The dermestid beetle (Dermestes maculatus) has become renowned in the laboratory as an aid to skeletonization of remains, both for the zoologist and the anthropologist. However, little attention has been paid to the potential effects these insects can have on hard tissues and whether their traces may be mistaken for trauma or the effects of taphonomic processes. The use of dermestid beetles in the anthropology laboratory was tested by a choice experiment based on ASTM D3345-17, examining the behavior of D. maculatus regarding Styrofoam, wood, and two types of bone. Specifically, this project tested 1.) whether the beetles showed preference for a particular material (of wood, Styrofoam, dry bone, or green bone), 2.) whether material(s) had an impact on survivorship from the larval to adult stages, and 3.) what traces dermestids leave on skeletal remains and the variation in form of those traces. Results suggest dermestid beetles will preferentially bore pupal chambers in softer materials (Styrofoam > wood > dry bone > green bone), but preference is not absolute, as pits appear in dry bone even when softer materials are present. Preference did not appear to impact survivorship. Information on the “typical” form of dermestid trace on materials is presented, as an aid to identifying these features as resulting from taphonomic processes, as opposed to ante- or perimortem processes/events.

De par leur diversité de mécanismes de déterminisme du sexe et de chromosomes sexuels, les poissons téléostéens représentent d’excellents modèles pour mieux comprendre les bases moléculaires et évolutives du contrôle du développement sexuel chez les vertébrés. Grâce à l’analyse de chromosomes artificiels bactériens couvrant les chromosomes sexuels du platy Xiphophorus maculatus, trois copies d’un nouveau gène nommé teximY ont été découvertes dans la région de déterminisme du sexe du chromosome Y mais pas du chromosome X. Un gène texim1 très apparenté à teximY ainsi que trois gènes plus divergents ont été identifiés sur les autosomes. Les gènes teximY sont préférentiellement exprimés dans les testicules, au niveau des cellules germinales lors des étapes tardives de la spermatogénèse, alors que texim1 est également transcrit dans les gonades femelles. Des gènes texim ont été détectés chez d'autres poissons téléostéens mais pas chez le poisson-zèbre, ainsi que chez des céphalocordés, des urocordés et des échinodermes mais pas chez les tétrapodes. Les gènes texim codent pour des estérases putatives à domaine SGNH apparentées à des protéines cellulaires procaryotes et eucaryotes ou codées par des retrotransposons animaux. Les gènes texim sont associés à des transposons Helitron chez les poissons mais pas chez les autres animaux, suggérant capture et mobilisation du gène ancestral texim par un transposon à la base de la radiation des téléostéens. TeximY pourrait jouer un rôle dans la transposition du transposon Helitron dans la lignée germinale mâle, ou correspondre à un gène de spermatogenèse mobilisé par le transposon Helitron sur les nouveaux chromosomes sexuels de poissons. / The molecular and evolutionary basis of sex determination in vertebrates needs to be unveiled via comparison of different systems. Fish exhibit hypervariability of sex determination mechanisms. Thanks to the analysis of the Bacterial Artificial Chromosome (BAC) library covering the sex chromosomes of the platyfish Xiphophorus maculatus (Rio Jamapa population, XX /XY), three copies of a new gene have been identified in the sex-determining region of the Y but not the X chromosome, and named teximY. Four autosomal counterparts of teximY have been also detected in the genome of the platyfish with one of them, texim1 presenting 95% of cDNA sequence identity with the Y-linked copies. RT-qPCR expression analyses have been performed for each copy in male and female tissues. Two