A seleção sexual atua de forma significativa na evolução de características reprodutivas. Os machos apresentam diversas estratégias para garantir a fertilização da fêmea, a qual, por sua vez, possui papel crucial em processos pré- e pós-copulatórios. Nesse contexto, cefalópodes apresentam aspectos reprodutivos peculiares, apresentando transferência de espermatozoides via espermatóforos e, em alguns casos, armazenamento desses gametas em órgãos especializados (i.e., receptáculos seminais). Entretanto, pouco se sabe sobre a morfologia, funcionamento e mecanismos de captação, armazenamento e liberação dos espermatozoides pelos receptáculos seminais. Neste contexto, a presente dissertação teve como objetivo investigar a estrutura e função do receptáculo seminal de Doryteuthis plei, espécie de lula adotada como modelo, como base para compreensão de mecanismos pós-copulatórios de seleção sexual em cefalópodes. Para atingir esse objetivo, a morfologia do receptáculo seminal foi analisada com base em microscopia integrativa (microscopia óptica com emprego de técnicas histoquímicas, microscopia eletrônica de varredura e de transmissão, microscopia confocal e microCT), visando a uma caracterização detalhada da estrutura do órgão. Além disso, a morfologia e histologia/histoquímica do receptáculo seminal foi analisada em três situações experimentais distintas: (1) antes de cópula recente, (2) após cópula recente (e antes da desova) e (3) após a desova, para investigar alterações morfológicas no órgão e no volume de espermatozoides armazenados, como base para compreensão dos mecanismos de captação, armazenamento e liberação de gametas masculinos pela fêmea. Os resultados revelaram enorme complexidade do receptáculo seminal com relação à diversidade de células secretoras e de fibras musculares associadas. Constatou-se que há alteração das células secretoras após a cópula recente e após a desova, com liberação do conteúdo de parte dessas células. Com base nos resultados obtidos, discute-se o papel dessas secreções na captação, armazenamento e liberação de espermatozoides. Além disso, a estrutura dos sistemas nervoso e muscular associados ao receptáculo seminal sugere que a fêmea tenha controle sobre deformações precisas do órgão relacionadas à captação e liberação de espermatozoides. Entretanto, outros mecanismos seriam igualmente possíveis, como atração química, natação ativa dos espermatozoides e ação ciliar do órgão. Alguns receptáculos seminais foram encontrados com espermatângios (i.e., espermatóforos evertidos) bloqueando total ou parcialmente sua abertura. Devido à frequência, posição e morfologia dos espermâtangios dentro do receptáculo seminal, propõe-se a hipótese de que essas estruturas poderiam atuar também como plugues copulatórios. Esses plugues seriam mais eficientes nas primeiras 24 horas, sua eficiência sendo gradualmente reduzida com o tempo após a cópula, devido ao esvaziamento do conteúdo espermático e consequente redução de sua turgidez. Embora plugues copulatórios sejam conhecidos em inúmeras espécies de animais, este é o primeiro registro para cefalópodes, sendo, portanto, marco importante nos estudos de seleção sexual para o grupo. Cefalópodes são considerados modelos interessantes para o estudo de seleção sexual e os resultados desta dissertação contribuem para compreensão dos complexos mecanismos pós-copulatórios em lulas, como escolha críptica da fêmea e competição espermática / Sexual selection exerts a significant pressure on the evolution of reproductive attributes. Males show a diverse array of strategies to gain advantage in mating and fertilization success, but females also play a crucial role in pre- and post-copulatory sexual selection processes. Within this context, cephalopod mollusks show peculiar reproductive strategies, including sperm transfer via spermatophores, and the presence of female sperm-storage organs (seminal receptacles). However, the knowledge of the functioning of the cephalopod seminal receptacles is scarce, the mechanisms involved with sperm uptake, storage and release being unknown. To shed light on post-copulatory mechanisms in cephalopods, the present dissertation aimed at investigating the structure and function of the seminal receptacle of the squid Doryteuthis plei. To achieve this goal, the morphology of the seminal receptacle was thoroughly analyzed applying integrative microscopy (light microscopy including histochemical techniques, scanning & transmission