Ex situ collection of Limonium spp.: cytogenetic and reproductive characterization for the conservation of rare species

Dissertação mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Reproductive biology of Macrobrachium surinamicum (Decapoda: Palaemonidae) in the Amazon River mouth

Macrobrachium surinamicum is an indigenous prawn distributed from the lower Amazon and Tocantins river basins to Venezuela in the Orinoco Delta region. It is common bycatch fauna of Macrobrachium amazonicum artisan fishing in the states of Pará and Amapá. The aim of this study was to investigate aspects on reproductive biology (reproductive period, size of sexual maturity population, fecundity, reproductive output and recruitment) of M. surinamicum from four important areas to artisanal prawn fishing located at the Amazon River mouth (Amapá and Pará). The specimens were captured using 20 handcrafted traps called "matapi". A number of 675 prawns were captured, 258 males, 409 females and eight juveniles, resulting in 1:1.6 (Male: Female) sex ratio. The reproductive peak period occurred from March to July, coinciding with the higher rainfall period. The juvenile prawn occurred only in May and July. Total length of egg-bearing females ranged from 12.12 to 38.30 mm, with mean female length at first maturity (L50) of 23.7 mm. Fecundity increased with prawn size and varied between 174 and 1780 eggs per female. Mean egg volume increased gradually from 0.031 (Stage I) to 0.060 mm³ (Stage III) during embryogenesis. Macrobrachium surinamicum depends on brackish water to complete the larval development. Irrespective of female size, reproductive output of M. surinamicum varied between 4.3 % and 35.5 % of their body weight for egg production. The knowledge of the reproductive biology reported in the present study is an important tool to define strategies to preserve M. surinamicum in Amazon River mouth.

Age, growth and reproductive biology of whiting Merlangius merlangus (Linnaeus 1758) in the Celtic Sea

Age, growth and reproductive biology were investigated for whiting (Merlangius merlangus) captured from the Celtic Sea (ICES division Vllg), for the period January 2001 to January 2002. Females dominated the sex ratio of 1: 2.25. The relative abundance of females exceeded the number of males in all length classes. The relationship between weight (g) and total length (cm) was the same for male and female whiting. A total of 973 fish were aged and the maximum age recorded was 11 years. Results from an intercalibration exercise showed 87% agreement in age readings between the author and an expert in ageing whiting at the Marine Institute. Females were dominated by 2 year olds, while males were dominated by 3 year olds. Lx was estimated as 38cm and a growth rate was calculated K = 0.3769 year'1. Females were fully recruited to the fishery at 3 years of age, while the age at full recruitment (tr) for males was 4 years. Female whiting spawned from late February to June 2001 and matured at a total length of 23 cm in their first year. Female whiting reached L5o at a total length of 28 cm and 2.7 years of age. Male whiting spawned from February to June 2001. They matured at a total length of 21 cm and in their first year. Male whiting reached L50 at a total length of 30.4 cm and 3.6 years of age. The following critical points should be taken into account in the management of the Celtic Sea whiting stock: An Fpa should be established in order to assess the current level of fishing mortality; The maturity ogives need further study; The extent of gutting of large fish before landing by fishers in the fleet should be investigated and the apparent decline in size of 4 - 7 year old fish in the Celtic Sea between 1996 and 2001 needs to be assessed.

Age, growth and reproductive biology of Plaice, (Pleuronectes platessa L.) in Irish waters, 2003-2005