Y-linked teximY copies were preferentially expressed in testis, whereas the autosomal copy texim1 showed preferential expression in male and female gonads. In situ hybridizations with a teximY/1 probe revealed expression in late spermatids and spermatozeugmata. Texim sequences were detected in several fish species, but not in zebrafish, as well as in cephalochordates, urochordates and sea echinoderms but not in tetrapods. Predicted Texim proteins are related to proteins from different origins. Interestingly, texim genes are associated with a Helitron transposon in fish but neither in cephalochordates nor in echinoderms, suggesting capture and mobilization of an ancestral texim gene at the base of the bony fish lineage. TeximY proteins may play a role in Helitron transposition in the male germ line in fish, or texim genes are spermatogenesis genes mobilized and spread by transposable elements in fish genomes.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-09-14T11:46:46Z No. of bitstreams: 1 2011 - Vivian Suane de Freitas Vieira.pdf: 2001045 bytes, checksum: 2cce4947186746927e3f6cc35826b411 (MD5) / Made available in DSpace on 2016-09-14T11:46:46Z (GMT). No. of bitstreams: 1 2011 - Vivian Suane de Freitas Vieira.pdf: 2001045 bytes, checksum: 2cce4947186746927e3f6cc35826b411 (MD5) Previous issue date: 2016-01-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / A list of metazoan species associated to Pimelodus Lac?p?de, 1803 genus has been prepared in this work. The total of 516 records in 10 host species of genus was compiled. Were included in the list of new geographic records monogeneans parasites of Pimelodus maculatus Lac?p?de, 1803 collected from Midwest, Southeast and South of Brazil. New morphological data of Cucullanus pinnai pinnai Travassos, Artigas & Pereira, 1928, parasite of P. maculatus collected in the Guandu river, in the State of Rio de Janeiro have been reported. This work noted that the position of the excretory pore and deirids of the specimens are at the level or posterior to the oesophagus- intestine junction in the most specimens and gubernaculums size is greater than in other previously studied of C. pinnai pinnai. Nematodes were studied using light and scanning electron microscopy (SEM) and differential interference contrast (DIC) and analyzes confirmed morphological details of the cephalic region and the caudal papillae that species. Comparative study of parasitic communities of P. maculatus was also performed. 50 specimens of the P. maculatus were collected from Guandu river, in the State of Rio de Janeiro and others 50 from Mogi-Gua?? river, State of S?o Paulo. In the samples from Guandu river were identified the following species: Ameloblastella paranaensis Fran?a, Isaac, Pavanelli & Takemoto, 2003 (P= 8%; MI= 1,0?0,0); A. satoi Monteiro, Kritsky, Brasil-Sato, 2010 (P= 8%; MI= 1,0?0,0); Demidospermus bidiverticulatum Suriano & Incorvaia, 1995 (P= 8%; MI= 1,0?0,0); D. paravalenciennesi Guti?rrez & Suriano, 1992 (P=90%; MI=14,77?5,04), D. uncusvalidus Guti?rrez & Suriano, 1992 (P=90%; MI=12,37?4,86), D. valenciennesi Guti?rrez & Suriano, 1992 (P=56%; MI=6,06?3,95) e Cucullanus pinnai pinnai Travassos, Artigas & Pereira, 1928 (P=64%; MI=3,28?4,87). In the rio Mogi-Gua?? the parasites identified were: Ameloblastella paranaensis (P= 8%; MI= 1,0?0,0), Ameloblastella satoi (P= 8%; MI= 1,0?0,0), Demidospermus paravalenciennesi (P=86%; MI=18,58?10,79), D. uncusvalidus (P=84%; MI=16,73?7,74), D. valenciennesi (P=52%; IM=6,84?4,55) e Cucullanus pinnai pinnai (P= 44%; MI= 3,88?4,87). The females of P. maculatus had higher weight and length of males in both samples. The sex and o length of the hosts did not influence the level of parasitism. Positive correlation was observed between weight of host and abundance of D. uncusvalidus only in the sample of the Guandu river. Aggregate distribution