electron microscopy, confocal microscpy and microCT). Moreover, to investigate morphological and sperm volume changes associated with possible mechanisms of sperm uptake, storage and release by the organ, the morphology and histology/histochemistry of the seminal receptacle was also analyzed under three distinct experimental manipulations: (1) before recent mating (2) after recent mating (and before egg release) and (3) after egg release. The results show a complex and striking diversity of secretory cells and associated muscle fibers in the seminal receptacle. There were changes in the composition and predominance of secretory cells between major reproductive events, suggesting a secretory activity associated with mating and spawning. The possible roles of these secretions in sperm uptake, storage and release are discussed in light of our data. Moreover, the structure of the nervous and muscular systems associated with the receptacle suggests that females have control over precise deformations of the organ, possibly related to sperm uptake and release. However, other mechanisms would be possible, such as chemical attraction, sperm active swimming, and ciliary action. Seminal receptacles were sometimes found with spermatangia (i.e., everted spermatophores) blocking totally or partially their openings. Given their frequency, position, and morphology, we hypothesize spermatangia might function as copulatory plugs that physically obstruct the female storage organ. Plug efficiency should be high within the first 24 hours after mating, gradually decreasing its efficiency with time, when spermatangia lose their turgidity by releasing part of their sperm content. Although copulatory plugs have been reported for numerous taxa, this is the first record for cephalopods, and as such this finding has an impact for sexual selection studies based on these mollusks. Cephalopods are considered interesting models to investigate sexual selection, and this study has cast some light on the understanding of complex post-copulatory mechanisms in squids, such as cryptic female choice and sperm competition

Die Brachyura umfassen ca. 7000 Arten. Die Variationsbreite ihrer morphologischen Merkmale spiegelt sich in den männlichen und weiblichen Reproduktionssystemen wider und machen sie zu einem herausfordernden Untersuchungsgegenstand. Obwohl sich zahlreiche Studien mit der Phylogenie der Brachyura befasst haben, fehlen eindeutige Ergebnisse. Manche Studien stützen sich weiterhin auf deren Teilung in die Podotremata, die Heterotremata und die Thoracotremata (die beiden letzteren bilden die Eubrachyura), die auf der Position der Gonoporen basiert. In dieser Arbeit wurden die männlichen Kopulations- und weiblichen Reproduktionssysteme von vier eubrachyuren Arten untersucht. Zur Analyse ihrer inneren Morphologie, wurden die Gonopoden in der ersten und zweiten Studie μCT-gescannt und 3D-rekonstruiert. Zusätzlich wurden in allen Studien rasterelektronenmikroskopische Untersuchungen angewendet, um Informationen über die Oberflächenstrukturen der Gonopoden zu erhalten. Alle Untersuchungen des weiblichen Systems wurden mit bewährten histologischen Methoden und Lichtmikroskopie durchgeführt. In der ersten und zweiten Studie wurde diese detaillierte Strukturanalyse durch 3D-Rekonstruktion ergänzt. Die Ergebnisse dieser Studien wurden in Zusammenhang mit der vorhandenen Literatur interpretiert, um Merkmale des männlichen und weiblichen Reproduktionssystems zu definieren und deren Potenzial für phylogenetische Untersuchungen zu diskutieren. Außerdem wird ein evolutives Scenario bezüglich der Transformation der hier vorgeschlagenen Merkmalszustände des weiblichen Reproduktionssystems diskutiert. Die Gonopoden sind wertvoll, um Artenzugehörigkeiten zu Brachyurengruppen zu identifizieren, sind aber für Untersuchungen großskaliger Brachyurenphylogenie ungeeignet. Einige der weiblichen Merkmale können ausschließlich heterotremen oder thoracotremen Weibchen zugeordnet werden. Die vorgeschlagenen Szenarien deuten darauf hin, dass einige dieser Charaktere mehrfach entstanden sind. / The Brachyura comprise approximately 7000 species. The variability of their morphological traits is reflected in the male copulatory and the female reproductive systems that make them a challenging object of investigation. Numerous studies addressed the brachyuran phylogeny but unambiguous results have yet to be presented. Some studies still rely on the division of Brachyura