes determined that neo-males in the lowest dose group (5 mg MT/kg diet) developed funnel-shaped testes similar to testes found in control males and the structures of testicular lobes were comparable to those of control males. However, neo-males in the 10 and 20 mg MT/kg groups formed oval-shaped testes and abnormal development of testicular tissue. These observations demonstrated that MT at the dose of 5 mg/kg diet effectively sex reversed genetic females to phenotypic males with the complete development of gonopodia and testes as normal males. The neo-males in the 5 and 10 mg MT/kg diet groups showed well-developed gonopodia and were subsequently used to determine their behavioural interactions and reproductive fitness with normal virgin females. Behavioural interactions, reproductive fitness assessment and sperm packet expression were conducted on the two groups of neo-males (fed on 5 and 10 mg MT/kg diet) and one control group. Fish from control or neo-male groups (test fish) were individually paired with a virgin female (stimulus fish) and their behaviours were recorded for 60 minutes using an in-front camera. The footage was analysed to observe behavioural interactions of the test fish and stimulus fish. After recording behaviour interactions, each pair of a control male/neo-male and a virgin female was kept together in an aquarium until the signs of female pregnancy were evident or at least 6 months before the females were separated to observe reproductive fitness. The results indicated that there were significant differences in median numbers of mating attempts between control and neo-male groups (Kruskal-Wallis H test, χ\(^2\)\(_{(2)}\) = 6.6, p = 0.036). Post hoc tests revealed that there was a significant difference in median numbers of mating attempts between neo-males of 5 mg MT/kg diet (42) and neo-males of 10 mg MT/kg diet (17, p = 0.043), but no significant difference was found between the neo-male groups and control group (p = 0.17 and p = 1.0 respectively for the neo-males of 5 and 10 mg MT/kg diet groups). Within the 60 minutes of behavioural observations, most control males and neo-males did not illustrate any aggressive behaviours to virgin females. The number of aggressive behaviours by the virgin females increased over the time. In terms of reproductive fitness, 60 % of virgin females paired with control males gave birth to young, while only 20% of virgin females paired with the neo-males of 5 mg MT/kg diet group parturated to immature neonates all of which died one day later. No virgin females paired with the neo-males of 10 mg MT/kg diet group gave birth or showed signs of pregnancy. Like control males, spermatozeugmata could be expressed from all neo-males in the 5 mg MT/kg treatment, but these sperm packets could be squeezed from only 55.6% of neo-males of 10 mg MT/kg diet group. The observations indicated that MT at the dose of 5 mg/kg diet effectively sex reversed genetic females to phenotypic males with behaviour interactions and production of sperms packets as normal males, but the results for their reproductive fitness was uncertain because of time limitation of the PhD project. Therefore, further investigations of the reproductive fitness of neo-males in this group are warranted. In conclusion, masculinization of G. holbrooki was successfully achieved by using MT, although a 100 % male population could not be attained. The study established a protocol to masculinize this species at optimum dose of MT through oral administration to neonates (5 mg/kg diet). The protocol and information produced in this recent study provides key knowledge towards producing functional neo-males for developing the TSC strategy to control and eradicate this pest species. The results presented in this thesis will also benefit other studies on fish sex differentiation especially in live-bearing species as well as research on biology, ecotoxicology and pest fish management.