was observed in all the prevalent species of metazoan parasites of P. maculatus in the two localities. D. paravalenciennesi showed a higher frequency of dominance and relative dominance in the samples of Guandu river and D. uncusvalidus in the samples of Mogi-Gua?? river. The results of the qualitative similarity index Jaccard (CJ) and quantitative Sorensen (CS) within the collected samples were homogenea. The Jaccard index indicated heterogeneity while Sorensen index indicated homogeneity between samples. The results demonstrated that the relationship between parasites and their hosts are constant even in different river basins. / Uma lista de esp?cies de metazo?rios associados ao g?nero Pimelodus Lac?p?de, 1803 foi elaborada neste trabalho. O total de 516 registros em 10 esp?cies de hospedeiros do g?nero foi compilado. Foram inclu?dos na lista novos registros de geogr?ficos de parasitos monogen?ticos de Pimelodus maculatus Lac?p?de, 1803 coletados nas regi?es Centro-oeste, Sudeste e Sul do Brasil. Novos dados morfol?gicos de Cucullanus pinnai pinnai Travassos, Artigas & Pereira, 1928, parasito de P. maculatus coletado no rio Guandu, no Estado do Rio de Janeiro foram relatados. Foi observado que a posi??o do poro excretor e deir?deos nos esp?cimes estudados s?o posteriores ao final do es?fago na maioria dos esp?cimes e o tamanho do gubern?culo ? maior do que em outros de C. pinnai pinnai estudados anteriormente. Os nematoides foram estudados utilizando microscopia eletr?nica de varredura (MEV) e contraste interferencial de fase (DIC) e as an?lises confirmaram detalhes morfol?gicos da regi?o cef?lica e as papilas caudais dessa esp?cie. Estudo comparativo das comunidades parasit?rias de P. maculatus tamb?m foi realizado. Foram coletados 50 esp?cimes de P. maculatus no rio Guandu, no Estado do Rio de Janeiro e outros 50 exemplares no rio Mogi-Gua??, no Estado de S?o Paulo, foram examinados para o estudo de sua parasitofauna, no per?odo de maio ? dezembro de 2012. No rio Guandu foram coletadas as seguintes esp?cies: Ameloblastella paranaensis Fran?a, Isaac, Pavanelli & Takemoto, 2003 (P= 8%; IM= 1,0?0,0); A. satoi Monteiro, Kritsky, Brasil-Sato, 2010 (P= 8%; IM= 1,0?0,0); Demidospermus bidiverticulatum Suriano & Incorvaia, 1995 (P= 8%; IM= 1,0?0,0); D. paravalenciennesi Guti?rrez & Suriano, 1992 (P=90%; IM=14,77?5,04), D. uncusvalidus Guti?rrez & Suriano, 1992 (P=90%; IM=12,37?4,86), D. valenciennesi Guti?rrez & Suriano, 1992 (P=56%; IM=6,06?3,95) e Cucullanus pinnai pinnai Travassos, Artigas & Pereira, 1928 (P=64%; IM=3,28?4,87). No rio Mogi-Gua?? os parasitos identificados foram: Ameloblastella paranaensis (P= 8%; IM= 1,0?0,0), Ameloblastella satoi (P= 8%; IM= 1,0?0,0), Demidospermus paravalenciennesi (P=86%; IM=18,58?10,79), D. uncusvalidus (P=84%; IM=16,73?7,74), D. valenciennesi (P=52%; IM=6,84?4,55) e Cucullanus pinnai pinnai (P= 44; IM= 3,88?4,87). As f?meas de P. maculatus apresentaram peso e comprimento maiores que dos machos nas duas amostras. O sexo e o comprimento dos hospedeiros n?o influenciaram o n?vel de parasitismo. Correla??o positiva foi observada entre o peso e a abund?ncia de D. uncusvalidus somente na amostra do rio Guandu. Foi observada distribui??o agregada de todas as esp?cies mais prevalentes de metazo?rios parasitos de P. maculatus nas duas bacias de coleta. D. paravalenciennesi apresentou maior freq??ncia de domin?ncia e domin?ncia relativa no rio Guandu e D. uncusvalidus no rio Mogi-Gua??. Os resultados dos ?ndices de similaridade qualitativa de Jaccard (CJ) e quantitativa de Sorensen (CS) dentro das amostras coletadas demonstraram homogeneidade. O ?ndice Jaccard indicou heterogeneidade e o ?ndice de Sorensen indicou homogeneidade entre as amostras. Os resultados demonstram que o relacionamento entre parasitos e seus hospedeiros s?o constantes, mesmo em diferentes bacias hidrogr?ficas.