into the Podotremata, the Heterotremata and the Thoracotremata (the latter two forming the Eubrachyura) that is based on the position of the male and female gonopores. In this work, the male copulatory and female reproductive systems of four eubrachyuran species were investigated. In the first and the second study, the gonopods were µCT-scanned and 3D-reconstructed to analyse their internal morphology. Additionally, in all studies scanning electron microscopy was used in order to obtain information about the surface structures of the gonopods. All investigations of the female system were conducted using approved histological methods and light microscopy. In the first and second study, this detailed structural analysis was complemented by 3D-reconstruction. The results of these studies are evaluated in comparison with the existing literature in order to define characters of the male copulatory and female reproductive system and discuss their potential for phylogenetic investigations. Additionally, an evolutionary scenario of the transformations of the herein proposed character states of the female reproductive system is discussed. Without additional information from the female reproductive system, the gonopod morphology is valuable to identify species affiliations to certain groups but remains inconclusive for large-scale brachyuran phylogeny. Some of the female characters found in these studies can explicitly be assigned to heterotreme or thoracotreme females. The proposed scenarios suggest, that some, if not all of these characters probably have evolved multiple times.

Information on the biosynthetic origin and functional advantage of marine mollusc natural products is not only essential to our understanding of chemical ecology, but to the development and responsible production of therapeutic agents. As demonstrating in situ activity is methodologically hindered, functions inferred by in vitro activity have been assumed for many secondary metabolites. The anatomical and ontogenetic distribution of natural products can not only provide information on the biosynthesis and storage of metabolites, but identify selective pressures likely to affect survivorship at a specific life stage. Thus, dissection and chemical analysis of distinct tissues, in combination with histochemistry may offer a valuable approach. Marine gastropods of the Muricidae are renowned for the ancient dye Tyrian purple, which evolves from choline esters of bromoindoxyl sulphate in the hypobranchial gland through a series of enzymatic and photo-oxidative reactions. Prochromogen hydrolysis by arylsulphatase liberates neuromuscular active choline esters and cytotoxic bromoindole precursors, which also occur in muricid egg masses. Although visual accounts of dye pigments in the muricid gonoduct suggest precursors may be incorporated into egg masses from a maternal source, their biosynthetic origin and the evolutionary significance of the hypobranchial gland is unknown. Thus, the Muricidae, and in particular Dicathais orbita upon which most previous research has been focused, is an ideal model for this novel approach to natural product research. To confirm observations of dye pigments in muricid gonoducts and gain an understanding of their anatomical distribution, a liquid chromatography-mass spectrometry (LC-MS) method was developed to simultaneously quantify pigments, precursors and the prochromogen, tyrindoxyl sulfate. The prochromogen was not only detected in albumen and capsule gland extracts, but bioactive intermediates and the dye 6,6’-dibromoindigo were also present in the latter. These findings provided preliminary evidence for the maternal provision of prochromogens in egg masses of D. orbita and identified regions within which to conduct histochemical investigations. Tyrindoxyl sulphate was also detected in male prostate gland extracts, along with the dibromoindigo isomer, 6,6’-dibromoindirubin and its oxidative precursor, 6-bromoisatin. This not only implies physiological differences exist between male and female gonoducts, but that these secondary metabolites are not solely intended for egg masses and may hold significance throughout the life cycle. Histomorphological inspection of the pallial gonoduct-hypobranchial gland complex was conducted over the annual cycle to determine a mechanism for precursor transfer between these structures. Although an anatomical connection was not detected, the secretions of two hypobranchial cell