The current study presents data on age and growth for plaice (Pleuronectes platessa L.) sampled between November 2003 and February 2005 in ICES areas Via (northwest coast of Ireland), Vila (Irish Sea), Vllg (Celtic Sea), VDj (southwest coast of Ireland) and VHb (west coast of Ireland), and data on the reproductive biology and maturity of plaice in ICES area Vllb (west coast of Ireland). This is the first detailed account of the biology of plaice for some of these areas. It is intended that this study will improve understanding of the life cycle of plaice and help fisheries scientists to better predict the effect of fishing effort on Irish plaice stocks. The overall length range found for plaice was 9-51.99cm TL, with a length range of 9-5 lcm TL for females and 9-40cm for males. In all ICES areas the length range for female fish was larger than for male fish. The age range of plaice sampled during this study was 1 to 16 years. In all ICES areas females had a greater range in ages and fish in the larger age groups. From analysis of length and age data it was concluded that there was a significant difference (P=0.000) in growth rate of males and females between ICES areas sampled in March 2004. The highest rate of fishing mortality was determined for ICES area Via (F=1.06) and the lowest for ICES area Vila (F=0.56). In each ICES area male and female plaice have fully recruited to the population by age 4, with the exception of females in ICES area Via, for which a tr value of 5 years was determined. Length at first maturity (L50%) was determined to be 23cm and 21cm for males and females respectively. Age at first maturity (A50%) was determined to be 3 years for both males and females. It was found that males and females in ICES areas Vllb, Vila and Via are well above the length and age at first maturity when they are recruited to the fishery. In ICES area Vllb female plaice spawn from November to March, with peak spawning occurring in February, and male plaice spawn from November to April, with peak spawning occurring in November. Spawning females had an age range of 2 to 10 years and spawning males had an age range of 2 to 7 years. From the oocyte length frequency distributions, it was determined that the plaice is a determinate batch spawner. During this investigation a total of 177 ovaries and 127 testes were staged using both macroscopic and histological criteria. The overall percentage of maturity stages which compared favorably between the two assessment methods was 22.03% for female plaice and 37.80% for male plaice. In general, the findings of this study indicate that there was a very poor match between the macroscopic and histological assessment methods. Given that the histological determination of these stages is based on the observation of a distinct set of developmental features, it is expected that it would be more accurate to use histologically assessed gonads to calculate the annual percentage maturity assessment. The biology of plaice in the areas studied is compared with previous studies of plaice in Irish and European waters.

Male-female interaction during breeding and non-breeding seasons in Akodon azarae (Rodentia, Muridae)

Dyad encounters between male and female adults of Akodon azarae (Fischer, 1829) were analyzed by means of observational techniques in a natural closure during the breeding and non-breeding seasons. The animals were held in observation during 21 days, with daily 15-minute recordings of interindividual separation distance, relative displacements, characteristics of the male-female interaction, copulation, and construction and exclusive or shared use of nests by each pair. The couples, which bred successfully, showed, on average, the longest separation distance between male and female allowed by the closure. During the first two weeks of gestation the females exhibited more displacements than their respective mates did. The male-pregnant female encounters were significantly more aggressive than those recorded between pairs which did not breed successfully. During the non-breeding season a shorter average distance between individuals and a frequent use of nests shared by the pair were recorded. The results obtained are discussed within the framework of the social system of A. azarae.

Reproductive biology of Hyla goiana (Anura, Hylidae)

Male-male and male-female interactions, reproductive habitat, and vocalizations of Hyla goiana B. Lutz, 1968 a member of H. polytaenia species group, are described. Three groups of calling males were surveyed along a small stream, at the Estação de Pesquisa e Desenvolvimento Ambiental de Galheiro, Perdizes municipality, State of Minas Gerais, Brazil. The mean distance between the nearest calling neighbors was 2.7 m, in accordance with an uniform distribution. Clutches (mean 180 eggs) were deposited in the stream, submerged and attached to plants. In two observed courtships the female moved towards a calling male that rapidly clasped her. In the male-male interaction, the males emitted advertisement and encounter calls and then engaged in physical combat. The males have a prepollical fang-like spine on each hand and several of them were observed with scratches on dorsum. The advertisement call consists of alternating harsh notes and a trilled of brief notes. The frequency of the call is lower than that of H. aff. polytaenia and H. cipoensis B. Lutz, 1968. The clutch characteristics of H. goiana are similar to those described for H. polytaenia Cope, 1870 and H. cipoensis.

Reproductive behavior of Leptodactylus mystacinus (Anura, Leptodactylidae) with notes on courtship call of other Leptodactylus species

Here we present data on the reproductive behavior of Leptodactylus mystacinus (Burmeister, 1861), including details on courtship behavior. We also describe and compared the courtship calls of L. mystacinus, L. furnarius Sazima & Bokermann, 1978 and Leptodactylus sp. (L. aff. andreae). Field works were conducted in Uberlândia (central Brazil). During courtship, a female approaches a calling male and is led to a previously excavated chamber; a female can approach a silent male that beat his hands and/or feet on the ground as well. The courtship call of L. mystacinus consists of one single arch-shaped note (duration = 0.04 s) repeated 258 times per minute; the courtship calls of L. furnarius (0.06 s, 84 times per minute) and Leptodactylus sp. (0.15 s, 5 times per minute) also are arch-shaped. The courtship behavior of L. mystacinus is similar to that of other species of the L. fuscus (Schneider, 1799) group; unique to it is that males can beat his hands and/or feet on the ground while courting. The male behavior of conducting the female to a previously excavates chamber and the arch-shaped courtship call may represent other shared derived features of members of the L. fuscus group, including the former Adenomera species.

