Sin, Yung Wa. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 105-112). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgements --- p.v / Contents --- p.vi / List of tables --- p.x / List of figures --- p.xii / Chapter Chapter 1 --- General introduction / Chapter 1.1 --- Introduction to loliginid squids --- p.1 / Chapter 1.1.1 --- Cephalopod taxonomy and fishery --- p.1 / Chapter 1.1.2 --- Family Loliginidae --- p.2 / Chapter 1.1.3 --- Genus Uroteuthis --- p.4 / Chapter 1.1.4 --- Uroteuthis chinensis and Uroteuthis edulis --- p.5 / Chapter 1.2 --- Introduction to morphological differentiation methods in cephalopods --- p.11 / Chapter 1.3 --- Introduction to genetic differentiation methods --- p.13 / Chapter 1.3.1 --- Molecular markers --- p.13 / Chapter 1.3.1.1 --- Animal mitochondrial DNA --- p.13 / Chapter 1.3.1.2 --- Microsatellite DNA --- p.14 / Chapter 1.3.2 --- Systematic studies of cephalopods using molecular markers --- p.15 / Chapter 1.3.3 --- Population genetic studies of cephalopods using molecular markers --- p.18 / Chapter 1.4 --- Objectives --- p.19 / Chapter Chapter 2 --- Morphological differentiation of Uroteuthis chinensis and Uroteuthis edulis --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and methods --- p.21 / Chapter 2.2.1 --- Specimens --- p.21 / Chapter 2.2.2 --- Morphometric characters --- p.22 / Chapter 2.2.3 --- Multivariate analysis of data --- p.23 / Chapter 2.3 --- Results --- p.24 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 3 --- Genetic differentiation of Uroteuthis chinensis and Uroteuthis edulis --- p.57 / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.2 --- Materials and methods --- p.58 / Chapter 3.2.1 --- Collection of specimens --- p.58 / Chapter 3.2.2 --- "DNA extraction, PCR amplification and sequencing" --- p.59 / Chapter 3.2.3 --- Sequence data analysis --- p.60 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Sequence data set --- p.62 / Chapter 3.3.2 --- Phylogenetic analysis --- p.63 / Chapter 3.3.3 --- Sequence divergence within species --- p.67 / Chapter 3.4 --- Discussion --- p.89 / Chapter 3.4.1 --- Genetic differentiation of U. chinensis and U edulis --- p.89 / Chapter 3.4.2 --- Population structuring within U. chinensis and U edulis --- p.91 / Chapter Chapter 4 --- Isolation of microsatellite loci for U. chinensis --- p.94 / Chapter 4.1 --- Introduction --- p.94 / Chapter 4.2 --- Materials and methods --- p.95 / Chapter 4.3 --- Results --- p.97 / Chapter 4.4 --- Discussion --- p.102 / Chapter Chapter 5 --- Conclusion --- p.103 / References --- p.105 / Appendix 1: Aligned COI sequences used in this study --- p.113 / Appendix 2: Aligned 16S rRNA sequences used in this study --- p.122

En el presente trabajo se evalúa la estructura histológica de las gónadas del calamar Loligo gahi como también su comportamiento reproductivo en determinados meses. De Enero a Marzo de 2007 se realizaron muestreos mensuales de 56 ejemplares de calamar Loligo gahi del muelle de la playa Pescadores de la costa de Lima, Perú. Fueron procesadas por técnica histológica convencional de hematoxilina–eosina para poder determinar las etapas de la ovogénesis, espermiogénesis y describir la organización histológica del ovario y del testículo. En las hembras de Loligo gahi la ovogénesis presenta 6 etapas o fases de desarrollo. En el macho la espermiogénesis constó de 5 etapas de desarrollo, lo que facilitará predecir adecuadamente el ciclo reproductivo. El análisis histológico se complementó con la obtención del índice gonadosomático, cuyos resultados permiten predecir que los meses de enero a marzo son los más adecuados para la maduración sexual y el desove en esta especie. The present work evaluates the histological structure of gonads of the squid Loligo gahi as well as their behavior in certain months. From January to March 2007 were conducted monthly sampling of 56 specimens of the squid Loligo gahi Beach pier fishermen along the coast of Lima, Peru. Were processed by conventional histological technique of hematoxylin-eosin to determine the stages of ovogénesis, espermiogénesis and describe the histological organization of the ovary and testis. In females of Loligo gahi 6 presents the ovogénesis phases or stages of development. In the male espermiogénesis consisted of 5 stages of development, which provide adequately predict the reproductive cycle. The histological analysis was supplemented by obtaining Gonadosomatic The results predict that the months of January through March are the most suitable for sexual maturation and spawning in this species