types thought to be involved in Tyrian purple synthesis were of remarkable biochemical similarity to those of various capsule and albumen gland lobes. Together these findings implied the potential for natural product synthesis within the pallial gonoduct of D. orbita. To establish the role of these glandular lobes in the incorporation of intracapsular fluid and capsule laminae, identical histochemical techniques were applied to transverse capsule wall sections. Biochemical correlations not only provided a simple method of deciphering the complex process of encapsulation in neogastropods, but effectively identified the destination of gonoduct secretions in egg capsules of D. orbita. Comparisons of capsule and gonoduct biochemistry revealed that the intracapsular fluid and inner capsule wall are secreted by the posterior capsule gland lobe, the middle lamina by the lateral lobes and the outer layers by the dorsal lobe, albumen and pedal glands. Investigation into the location of regulatory enzymes and precursors was conducted to establish the biosynthetic origin of Tyrian purple prochromogens and mechanisms governing bioactive precursor synthesis. Novel histochemical techniques for the localization of bromoperoxidase, the enzyme thought to facilitate prochromogen bromination, and tyrindoxyl sulphate were developed and applied to gonoduct, hypobranchial gland, and encapsulated larvae sections. Standard staining reactions for the indole precursor, tryptophan, and arylsulphatase were also applied. The histochemical approach adopted revealed that tyrindoxyl sulphate is de novo biosynthesized through the post-translational bromination of dietary derived tryptophan. Two biosynthetic sites were identified, one related to hypobranchial secondary metabolism and the second of significance to the presence of bioactive precursors in muricid egg masses. Tryptophan is stored within secretory cells of the lateral hypobranchial epithelium and once exocytosed, is united with bromoperoxidase from supportive cells to form tyrindoxyl sulphate. Prochromogen synthesis also occurs in the subepithelial vascular sinus for storage and secretion by medial hypobranchial secretory cells. Bioactive precursor synthesis on the epithelial surface is regulated by the liberation of arylsulphatase from adjacent supportive cells. These findings not only provide evidence for de novo biosynthesis of Tyrian purple precursors, but are first account of natural product biosynthesis within the gastropod hypobranchial gland. Together these findings imply a naturally selected function for the synthesis of bioactive indoles in hypobranchial gland secretions of the Muricidae and Gastropoda. Tyrindoxyl sulphate is also transported within the vascular sinus to lateral and dorsal capsule gland lobes where bromoperoxidase and arylsulphatase also occur. Arylsulphatase was also detected within the albumen gland, which along with the posterior capsule gland lobe, acts as a storage site for dietary tryptophan. Thus, tyrindoxyl sulphate and the constituents for prochromogen and precursor biosynthesis are introduced to intracapsular fluid and capsule laminae by the capsule gland. Histochemistry in combination with LC-MS revealed an identical biosynthetic profile within larval vitellus, which is elaborated during oogenesis and may also receive secretions from the albumen gland. Due to the absence of a hypobranchial gland in veligers, it appears that pelagic larvae rely on vitelline natural products until settlement and metamorphous. These findings together with the in situ antimicrobial activity of bromoindoles suggest Tyrian purple precursors are incorporated into muricid egg masses as a maternal investment in larval defence against pathogens. The results of this investigation clearly highlight the benefits of adopting a histochemical approach to natural product research. This novel alternative to radioisotopes and in situ demonstration of bioactivity, can not only aid in the elucidation of secondary metabolic pathways and chemically mediated interactions, but identify mechanisms of metabolite regulation and differentiate between biosynthetic and storage tissues. Apart from providing insight into the ecological significance of muricid secondary metabolites, the biosynthetic information provided is valuable to our understanding of chemical phylogeny and biosynthetic enzyme sequencing for the environmentally sound development of natural products as biomedical agents.