Reproductive traits of Gymnogeophagus labiatus (Teleostei, Cichlidae), in an upper stretch of Sinos river, Caraá, Brazil

This work describes the reproduction of Gymnogeophagus labiatus (Hensel, 1870) from an upper stretch of Sinos river, southern Brazil, based on the analysis of 174 males and 132 females captured in monthly samples taken from January to December 2007. Results showed that reproductive activity occur in spring and summer although ripe males were found along the year. The standard length of the smallest ripe male was 104.74 mm (Lt) and the smallest ripe female was 55.00 mm (Lt). There was a significant difference in total sex ratio, with 1.32 males to each female (χ2 = 5.76). Males were much more abundant in March (1.75 males: 1 female) and December (5 males: 1 female). Females were more abundant in the 62├77 mm interval (1 male: 2.36 female) while males were more abundant in the 77├92 mm size interval (2.57 males: 1 female). The largest length intervals were composed of only males. Mean absolute fecundity was 113.4 (± 31.24 sd) and mean relative fecundity was 0.0125 (± 0.0026 sd) oocytes/mg. In ripe ovaries, small-diameter oocytes were observed at high frequencies while larger ones occurred at lower frequencies. This pattern is common in fishes with asynchronous oocyte development. Characteristics of G. labiatus, such as low fecundity, asynchrony in oocyte development, multiple spawning, and its well-known parental care behavior, are consistent with an equilibrium strategy, as proposed for other cichlids.

Reproductive aspects of Dendropsophus sanborni (Anura, Hylidae) in northeastern Argentina

Reproductive aspects of Dendropsophus sanborni (Anura, Hylidae) in northeastern Argentina. The aim of this investigation was to characterize reproductive aspects of Dendropsophus sanborni (Schmidt, 1944) (Anura, Hylidae) in the northwest of Corrientes province, in northeastern Argentina. The reproductive cycles of males and females, the existence of sexual dimorphism and the male’s vocalization period were analyzed. Samplings were made from September 2011 to August 2012. Dendropsophus sanborni showed a prolonged reproductive pattern because reproductive activity was observed during most part of the year. Calling males were recorded all months except in July. The spermatogenic cycle can be characterized as potentially continuous. In males, snout-vent length was significantly correlated with mean testicular volume. The species showed sexual dimorphism in body mass, with females larger than males. Post-ovulatory females were found from March to June and most females had oocytes at different stages of maturity, with abundant post-vitellogenic oocytes from August to December.

Morphology and dynamics of male gametogenesis in Sympterygia bonapartii (Chondrichthyes, Rajidae) from Northern Patagonia

ABSTRACT The smallnose fanskate, Sympterygia bonapartii Müller & Henle, 1841 is one of the most disembarked items in commercial harbors in Argentina. In this work, the microscopic architecture of mature male gonads and the dynamics of cysts development are analyzed as a contribution to awareness of the reproductive biology of the species. Some biological data related to reproduction are given as well. Two seasons were sampled (fall and spring) and length classes's frequency distribution and maturity stages frequency distribution are given. Size at first sexual maturation for males was estimated at 57 cm of total length. Testes are symmetric, peer, lobed, with several germinal zones. Inside the gonads, there are many spermatocysts, containing reproductive cells at the same developmental stage. On the basis of their cytological and microanatomical features, several maturative degrees of the spermatogenic series were differentiated. Few Leydig cells were recognized at the interstitial tissue among cysts. The microscopic and semiquantitative analysis performed in this work provides morphological information about male gametogenesis and some biological data for the North Patagonian population of this economically and ecologically important species.

Inbreeding avoidance through kin recognition: choosy females boost male dispersal.

Inbreeding avoidance is predicted to induce sex biases in dispersal. But which sex should disperse? In polygynous species, females pay higher costs to inbreeding and thus might be expected to disperse more, but empirical evidence consistently reveals male biases. Here, we show that theoretical expectations change drastically if females are allowed to avoid inbreeding via kin recognition. At high inbreeding loads, females should prefer immigrants over residents, thereby boosting male dispersal. At lower inbreeding loads, by contrast, inclusive fitness benefits should induce females to prefer relatives, thereby promoting male philopatry. This result points to disruptive effects of sexual selection. The inbreeding load that females are ready to accept is surprisingly high. In absence of search costs, females should prefer related partners as long as delta<r/(1+r) where r is relatedness and delta is the fecundity loss relative to an outbred mating. This amounts to fitness losses up to one-fifth for a half-sib mating and one-third for a full-sib mating, which lie in the upper range of inbreeding depression values currently reported in natural populations. The observation of active inbreeding avoidance in a polygynous species thus suggests that inbreeding depression exceeds this threshold in the species under scrutiny or that inbred matings at least partly forfeit other mating opportunities for males. Our model also shows that female choosiness should decline rapidly with search costs, stemming from, for example, reproductive delays. Species under strong time constraints on reproduction should thus be tolerant of inbreeding.