The chokka squid, Loligo vulgaris reynaudii, is a commercially important species supporting a large, high-value export fishery for South Africa. This study reviewed its population dynamics and reproductive biology. Biological information collected on biannual research cruises from September 1986 to April 1999 on the Agulhas Bank as well as information from commercial vessels operating in the inshore environment collected between April 1988 and July 1989 and again between June 1999 and May 2000 were analysed. Size ranges of juveniles, subadults and adults were calculated and used to determine seasonal distribution and abundance patterns. Based on Gonadosomatic Indices (GSI) and percentages of mature squid in each month, two peak reproductive seasons in summer and winter were identified although mature squid were present all year round. Seasonal peaks in sex ratio were also apparent with males outnumbering females by up to 4:1 in the peak-breeding season. The size at which squid matured, demonstrated both spatial and temporal patterns. Squid caught in spring matured at a smaller size than squid caught in autumn and at successively smaller sizes from west to east across the Agulhas Bank. Size at maturity was highly variable especially in males where “sneaker males” were evident in both seasons. Length-to-weight linear regression revealed significant differences between maturity stages and between sexes. Females demonstrated steeper length-to-weight regression slopes than males in the peak-breeding seasons. Aspects of squid biology pertinent to fishery management were highlighted as well as potential areas where research should be directed in order to develop future stock assessment models.

Cefalópodes coleóides (lulas, sépias e polvos) produzem espermatóforos muito elaborados, os quais são transferidos à fêmea durante a cópula por meio de um apêndice modificado nos machos. Durante a transferência à fêmea, os espermatóforos sofrem, de forma autônoma, drásticas modificações na chamada \"reação espermatofórica\", complexo processo de evaginação do aparato ejaculatório, que conduz à exteriorização da massa espermática e corpo cimentante. Poucos trabalhos abordam com detalhes a morfologia e anatomia funcional dos espermatóforos dos cefalópodes, grande parte do conhecimento acerca da estrutura do espermatóforo tendo sido gerada por trabalhos clássicos do século XIX e início do século XX. Investigações acerca do funcionamento dos espermatóforos são consideravelmente mais raras, estando o conhecimento básico sobre a reação espermatofórica restrito a apenas 20 espécies de coleóides. Como o funcionamento extracorpóreo dos espermatóforos depende exclusivamente da intrincada estrutura e organização de seus componentes (e.g., membranas e túnicas), somente investigações detalhadas dessas estruturas proverão as bases para a compreensão do funcionamento e da exata função do complexo espermatóforo dos coleóides. Nesse contexto, a presente Tese, organizada em cinco capítulos, teve como objetivo principal investigar a estrutura e o funcionamento dos espermatóforos da lula Doryteuthis plei (Blainville, 1823). No primeiro capítulo, com o objetivo de se estudar a fundo a organização estrutural dos espermatóforos da espécie, diversas técnicas de microscopia foram testadas e empregadas. Como resultado da combinação de diferentes ferramentas de análise, a estrutura do espermatóforo revelou-se ainda mais complexa, sendo as principais descobertas referentes à: 1) elaborada estrutura da membrana mediana, organizada em camadas e apresentando um segmento aboral quimicamente distinto, que envolve parte do corpo cimentante; 2) presença de um material reticulado preenchendo o espaço entre a túnica interna e a membrana mediana (discute-se a possibilidade do mesmo consistir em um fluido viscoso em espermatóforos intactos); 3) presença de espículas intimamente associadas à membrana interna na região do corpo cimentante (além das espículas embebidas no filamento espiral); 4) presença de extensões membranosas que delimitam uma câmara pré-oral na região do capuz; e 5) complexa organização estrutural do corpo cimentante, delimitado por duas camadas e contendo substâncias de distintas propriedades químicas. Uma avaliação cuidadosa da literatura permite sugerir que pelo menos parte dessas características deva ser comum aos espermatóforos de outros loliginídeos, e, em alguns casos, de outros grupos de coleóides. Como parte da investigação acerca da reação espermatofórica e dos mecanismos envolvidos na fixação da massa espermática no corpo da fêmea, constatou-se que, sob condições artificiais, espermatóforos em evaginação