Made available in DSpace on 2014-12-17T14:03:30Z (GMT). No. of bitstreams: 1 RodrigoOA.pdf: 1265658 bytes, checksum: 99f4f1b083a5b39748d0fb4e05fcf79f (MD5) Previous issue date: 2009-02-06 / Chitin is an important structural component of the cellular wall of fungi and exoskeleton of many invertebrate plagues, such as insects and nematodes. In digestory systems of insects it forms a named matrix of peritrophic membrane. One of the most studied interaction models protein-carbohydrate is the model that involves chitin-binding proteins. Among the involved characterized domains already in this interaction if they detach the hevein domain (HD), from of Hevea brasiliensis (Rubber tree), the R&R consensus domain (R&R), found in cuticular proteins of insects, and the motif called in this study as conglicinin motif (CD), found in the cristallography structure of the β-conglicinin bounded with GlcNac. These three chitin-binding domains had been used to determine which of them could be involved in silico in the interaction of Canavalia ensiformis and Vigna unguiculata vicilins with chitin, as well as associate these results with the WD50 of these vicilins for Callosobruchus maculatus larvae. The technique of comparative modeling was used for construction of the model 3D of the vicilin of V. unguiculata, that was not found in the data bases. Using the ClustalW program it was gotten localization of these domains in the vicilins primary structure. The domains R&R and CD had been found with bigger homology in the vicilins primary sequences and had been target of interaction studies. Through program GRAMM models of interaction ( dockings ) of the vicilins with GlcNac had been gotten. The results had shown that, through analysis in silico, HD is not part of the vicilins structures, proving the result gotten with the alignment of the primary sequences; the R&R domain, although not to have structural similarity in the vicilins, probably it has a participation in the activity of interaction of these with GlcNac; whereas the CD domain participates directly in the interaction of the vicilins with GlcNac. These results in silico show that the amino acid number, the types and the amount of binding made for the CD motif with GlcNac seem to be directly associates to the deleterious power that these vicilins show for C. maculatus larvae. This can give an initial step in the briefing of as the vicilins interact with alive chitin in and exert its toxic power for insects that possess peritrophic membrane / A quitina (homopol?mero linear contendo res?duos de β-1,4-N-acetil-D-glicosamina (GlcNac) ? um importante componente estrutural da parede celular de fungos e exoesqueletos de muitos invertebrados pragas, tais como insetos e nemat?ides. Em sistemas digest?rios de insetos forma uma matriz denominada de membrana peritr?fica. Um dos mais estudados modelos de intera??o prote?na-carboidrato ? o modelo que envolve as prote?nas ligantes ? quitina. Dentre os motivos j? caracterizados envolvidos nesta intera??o se destacam o motivo heve?na (HD), obtida de Hevea brasiliensis (Seringueira), o motivo R&R consenso (R&R), encontrado em prote?nas cuticulares de insetos, e o motivo denominado neste estudo como motivo conglicinina (CD), encontrado na estrutura cristalogr?fica da β-conglicinina complexada com GlcNac. Estes tr?s motivos de liga??o ? quitina foram usados para determinar qual(is) deles poderia(m) estar envolvido(s) in silico na intera??o das vicilinas de Canavalia ensiformis e Vigna unguiculata com quitina, como tamb?m associar estes resultados com o WD50 destas vicilinas para larvas de Callosobruchus maculatus. A t?cnica de modelagem comparativa foi utilizada para constru??o do modelo 3D da vicilina de V. unguiculata, que n?o foi encontrada nos bancos de dados. Atrav?s do programa ClustalW obteve-se a localiza??o destes dom?nios na estrutura prim?ria das vicilinas. Os dom?nios R&R e CD foram encontrados com maior homologia nas seq??ncias prim?rias das vicilinas e foram alvos de estudos de intera??o. Atrav?s do programa GRAMM foram obtidos modelos de intera??o ( dockings ) das vicilinas com GlcNac. Os resultados mostraram que, atrav?s de an?lises in silico, o motivo HD n?o faz parte da estrutura das vicilinas, comprovando o resultado obtido com o alinhamento das seq??ncias prim?rias; o motivo R&R, apesar de n?o ter semelhan?a estrutural nas vicilinas, provavelmente tem uma participa??o na atividade de intera??o destas com GlcNac; enquanto que o motivo CD participa diretamente na intera??o das vicilinas com GlcNac. Estes resultados in silico mostram que o n?mero de amino?cidos, os tipos e a quantidade de liga??es feitas pelo motivo CD com GlcNac parecem estar diretamente associados ao poder delet?rio que essas vicilinas possuem para larvas de C. maculatus. Isso pode constutuir um passo inicial na elucida??o de como as vicilinas interagem com quitina in vivo e exercem seu poder t?xico para insetos que possuem membrana peritr?fica

Divergence in the face of gene flow is perhaps the most wildly disputed subject among researchers through time. The debate is an old one and we find its origin as far back as the era of Darwin. The theories dealing with sympatric and parapatric speciation, its processes and ecological conditions, are numerous and the empirical data supporting the ideas is constantly growing. However, the reach of a consensus almost seem as distant as ever. Two fundamental prerequisites can be identified for the evolution of divergence with gene flow, the act of disruptive selection, and the development of assortative mating. A set of models in which speciation with gene flow seem particularly likely is when a shift occurs in host preference in phytophagous insects and mating takes place on the host. In the work behind this thesis, the role of assortative mating in the initial stages of sympatric and parapatric speciation has been studied, as has the interaction between assortative mating and inbreeding and how it effects speciation in small sympatric populations, an aspect not much attended to earlier in the literature. My results show that assortative mating based on resource preference, can evolve rapidly upon secondary contact, and even in parapatric populations with a migration rate of 8% (13-15 individuals) per generation. However for assortative mating to be maintained selection against hybrids is needed. My results also suggests that small inbred populations have a hard time coping with strong assortative mating an as a consequence tend to relax their mating preferences to avoid inbreeding depression. Based on these results, I advocate for the importance of considering not only assortative mating in itself, but also the joint effects of assortative mating and inbreeding when dealing with theories of speciation with gene flow.