Research Doctorate - Doctor of Philosophy (PhD)
The myriad of concurrent threats driving species declines make it difficult for managers to identify causal agents and preserve threatened populations. The decline of the green and golden bell frog (Litoria aurea) in Australia is an example of how multiple threatening agents such as the novel pathogen, Batrachochytrium dendrobatidis (Bd), invasive mosquitofish Gambusia holbrooki, and habitat degradation, can act independently or synergistically to rapidly decimate populations and prove difficult to manage. The broad focus of this study was to assess the impact of these threats on L. aurea, with the aim of improving current management techniques. Multi-state mark recapture analysis of three years of monitoring data from an extant and released population of L. aurea revealed that Bd significantly reduced survival probability, even within a habitat where the frogs persist. Analysis of data from the released population suggests the severity of Bd was affected by host age and condition, temperature, density, and food availability, and that both nutrition and Bd likely contributed to a lack of reproduction. Increased salinity may be tool for passively reducing Bd severity, as frogs used waterbodies of 3 parts per thousand (ppt), but care needs to be taken as salinities above 5 ppt elicited avoidance behaviour. Generalised linear models showed a marked separation between G. holbrooki and tadpole occupied ponds with a lack of avoidance by adult frogs. These patterns suggest that adults are naïve to G. holbrooki and the fish likely reduces recruitment through larval predation. A laboratory experiment identified few synergistic effects of Bd and G. holbrooki on tadpoles, although we argue that this may change in the wild. This thesis affirms the negative impact Bd and G. holbrooki have on L. aurea, and suggests why our study population persists in the face of these threats. We highlight factors that can improve in situ conservation projects of this nature, such improvements to habitat that may mitigate Bd, the importance of habitat that promotes a healthy abundance of prey species to supply nutrients for immune function and breeding, the importance of excluding G. holbrooki from protected sites, and the importance of habitat close enough to extant sites or large enough to support dispersal behaviours; all potentially achievable goals that may greatly benefit many threatened amphibians.

This study investigated interactions between native Galaxias maculatus and invasive Gambusia holbrooki, from the freshwater and tidally influenced wetlands of the kanamaluka/Tamar River system in cool temperate northern Tasmania. Galaxias maculatus is a valued species for human consumption and as a member of the ecosystem, and Gambusia is the target of an eradication program since its illegal introduction into the area in 1992, due to its detrimental impacts on the native habitat and the native fish and frog fauna. In addition to manual removal methods, the approach being developed (Trojan Y) aims to eradicate the population through the release of genetically manipulated Trojan Y fish which, through breeding with wild fish, would bias the sex ratio of the population to males. To maximise efficacy, we need to understand the behaviour of released fish and how they interact with wild fish. This study examines sublethal effects of predation and competition, including changes in habitat use, social interaction, and behaviour, with implications for interrupted feeding, potentially decreasing tolerance and raising vulnerability to other predators. Cover was chosen for the main habitat variable as it is important in the field and often used in habitat restoration. As these effects can have as strong an impact on the affected species’ fitness as lethal effects, this study will inform G. maculatus management and G. holbrooki eradication, including providing a baseline against which the behaviour of sex-reversed Trojan Y fish can be compared. To examine how sexual dimorphism affects G. holbrooki behaviour, all-male, all-female, and mixed sex groups of fish were tested in experimentally manipulated tanks for changes in aggression, schooling, and position in the water column. Male G. holbrooki were more aggressive, but there was more display behaviour in all-female groups. Fish were mostly solitary, which was more marked in males. Males used surface water less than females, even when in single sex groups. The effect of cover on social, aggressive, and protective behaviours was tested separately for each species. Galaxias maculatus had low rates of conflict, usually formed schools, and used cover regularly, which allowed them to increase swimming activity (swimming in and out of cover), presumably due to the protection it provided reducing the need to use relative immobility as a defence. Conversely, G. holbrooki rarely used