são capazes de penetrar musculatura exposta, de forma similar ao fenômeno de \"implante profundo\" observado naturalmente em algumas lulas oceânicas. Esse resultado foi descrito no segundo capítulo, no qual foi levantada a hipótese de que um mecanismo de perfuração seria inerente à estrutura dos espermatóforos dos coleóides. Dando continuidade ao estudo da morfologia funcional dos espermatóforos de D. plei, o terceiro capítulo apresenta os resultados obtidos a partir da investigação do funcionamento do espermatóforo e da morfologia dos espermatângios (i.e, espermatóforos evertidos) obtidos in vitro, bem como daqueles naturalmente fixados na fêmea. As evidências reunidas permitem afirmar que o processo de fixação compreende distintas fases desempenhadas por diversos componentes do espermatóforo, contrariamente a um conceito anterior de que a fixação seria realizada somente por substâncias adesivas do corpo cimentante. Durante a reação espermatofórica, o aparato ejaculatório e respectivo filamento espiral são capazes de perfurar superficialmente ou escarificar o tecido-alvo. Subseqüentemente, o corpo cimentante sofre drástica modificação estrutural, resultando na extrusão de parte do conteúdo cimentante, o qual é injetado diretamente sobre o tecido perfurado. Além disso, o corpo cimentante é exteriorizado com uma extremidade afilada que, em alguns casos, foi encontrada firmemente implantada no tecido da fêmea, juntamente com as substâncias cimentantes. Concomitantemente ao processo de reconfiguração do corpo cimentante, a região da membrana interna que contém as espículas no espermatóforo intacto é evertida e estirada sobre a base do espermatângio, sugerindo um papel auxiliar no processo de fixação. Com base em evidências da literatura, bem como nas obtidas no âmbito da presente Tese, no quarto capítulo propõe-se um modelo teórico para explicar como o aparato ejaculatório em evaginação seria capaz de perfurar e implantar-se no corpo da fêmea durante a reação espermatofórica. Sugere-se que a perfuração seria mecânica e resultado da ação conjunta do aumento gradual do diâmetro dos anéis do filamento espiral e da distância entre os mesmos, bem como do poder de ancoragem proporcionado pelas respectivas espículas. Finalmente, o quinto capítulo apresenta uma revisão da literatura acerca do fenômeno de implante de espermatóforos em Decapodiformes, e reúne evidências que corroboram o modelo teórico proposto. Neste capítulo, é apresentada também uma reinterpretação da função da reação espermatofórica em Octopodiformes. Com base no levantamento de diversos caracteres reprodutivos, foi possível testar hipóteses de evolução da estrutura do espermatóforo e do sistema de implante dos espermatângios, bem como hipóteses de co-evolução de estruturas envolvidas no processo de transferência e armazenamento de espermatozóides. Duas hipóteses principais acerca da evolução do sistema de implante dos espermatângios são propostas. / Male coleoid cephalopods produce elaborate spermatophores, which are transferred to the female during mating. These spermatophores are capable of functioning autonomously and extracorporeally, undergoing complicated changes during the so-called spermatophoric reaction, i.e., a complex process of evagination of the spermatophoric tunics and membranes that, ultimately, leads to the extrusion and attachment of the sperm mass on the females body. Few detailed morphological studies regarding this structure have yet been conducted, and much of the knowledge on the coleoid spermatophore was generated by classical studies of the 19th and early 20th centuries; furthermore, investigations on the functioning of this structure are even rarer. Since the extracorporeal functioning of coleoid spermatophores must rely entirely on the intricate structure and organization of the tunics, membranes, and other structures composing the spermatophore, only detailed investigations of these components would provide the basis for comprehending its mechanics. On these grounds, the present five-chapter Thesis aimed to provide solid evidence that could allow for postulating hypothesis on the functioning and evolution of this unique structure. In the first chapter, an investigation of the morphology of the spermatophore of Doryteuthis plei (Blainville, 1823) applying several microscopy techniques was carried on. A much more complex structural arrangement was revealed for the loliginid spermatophore, the most striking findings being: 1) the complex, layered structure of the middle membrane, which bears an additional, chemically distinct segment surrounding part of