Sexual conflict over mating duration drives the evolution of male and female adaptations that facilitate the manipulation of mating interactions in their favor. This conflict drives the evolution of traits that improve the fitness of the focal sex despite inflicting costs on mates. However, males can express multiple traits that increase and decrease female fitness simultaneously. When the effects of male traits on female fitness increase or decrease with duration of exposure, females traits that influence mating duration are selected upon. Females of Callosobruchus maculatus, a bruchid bean beetle, kick mates to forcefully end copulation. Although both negative effects of male genital spines and positive of effects ejaculatory materials on female fitness have been documented, it is not yet clear how these male traits interact to influence the timing of female kicking. In this study, we observed the effect of male genital spine size, ejaculate size and mating history, and manipulated mating duration to disentangle the effects of male traits on the timing of female kicking behavior. We found that male mating history and mate body size dimorphism predicted the timing and duration of female kicking, but that male ejaculate size and spine length did not predict female kicking timing.

Speciation is the process describing the formation of new species and is at the heart of evolutionary biology. According to the biological species concept only reproductively isolated forms are regarded good species. This thesis aims at identifying evolutionary processes that cause population divergence and, ultimately, speciation. Natural and sexual selection are two major candidates driving changes in traits that could render populations reproductively incompatible. In recent years, biologists have recognized that most animal species are polygamous. Therefore, sexual selection does not end at mating but continues to include interactions between individuals after mating has occurred but before zygote formation. Male-male sperm competition and cryptic female choice are two main forms of postmating sexual selection shaping reproductive traits like behaviour. The studies presented in this thesis focus on laboratory experiments attempting to identify the role of postmating sexual selection in causing reproductive divergence across populations in beetles. The majority of studies were performed using a Bruchid beetle, Callosobruchus maculatus, a common, worldwide pest on stored leguminose seeds. I used two major methodological approaches. One approach seeks to identify the traces left by past selection among extant conspecific populations, by assessing the pattern of female reproductive responses to mating with males of decreasing relatedness. Second, I used a selection experiment to disentangle the joint effect of natural and sexual selection acting simultaneously on diverging replicated selection lines. In general, these experiments revealed that postmating sexual selection can be a powerful engine of incipient divergence between allopatric populations. Changes in traits underlying variables such as female reproductive output, female mating rate or male success in sperm competition evolved rapidly and could in some cases effectively reduce gene flow between conspecific populations. While postmating sexual selection per se can drive divergence, I found that interactions with natural selection can limit divergence in reproductive characters. Sexual selection tended to reinforce natural selection under strong directional selection. In contrast, sexual selection inflicted a reproductive load on populations under weak natural selection. Thus, the joint effects of natural and sexual selection on allopatric populations are non-trivial and should be considered in greater detail in future studies of early divergence.