cover, and both sexes were mostly solitary with high aggression towards other fish, with higher rates in males. The role of different levels of cover and G. holbrooki sex ratios were tested with the two species combined, focussing on interspecific aggression, avoidance, and co-existence. Gambusia holbrooki positioned themselves higher in the water column than G. maculatus when both species were present, similar to when each species was in mono-specific groups. Galaxias maculatus used cover less than without G. holbrooki, but still increased activity when cover was present. Aggression by G. holbrooki was lower than when in mono-specific groups, but the rate of aggression was not reduced by cover. The presence of G. holbrooki, especially females, greatly reduced G. maculatus schooling, while Galaxias aggression, both towards conspecifics and G. holbrooki, increased in the presence of G. holbrooki males. Social, aggressive, and defensive behaviours were examined for adult and immature G. maculatus to test if these behaviours changed with size in G. maculatus and whether there was greatervulnerability in smaller fish. It was found that behaviours were generally similar with size, with the only major difference being that large fish swam in small groups more often than small fish. Although interspecific aggression was much rarer than intraspecific aggression, G. holbrooki primarily affected G. maculatus behaviour by reducing schooling and cover use. In addition, G. maculatus increased intraspecific aggression when G. holbrooki was present, whereas G. holbrooki decreased their intraspecific aggression in the presence of G. maculatus. This research provides the most detailed baseline of behaviour in G. maculatus and G. holbrooki in for both monospecific groups and interspecific interactions; and adds to the understanding of the behaviour of these species in temperate conditions. This can be used to evaluate the behaviour of sex-reversed (Trojan Y) fish. Behavioural changes play a major role in G. maculatus impacts, suggesting that habitat restoration alone may not be sufficient to mitigate G. holbrooki impacts on G. maculatus. The observed higher intra- and lower inter-specific aggression in male G. holbrooki suggests that a male-biased ratio, a potential outcome of a proposed control program (Trojan Y), may be positive for native fish populations.

Primordial germ cells (PGC) are the progenitors of life; they develop into gametes and safeguard gonad function in sexually reproducing animals. By virtue of diverse reproductive strategies, fishes provide an invaluable field to study germ cell emergence and fate. The current knowledge of PGC development in live-bearing fish (poeciliids) is limited and instead it is predominantly restricted to well-studied oviparous teleost models such as zebrafish, Danio rerio and medaka, Oryzias latipes. To address this basic knowledge gap and to assist future development of species-specific reproductive biotechnologies to control invasive populations, this thesis focused on Gambusia holbrooki, a notorious pest fish. Beginning with a review of current knowledge, the developmental dynamics of germ cells in this species was evaluated employing the spatio-temporal expression of key germline markers and screening the morphological changes of gonads during the early stages of development. Also, this study discussed the potency of G. holbrooki as a model organism for fertility research, based on its developmental similarities with mammalian models. First, the study systematically reviewed the available literatures on PGC development, with a particular emphasis on teleosts, to synthesise evolutionary origin and mechanism of germ plasm (Gp) acquisition in fish taxa (Chapter 1). Specifically, the review identified two modes of germ cell development as distinguished by PGC specification of being either maternal (e.g., in teleost) or zygotic (e.g., in mammals) origin based on the role of epigenetic machinery in teleostean PGC emergence and fate. Further, prior knowledge of the interplay between molecular pathways and effector genes involved in germ cell signalling, mobilization and homing, and the regulatory RNA interference (RNAi) mechanisms leading to PGC specificity, their en routemigration and homing were distilled. These along with the role of non-coding RNAs (ncRNA), e.g., mi-RNA and pi-RNA, in germ line development was graphically illustrated to identify crucial roles and pathways that occur in fish PGC development. The contextual role of the nested germ cells in determining sexual fate of gonad and gonado-soma sexual identity via cell-cycle decisions and the timing of sex-biased germline stem cell (GSC) differentiation was also reviewed. This review formed the basis to address critical questions, including PGC specification mode, the pattern of germline migration, the temporal and spatial expression of key PGC markers