the cement body; 2) the presence of a space between the inner tunic and middle membrane filled with a fine reticulated material, presumably a viscous fluid in the fresh state; 3) the presence of stellate particles not only embedded in the spiral filament, but also closely applied to the inner membrane at the level of the cement body; 4) the presence of a pre-oral chamber in the cap region; and 5) the complex organization of the cement body, formed by two distinct layers encompassing contents of different chemical and textural properties. Careful literature reassessment suggests several of these features are common to loliginids, and to some extent to other squids. Their possible functional implications are discussed in light of our knowledge on the spermatophoric reaction mechanics. As part of the investigation on the spermatophoric reaction, and the mechanisms involved in the attachment of the sperm mass on the females body, it was found that the everting spermatophore, when directed towards the incised region of an experimental tissue sample, was able to readily penetrate the artificially exposed musculature, almost resembling natural deep implantation observed in some oceanic and deep-sea squids; this finding is reported in the second chapter, where it was hypothesized that the mechanism involved in deep implantation could be inherent to the 215 spermatophore structure of all squids. The third chapter investigated the functional morphology of the spermatophore of the squid D. plei applying in vitro analysis of the reaction, as well as light and electron microscopy investigation of spermatangia (everted spermatophores containing the sperm mass) obtained either in vitro or naturally attached on the female. Hitherto unnoticed functional features of the loliginid spermatophore revealed herein required a reappraisal of some important processes involved in the spermatophoric reaction, as well as the proposal of new hypotheses to explain their mechanics. The most striking findings concern to the attachment mechanism, which is not carried out solely by cement adhesive material, as previously believed, but rather by a complex process performed by multiple structures that lead to the implantation of the base of the spermatangium into the female body. Firstly, the everting ejaculatory apparatus is presumably able to superficially puncture the female tissue. Subsequently to this process, the cement body passes through a complex structural rearrangement, which leads to the injection of both its viscid cement contents and pointed oral region through the puncture into the female tissue. When the inner membrane at the oral region of the cement body is everted, its sharp stellate particles are exposed, presumptively adhering to the scarified tissue and augmenting attachment by assuring the injection of the cement material inside the superficial hole. The functioning of the loliginid spermatophore is revisited in light of these findings. The forth chapter, building upon evidence from the literature along with evidence from these experiments, proposes a theoretical model to explain how the everting ejaculatory apparatus would be able to mechanically perforate, and concomitantly implant the spermatophore into the female body during the spermatophoric reaction. It is proposed that this process is achieved chiefly through the combination of 1) an \"evaginating-helix\" mechanism performed by the everting ejaculatory apparatus\' spiral filament, and 2) the anchorage provided by its numerous, minute sharp stellate particles. Finally, the fifth chapter reviews the literature concerning the phenomenon of implantation of spermatophores in decapodiforms, and presents evidence corroborating the proposed theoretical model ascribing the role of implantation to the mechanical perforation performed by the spiral filament. The mechanisms of spermatophore transfer are also reviewed for octopodiforms, and a reinterpretation of the function of the spermatophoric reaction in this case is provided. In light of parsimonious character optimizations performed onto recently published phylogenetic trees, a complete ejaculatory apparatus with a spiral filament, as well as the spermatophoric reaction, apparently emerged once and early in the evolution of the Coleoidea. This novelty possibly provided an efficient attachment mechanism and presumably countered the changes associated with the adoption of an active mode of life by coleoids, augmenting fertilization success. Two main hypotheses for the evolution of the complex spermatophore within Coleoidea and Decapodiformes are proposed.