Inanga (Galaxias maculatus) is an amphidromous fish species that is able to successfully inhabit a variety of salinities. Using an integrated approach this thesis has characterised for the first time the physiological characteristics that facilitate acclimation in inanga. Structural studies using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) revealed freshwater-acclimated inanga have a high density of apical pits and freshwater-type mitochondria-rich cells (MRCs) that can facilitate ion absorption from the hypo-osmotic environment. In seawater, inanga remodel their gills by increased proliferation of seawater-type MRCs to facilitate ion secretion in the hyper-osmotic environment. Concentration-dependent sodium (Na+) kinetic analysis revealed that at a whole body level, inanga regulate Na+ using a saturable, high affinity, low capacity uptake system which makes them extremely adept at extracting Na+ from very dilute freshwater environments. In fact inanga displayed an uptake affinity (Km) of 52 ± 29 µM, which is one of the lowest ever recorded in freshwater fish. The sodium/potassium ATPase transporter (NKA) is central to Na+ regulation within the gill. In high salinties inanga displayed increased NKA activity (6.42 ± 0.51 µmol ADP mg protein-1 h-1) in an effort to excrete the excess Na+, diffusively gained from the hyper-osmotic environment. This increase in NKA was most likely a reflection of the proliferation of NKA-containing MRCs. The NKA activities seen in freshwater- and 50% seawater-acclimated inanga were similar (2.54 ± 0.19 and 2.07 ± 0.22 µmol ADP mg protein-1 h-1 respectively) to each other suggesting the inanga gill is capable of supporting ion regulation in brackish waters without a significant increase in NKA activities, and the energetically-expensive changes in gill structure and function that accompany such a change. Molecular investigation of NKA isoform expression using quantitative PCR (qPCR) showed that inanga displayed salinity-induced changes in the expression of the three α NKA isoform variants investigated. Isoform α1a exhibited a pattern consistent with an important role in freshwater, confirming results from other fish species. While it is generally accepted that α1b isoform is the predominant NKA isoform in seawater, inanga did not display this pattern with a freshwater dominance seen. None of the salinity-induced changes could quantitatively explain the increased NKA activity in seawater suggesting that different isoforms may convey different activities, that there is also regulation of NKA at a post-transcriptional level, and/or other isoforms or subunits may have a significant role. The importance of the osmoregulatory hormone cortisol and prolactin is widely accepted and inanga were treated with cortisol, prolactin and a combination of the two in an effort to further elucidate their role. NKA activity and NKA isoform expression were assessed but no specific patterns were deduced, except for a decrease in both NKA activity and isoform expression in 100% seawater-acclimated inanga treated with cortisol and prolactin. The reasons for this decrease were not evident, although the impact of stress induced by the injection protocol was likely to be a confounding factor. The development of a new confocal-based technique in this study was able to describe, for the first time, intracellular sodium levels ([Na+]i) as a function of salinity in an intact euryhaline fish gill cell. Using the fluorescent Na+ indicator dye CoroNa Green this study demonstrated the ability of inanga gill cells to maintain [Na+]i in the face of environmental change. Freshwater-acclimated inanga displayed basal [Na+]i of 5.2 ± 1.8 mM, with 12 ± 2.3 mM and 16.2 ± 3.0 mM recorded in 50% seawater- and 100% seawater-acclimated cells, respectively. Low [Na+]i is advantageous in hypo-osmotic environments as it provides a gradient between the cell and the blood which is essential for generating electrochemical gradients cell volume regulation and other cellular homeostatic mechanisms. A slightly elevated [Na+]i seen at the higher sanities would help minimise the diffusive gradient for passive influx from the environment which would be of benefit in hyper-osmotic environments. Upon salinity challenge 50% seawater cells were equally adept at maintaining a constant [Na+]i at any salinity, suggesting these cells are have the necessary constituents to regulate Na+ in both lower and higher salinities. This novel LSCM approach is advantageous relative to existing transport models as it will allow the observation of cellular ion transport in real time, within a native filament structure displaying functional interaction of different cell types. The extreme ion uptake characteristics of the inanga and their amenability to in situ confocal-based studies demonstrated in this study, confirm inanga as a valuable model species for future research.