during germline formation, and early gonadogenesis in G holbrooki. Spatio-temporal expression of germline markers is a powerful tool to investigate the processes of gonad formation and gametogenesis. Correspondingly, cloning, and characterisation of the target genes is a fundamental need when investigating non-model organisms whose genetic information is scarce and patchy. To address this, the full-length cDNA of the key germline markers including cxcr4, dazl, dnd, nanos1, piwi II and vasa in G. holbrooki were cloned and their identity established (Chapter 2). Standard rapid amplification of cDNA ends (RACE) techniques were used to establish testis and ovary specific libraries, followed by amplification of G holbrooki homologues using degenerate primers. Subsequently, the full-length cDNAs were cloned, characterised, the functional domains annotated and their evolutionary relationship to other poeciliid homologues established. The Dazl exhibited RNA recognition motif (RRM) and poly(A) binding protein (PABP) domains, while Deadend (Dnd) was encoded by two alternatively spliced variants, with conserved RRM and DSRM-DND domains in the predicted polypeptide. As expected, Piwi II had PAZ domain contained nucleic acid-binding interface in N-terminus and 5′ RNA guide anchoring site at C-terminus. DEAD-box helicase motif was also conserved in predicted Vasa containing RNA- and ATP-binding sites. Similarly, chemokine binding site and zinc-finger motif in predicted Cxcr4 and Nanos1, respectively were present. Overall, the predicted amino acids of the studied genes showed a strong similarity with respective homologues of other poeciliids. In teleosts with preformation mode of PGC specification, maternal inheritance of Gp provides sufficient factors to form the germline fate of nascent PGCs protecting their identity, in contrast to the surrounding soma, right from post zygotic activation. Later, during embryonic patterning and cell determination, the Gp components are involved in proliferation, migration, and colonisation of germ cell precursors. To understand how and when PGC are formed, migrate, and colonise putative gonad in G holbrooki and how well these events are conserved across teleosts, in situ hybridisation and quantitative expression profiles of five Gp markers, namely dazl, dnd1, nanos, piwi II and vasa were studied (Chapter 3). Based on expression of PGC markers, a group of Gp components was detected in the ovum suggesting maternal inheritance mode of PGC specification in G. holbrooki. Subsequent to fertilisation, the results showed that vasa positive cells were detectable from late cleavage, later forming as two PGC clusters at the early gastrula stage, migrating anteriorly during somitogenesis and eventually colonised at the genital ridge before the pharyngula stage. Of all the markers only vasa and a dnd variant signals were restricted to PGCs. The embryonic expression of piwi II, dazl dnd-β were detected in PGC clusters as well as the central nervous system. Interestingly, the embryonic expression of nanos1an indispensable PGC marker in zebrafish, was only found in neural tube. The quantitative gene expression profiles showed a transient female-biased surge of the PGC markers at the gastrula stage, suggesting an early onset of PGC proliferation in females. Strictly zygotic expression pattern of dnd-β and nanos1 also showed maternal to zygotic transition (MZT) occurs before the mid-cleavage stage, in G. holbrooki. Collectively, the results suggest that despite sharing preformation mode of PGC specification, their migration pattern in G. holbrooki is unlike any other fish species studied so far. Instead, somatic expression of teleostean PGC markers (e.g., piwi II, nanos1 and dazl), splicing variation (e.g., dnd) and early MZT appear more similar to those of mammalian models. Gp is an electron dense body in ooplasm involved in germline determination of organisms with preformation mode of PGC specification including teleost. However, the expression patterns of Gp factors during gonadogenesis and their storage in gametes of poeciliids are unknown. To address this, quantitative gene expression of six markers of Gp components namely cxcr4, dazl, dnd-α, piwi II, tdrd6 and vasa, with essential roles in gonad function among vertebrate models were evaluated for their expression profile in pre- (i.e., the pharyngula stage and right before parturition) and post-natal (i.e.,juvenile, and adult phase) stages of genetic male and female G. holbrooki (chapter 4). The overall results showed the relative accumulation of Gp components were comparable in prenatal stages of gonadogenesis between genetic males and females. However, their post-natal expression in gonads was female-biased (p<0.05), particularly as the animal reached puberty. Interestingly, the existing Gp transcripts and their abundance showed dissimilar patterns between ovum and spermatozoa of