The role of the hypothalamic kisspeptin/GPR54 system in the control of GnRH neurons

Summary : The hypothalamus represents less than 1 % of the total volume of the brain tissue, yet it plays a crucial role in endocrine regulations. Puberty is defined as a process leading to physical, sexual and psychosocial maturation. The hypothalamus is central to this process, via the activation of GnRH neurons. Pulsatile GnRH secretion, minimal during childhood, increases with the onset of puberty. The primary function of GnRH is to regulate the growth, development and function of testes in boys and ovaries in girls, by stimulating the pituitary gland secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several factors contribute to the timing of puberty, including sex and ethnicity, genetics, dietary intake and energy expenditure. Kisspeptins constitute a family of small peptides arising from the proteolytic cleavage of metastin, a peptide with 54 amino acids initially purified from human placenta. These kisspeptins were the subject of much attention following their discovery because of their antimetastatic properties, but it was more recently that their determining role in the reproductive function was demonstrated. It was shown that kisspeptins are ligands of a receptor, GPR54, whose natural inactivating mutation in humans, or knockout in the mouse, lead to infertility. GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH release and GnRH neuron firing activity, but the neurobiological mechanisms for these actions are unknown. Gprotein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting that both direct and indirect effects are possible. In the first part of my thesis, we investigated a possible connection between the acceleration of sexual development induced by leptin and hypothalamic metastin neurons. However, the data generated by our preliminary experiments confirmed that the commercially available antibodies are non-specific. This finding constituted a major drawback for our studies, which relied heavily upon the neuroanatomical study of the hypothalamic metastinergic pathways to elucidate their sensitivity to exogenous leptin. Therefore, we decided to postpone any further in vivo experiment until a better antibody becomes available, and focused on in vitro studies to better understand the mechanisms of action of kisspeptins in the modulation of the activity of GnRH neurons. We used two GnRH-expressing neuronal cell lines to investigate the cellular and molecular mechanisms of action of metastin in GnRH neurons. We demonstrated that kisspeptin induces an early activation of the MAP kinase intracellular signaling pathway in both cell lines, whereas the SAP/JNK or the Akt pathways were unaffected. Moreover, we found an increase in GnRH mRNA levels after 6h of metastin stimulation. Thus, we can conclude that kisspeptin regulates GnRH neurons both at the secretion and the gene expression levels. The MAPK pathway is the major pathway activated by metastin in GnRH expressing neurons. Taken together, these data provide the first mechanism of action of kisspeptin on GnRH neurons. Résumé : L'hypothalamus est une zone située au centre du cerveau, dont il représente moins de 1 du volume total. La puberté est la période de transition entre l'enfance et l'age adulte, qui s'accompagne de transformations somatiques, psychologiques, métaboliques et hormonales conduisant à la possibilité de procréer. La fonction principale de la GnRH est la régulation de la croissance, du développement et de la fonction des testicules chez les hommes, et des ovaires chez les femmes en stimulant la sécrétion de l'hormone lutéinisante (LH) et de l'hormone folliculostimulante (FSH) par la glande hypophysaire. Plusieurs facteurs contribuent au déclanchement de la puberté, y compris le sexe et l'appartenance ethnique, la génétique, l'apport alimentaire et la dépense énergétique. Les Kisspeptines constituent une famille de peptides résultant de la dissociation proteolytique de la métastine, un peptide de 54 acides aminés initialement purifié à partir de placenta humain. Ces kisspeptines ont fait l'objet de beaucoup d'attention à la suite de leur découverte en raison de leurs propriétés anti-metastatiques, et c'est plus récemment que leur rôle déterminant dans la fonction reproductive a été démontré. Les kisspeptines sont des ligands du récepteur GPR54, dont la mutation inactivatrice chez l'homme, ou le knockout chez la souris, conduisent à l'infertilité par hypogonadisme hypogonadotrope. Les neurones à GnRH jouent un rôle central dans le règlement des fonctions reproductrices et la kisspeptine stimule l'activité des neurones à GnRH et la libération de GnRH par ces neurones. Toutefois, les mécanismes neurobiologiques de ces actions ne sont pas connus. Dans la première partie de ma thèse, nous avons étudié le lien potentiel entre l'accélération du développement sexuel induite par la leptine et les neurones hypothalamiques à metastine. Les données générées dans cette première série d'expériences ont malheureusement confirmé que les anticorps anti-metastine disponibles dans le commerce sont aspécifiques. Ceci a constitué un inconvénient majeur pour nos études, qui devaient fortement s'appuyer sur l' étude neuroanatomique des neurones hypothalamiques à metastine pour évaluer leur sensibilité à la leptine exogène. Nous avons donc décidé de focaliser nos travaux sur une étude in vitro des mécanismes d'action de la kisspeptine pour moduler l'activité des neurones à GnRH. Nous avons utilisé deux lignées de cellules neuronales exprimant la GnRH pour étudier les mécanismes d'action cellulaires et moléculaires de la metastine dans des neurones. Nous avons ainsi pu démontrer que la kisspeptine induit une activation précoce de la voie f de signalisation de la MAP kinase dans les deux lignées cellulaires, alors que nous n'avons observé aucune activation de la voie de signalisation de la P13 Kinase et de la SAP/JNK. Nous avons en outre démontré une augmentation de l'expression de la GnRH par la stimulation avec la Kisspeptine. L'ensemble de ces données contribue à élucider le mécanisme d'action avec lequel la kisspeptine agit dans les neurones à GnRH, en démontrant un effet sur l'expression génique de la GnRH. Nous pouvons également conclure que la voie de la MAPK est la voie principale activée par la metastine dans les neurones exprimant la GnRH.