Salinity and oxygen availability have long been recognised as important factors influencing animal physiology and therefore species distribution. The maintenance of appropriate cellular ion levels is critical for many essential physiological processes, but at the same time is energetically expensive. Since hypoxia is likely to impose aerobic limitations for ATP generation, the maintenance of salt and water homeostasis could be at risk during hypoxia. The amphidromous inanga (Galaxias maculatus) is well known for its salinity tolerance and its life cycle that involves several salinity related migrations. During these migrations inanga also frequently encounters hypoxic waters, and therefore must maintain energy homeostasis when aerobic metabolism may be compromised. The present study has investigated behavioural, physiological, biochemical and molecular mechanisms by which inanga tolerate changes in salinity and hypoxia. After 14 days of acclimation to salinities ranging from freshwater to 43‰, inanga showed physiological acclimation. This was evident by no changes in metabolic rates or energy expenditures through this salinity range. Energy balance seemed to be tightly and efficiently controlled by changes in the proportion of protein and lipids used as energy substrate. No mortalities and only minor changes in plasma osmolality also indicated salinity acclimation. The remarkable osmoregulatory capacity of inanga was also evidenced after a seawater challenge. The osmotic balance of inanga was only disrupted during the first 24 hours after the challenge, evidenced by an increase in plasma osmolality and plasma Na+, and a decrease in muscle water content. These physiological changes were correlated with changes at the molecular level. Different isoforms of the catalytic subunit of the Na+,K+-ATPase (NKA) were isolated, partially sequenced and identified in inanga. Phylogenetic analysis grouped inanga isoforms (α-1a, α-1b, α-1c) with their respective homologues from salmonids. Patterns of mRNA expression were also similar to salmonids, with α-1a being downregulated and α-1b being up-regulated following seawater challenge. Previous to this study, NKA isoform switching was reported to occur only in salmonids and cichlids. The presence of NKA subunits that change with environmnetal salinity in inanga indicates that this isoform switching phenomenon is much more widespread among teleost lineages than previously thought. Aiming to elucidate the hypoxia tolerance of inanga, oxygen consumption rate as a function of decreasing external PO2 was evaluated. At no point did inanga regulate oxygen consumption, suggesting that this species is an oxyconformer. This is the first robust demonstration of the existence of oxyconforming in fish. Evaluation of the scaling relationship between oxygen consumption and fish size in normoxia, showed that the exponent of this relationship fell within the range previously reported for fish. However, in hypoxic conditions the scaling relationship was less clear suggesting different size-related mechanisms for tolerating hypoxia. Analysis of the aerobic and anaerobic metabolism of small and large fish, showed that smaller inanga were able to sustain aerobic metabolism for longer than larger inanga, which instead relied on anaerobic metabolism for extending their survival. This knowledge is likely to be of value for the conservation of this iconic fish species, by incorporating these size related differences in hypoxia tolerance in streams management. In light of the unusual oxyconforming response of inanga, a study examining the behavioural responses of this species to declining dissolved oxygen was performed. Inanga did not display a behaviour that might reduce energy expenditure during oxygen limitation; instead swimming activity and speed were elevated relative to normoxia. As hypoxia deepened inanga leaped out of the water, emersing themselves on a floating platform. Once emersed, fish exhibited an enhanced oxygen consumption rate compared to fish that remained in hypoxic water. Although this emersion behaviour was hypothesised to be of physiological advantage, both aquatic hypoxia and emersion resulted in similar physiological and biochemical consequences in inanga. While in hypoxic water oxygen availability seemed to be the limiting factor, in air failure of the circulatory system was hypothesised to be the cause of a similar metabolic signature to that found in aquatic hypoxia. Overall, inanga seemed to be not particularly well adapted to tolerate aquatic hypoxia. In light of the increasing likelihood of anthropogenic-induced hypoxia in inanga habitats, this is likely to have negative consequences for the future of inanga populations in the wild. Although this study provides the mechanisms behind the exceptional salinity tolerance of inanga, its susceptibility to hypoxia is likely to impose further constraints for the osmoregulatory processes that guarantee inanga survival during life cycle migrations. The results of the present study are relevant for understanding and managing the fishery of this economically- and culturally important fish species.