G. holbrooki. Collectively, this study, for the first time, revealed the Gp components involved in early germline determination are detectable not only in ovum, but also in mature spermatozoa indicating a potency for the contribution of paternal factors in PGC specification and function. Gonadogenesis is triggered with successful germ cell colonisation and gonadosoma emergence at the genital ridge followed by sexual identity acquisition through sex determination mechanisms or environmental cues. Among vertebrates, teleosts are known to exhibit diversified plans of gonadogenesis and sex differentiation, however, little is known in poeciliids. To ascertain when and what cellular and molecular determinants trigger sex differentiation in G. holbrooki, this study (Chapter 5) also documented critical events during early gonadogenesis using gonad histology, in situ hybridisation of germ cell markers and quantitative expression of gonadosoma markers. The histological observations showed that germ cells were first colonised at the genital ridge prior to complete somitogenesis to form presumptive gonads. Thereafter, germ cells underwent mitotic proliferation before acquisition of their sexual identity and this pattern was sex-dimorphic in G. holbrooki. Moreover, it was shown that sex differentiation of early gonads in G. holbrooki occurs right before parturition evinced from emerging meiotic primary oocytes stage I in ovary and formation of spermatogonial stem cell cysts in testis. Gonad histology and germ cell labelling showed that presumptive gonad in G. holbrooki undergoes major morphological transformation during its early development. Specifically, presumptive gonads first developed as two distinctive lobes, however, the lobes grew convergently and fused around the parturition stage to form single lobed testis or ovary, as occurs in adults. The quantitative expression pattern of a group of gonadosoma markers, foxl2, cyp19α, amh, dmrt1, in pre- and post-natal developmental stages was consistent with morphological changes in early gonad; they were activated at the onset of gonad formation followed by sex-dimorphic expression pattern concurrent with sex differentiation of gonads. In conclusion, this study enlightened aspects of PGC development, their developmental dynamics and events involved in gonadogenesis of an invasive poeciliid fish, G. holbrooki. In concurrence with its ovo-viviparity, it also revealed resemblance between some aspects of its PGC development and those of mammalian models. Interestingly, the study for the first time provided evidence for “paternal origin” of Gp markers, suggesting that both parents contribute to germ line specification in this species, an aspect that might be shared by all teleosts. Moreover, the critical timepoints in gonadogenesis i.e., germ cell colonisation and sex differentiation were ascertained, with further evaluation of the sex-specificity of gonadosoma markers. In doing so, the study generated resources, reagents, and established germline specificity of PGC markers that can assist targeted ablation of germ cell function to produce sterile or sex reversed animals as may be desired to engineer genetic control options to manage this pest species. The shared features of PGC development with both of oviparous and viviparous vertebrates, suggest the species could serve as a key model to study vertebrate reproduction and reproductive conditions such as sterility.

Tese de doutoramento em Biologia (Ecologia) apresentada à Fac. de Ciências e Tecnologia da Univ. de Coimbra
A aplicação do surfactante Genapol OXD-080, não-iônico e biodegradável, nos arrozais, tem sido considerado como parte de uma abordagem ecotecnológica para mitigar os danos causados pelo lagostim vermelho da Louisiana, Procambarus clarkii, na produção de arroz. O Genapol OXD-080 apresenta-se como menos tóxico que os pesticidas (ex: paratião) comumente usados, embora ilegalmente, pelos orizicultores para controlar a actividade do lagostim nos campos de arroz. De uma forma geral, os objectivos de tal abordagem consistem em auxiliar a cultura do arroz e, simultaneamente, permitir a produção de lagostim não contaminado nos arrozais, proporcionando benefícios para ambas as actividades sem afectar significativamente outras populações animais. Por esta razão, as relações entre um peixe predador chave, a Gambusia (Gambusia holbrooki), e as suas principais presas foram estudadas no âmbito da cadeia trófica dos arrozais. O conhecimento da influência de certos factores ambientais e biológicos nestas interacções tróficas, em combinação com a informação relativa ao ciclo de vida e dinâmica populacional de G. holbrooki, bem como relativa à; determinação quantitativa dos efeitos do Genapol, permitiram avaliar (por intermédio de um modelo) a viabilidade ecológica da utilização deste tipo de surfactantes no controlo das populações de lagostim nos arrozais.