Establishing an association between a peri-operative perfusion score system (PerfSCORE) and post-operative patient morbidity/mortality during CPB cardiac surgery.

BACKGROUND: To date, there is no quality assurance program that correlates patient outcome to perfusion service provided during cardiopulmonary bypass (CPB). A score was devised, incorporating objective parameters that would reflect the likelihood to influence patient outcome. The purpose was to create a new method for evaluating the quality of care the perfusionist provides during CPB procedures and to deduce whether it predicts patient morbidity and mortality. METHODS: We analysed 295 consecutive elective patients. We chose 10 parameters: fluid balance, blood transfused, Hct, ACT, PaO2, PaCO2, pH, BE, potassium and CPB time. Distribution analysis was performed using the Shapiro-Wilcoxon test. This made up the PerfSCORE and we tried to find a correlation to mortality rate, patient stay in the ICU and length of mechanical ventilation. Univariate analysis (UA) using linear regression was established for each parameter. Statistical significance was established when p < 0.05. Multivariate analysis (MA) was performed with the same parameters. RESULTS: The mean age was 63.8 +/- 12.6 years with 70% males. There were 180 CABG, 88 valves, and 27 combined CABG/valve procedures. The PerfSCORE of 6.6 +/- 2.4 (0-20), mortality of 2.7% (8/295), CPB time 100 +/- 41 min (19-313), ICU stay 52 +/- 62 hrs (7-564) and mechanical ventilation of 10.5 +/- 14.8 hrs (0-564) was calculated. CPB time, fluid balance, PaO2, PerfSCORE and blood transfused were significantly correlated to mortality (UA, p < 0.05). Also, CPB time, blood transfused and PaO2 were parameters predicting mortality (MA, p < 0.01). Only pH was significantly correlated for predicting ICU stay (UA). Ultrafiltration (UF) and CPB time were significantly correlated (UA, p < 0.01) while UF (p < 0.05) was the only parameter predicting mechanical ventilation duration (MA). CONCLUSIONS: CPB time, blood transfused and PaO2 are independent risk factors of mortality. Fluid balance, blood transfusion, PaO2, PerfSCORE and CPB time are independent parameters for predicting morbidity. PerfSCORE is a quality of perfusion measure that objectively quantifies perfusion performance.

A new species of culex (Melanoconion) from atlantic tropical system of Souther Brazil (Diptera: Culicidae)

The male adult of Culex (Melanoconion) anoplicitus, a new species from Southern Tropical Atlantic System of Brazil, South America, is described and illustrated. Identification may be made by characteristic morphological aspects of genitalia which are peculiar to this species.