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-07-25T12:33:24Z No. of bitstreams: 1 texto completo.pdf: 655443 bytes, checksum: 60ce546b9212c90a2aea5ec4beba51ec (MD5) / Made available in DSpace on 2016-07-25T12:33:25Z (GMT). No. of bitstreams: 1 texto completo.pdf: 655443 bytes, checksum: 60ce546b9212c90a2aea5ec4beba51ec (MD5) Previous issue date: 2015-12-15 / O feijão-caupi, Vigna unguiculata, (L.), Walp. é uma leguminosa de ampla distribuição mundial, encontrada principalmente nas regiões tropicais. Os grãos de feijão-caupi apresentam problemas sérios de perdas pós-colheita, grande parte ocorrendo em razão do armazenamento inadequado e do ataque por insetos-praga, destacando-se o Callosobruchus maculatus (Coleoptera: Bruchidae). O controle de C. maculatus em feijão-caupi armazenado é feito principalmente com o fumigante fosfina, no entanto, o uso em longo prazo de um único inseticida aumenta o risco do crescimento de populações resistentes. Como alternativa, tem sido pesquisado o uso de inseticidas de origem vegetal que têm relativa toxicidade a diversas espécies de insetos. Este trabalho foi conduzido com o objetivo de avaliar a atividade fumigante do óleo essencial de salsa, Petroselinum sativum, (Mill.) Fuss, sobre C. maculatus, em feijão-caupi. A toxicidade do óleo essencial e do gás fosfina (controle positivo) foi avaliada para estimar as concentrações letais de 50 e 95% (CL 50 e CL 95 ). As unidades experimentais foram constituídas por frascos de vidro com 0,8 L de capacidade, contendo 100 g de feijão-caupi e 20 insetos adultos com idade de 1 a 3 dias. O óleo essencial foi aplicado em recortes de papel colocados dentro de sachês de organza e colados nas tampas dos frascos. A toxicidade foi avaliada 48 h após a exposição dos insetos ao óleo essencial e à fosfina. A taxa instantânea de crescimento populacional dos insetos na presença do óleo essencial e da fosfina foi determinada utilizando as concentrações letais CL 10 , CL 30 , CL 50 , CL 70 e CL 90 obtidas nos bioensaios de toxicidade. O experimento foi montado no delineamento inteiramente casualizado com 5 repetições. A progênie adulta foi contabilizada após 45 dias. Os resultados da toxicidade indicaram CL 50 de 489,5 μL L -1 e CL 95 de 635,8 μL L -1 para o óleo essencial de salsa e CL 50 de 35,7 μL L -1 e CL 95 de 68,5 μL L -1 para a fosfina. Verificou-se que o óleo essencial de salsa apresenta efeito inseticida fumigante no controle de adultos de C. Maculatus e depende da concentração aplicada. Além disso, a perda de massa do feijão-caupi tratado com óleo essencial de salsa e com o gás fosfina foi menor em relação ao controle negativo (sem tratamento), indicando que a exposição dos insetos ao óleo essencial e ao gás fosfina reduziu o número de insetos de C. maculatus. O poder germinativo dos grãos de feijão- caupi foi diretamente proporcional às concentrações do óleo essencial e do gás fosfina. / Cowpea, Vigna unguiculata (L.) Walp. is a legume of worldwide distribution, mainly found in tropical regions. The cowpea grains has serious problems of post-harvest losses, largely occurring due to improper storage and attack by insect pests, highlighting the Callosobruchus maculatus (Coleoptera: Bruchidae). The control of C. maculatus in stored cowpea is done primarily with the phosphine fumigation, however, the long term use of a single insecticide increases the risk of resistant populations development. Alternatively, it has been investigated the use of insecticides of plant origin that have relative toxicity to various insect species. This work was carried out to evaluate the fumigant activity of the essential oil of parsley, Petroselinum sativum (Mill.) Fuss, on C. maculatus in cowpea. Toxicity of essential oil and phosphine gas (positive control) was evaluated to estimate the lethal concentrations of 50 and 95% (LC 50 and LC 95 ). The experimental units consisted of glass bottles with 0.8 liter capacity containing 100 g of cowpea and 20 adult insects aged 1-3 days. The essential oil was applied to paper cutouts placed inside organza sachets and glued to the bottle caps. Toxicity was assessed 48 h after exposure of the insects to the essential oil and phosphine. The instantaneous rate of growth of the insects in presence of the essential oil and phosphine gas was determined using lethal concentration LC 10 , LC 30 , LC 50 , LC 70 and LC 90 obtained in bioassays of toxicity. The experiment was conducted in a completely randomized design with 5 repetitions. The adult progeny was recorded after 45 days. The results of toxicity indicated 489.5 μL -1 to LC 50 and 635.8 μL -1 to LC 95 for essential oil of parsley and 35.7 μL -1 to LC 50 and 68.5 μL -1 to LC 95 for phosphine. It was found that the essential oil of parsley has a fumigant insecticidal effect in controlling adults of C. maculatus which effectiveness depends on the applied concentration. Moreover, the weight loss of the cowpea treated with essential oil of parsley and with phosphine gas was lower compared to the negative control (no treatment), indicating that exposure of the insects to the essential oil and phosphine gas reduced the number of C. maculatus. The germination of cowpea grain was directly proportional to the concentrations of the essential oil and phosphine gas.