Cefalópodes coleóides (lulas, sépias e polvos) produzem espermatóforos muito elaborados, os quais são transferidos à fêmea durante a cópula por meio de um apêndice modificado nos machos. Durante a transferência à fêmea, os espermatóforos sofrem, de forma autônoma, drásticas modificações na chamada \"reação espermatofórica\", complexo processo de evaginação do aparato ejaculatório, que conduz à exteriorização da massa espermática e corpo cimentante. Poucos trabalhos abordam com detalhes a morfologia e anatomia funcional dos espermatóforos dos cefalópodes, grande parte do conhecimento acerca da estrutura do espermatóforo tendo sido gerada por trabalhos clássicos do século XIX e início do século XX. Investigações acerca do funcionamento dos espermatóforos são consideravelmente mais raras, estando o conhecimento básico sobre a reação espermatofórica restrito a apenas 20 espécies de coleóides. Como o funcionamento extracorpóreo dos espermatóforos depende exclusivamente da intrincada estrutura e organização de seus componentes (e.g., membranas e túnicas), somente investigações detalhadas dessas estruturas proverão as bases para a compreensão do funcionamento e da exata função do complexo espermatóforo dos coleóides. Nesse contexto, a presente Tese, organizada em cinco capítulos, teve como objetivo principal investigar a estrutura e o funcionamento dos espermatóforos da lula Doryteuthis plei (Blainville, 1823). No primeiro capítulo, com o objetivo de se estudar a fundo a organização estrutural dos espermatóforos da espécie, diversas técnicas de microscopia foram testadas e empregadas. Como resultado da combinação de diferentes ferramentas de análise, a estrutura do espermatóforo revelou-se ainda mais complexa, sendo as principais descobertas referentes à: 1) elaborada estrutura da membrana mediana, organizada em camadas e apresentando um segmento aboral quimicamente distinto, que envolve parte do corpo cimentante; 2) presença de um material reticulado preenchendo o espaço entre a túnica interna e a membrana mediana (discute-se a possibilidade do mesmo consistir em um fluido viscoso em espermatóforos intactos); 3) presença de espículas intimamente associadas à membrana interna na região do corpo cimentante (além das espículas embebidas no filamento espiral); 4) presença de extensões membranosas que delimitam uma câmara pré-oral na região do capuz; e 5) complexa organização estrutural do corpo cimentante, delimitado por duas camadas e contendo substâncias de distintas propriedades químicas. Uma avaliação cuidadosa da literatura permite sugerir que pelo menos parte dessas características deva ser comum aos espermatóforos de outros loliginídeos, e, em alguns casos, de outros grupos de coleóides. Como parte da investigação acerca da reação espermatofórica e dos mecanismos envolvidos na fixação da massa espermática no corpo da fêmea, constatou-se que, sob condições artificiais, espermatóforos em evaginação são capazes de penetrar musculatura exposta, de forma similar ao fenômeno de \"implante profundo\" observado naturalmente em algumas lulas oceânicas. Esse resultado foi descrito no segundo capítulo, no qual foi levantada a hipótese de que um mecanismo de perfuração seria inerente à estrutura dos espermatóforos dos coleóides. Dando continuidade ao estudo da morfologia funcional dos espermatóforos de D. plei, o terceiro capítulo apresenta os resultados obtidos a partir da investigação do funcionamento do espermatóforo e da morfologia dos espermatângios (i.e, espermatóforos evertidos) obtidos in vitro, bem como daqueles naturalmente fixados na fêmea. As evidências reunidas permitem afirmar que o processo de fixação compreende distintas fases desempenhadas por diversos componentes do espermatóforo, contrariamente a um conceito anterior de que a fixação seria realizada somente por substâncias adesivas do corpo cimentante. Durante a reação espermatofórica, o aparato ejaculatório e respectivo filamento espiral são capazes de perfurar superficialmente ou escarificar o tecido-alvo. Subseqüentemente, o corpo cimentante sofre drástica modificação estrutural, resultando na extrusão de parte do conteúdo cimentante, o qual é injetado diretamente sobre o tecido perfurado. Além disso, o corpo cimentante é exteriorizado com uma extremidade afilada que, em alguns casos, foi encontrada firmemente implantada no tecido da fêmea, juntamente com as substâncias cimentantes. Concomitantemente ao processo de reconfiguração do corpo cimentante, a região da membrana interna que contém as espículas no espermatóforo intacto é evertida e estirada sobre a base do espermatângio, sugerindo um papel auxiliar no processo de fixação. Com base em evidências da literatura, bem como nas obtidas no âmbito da presente Tese, no quarto capítulo propõe-se um modelo teórico para explicar como o aparato ejaculatório em evaginação seria capaz de perfurar e implantar-se no corpo da fêmea durante a reação espermatofórica. Sugere-se que a perfuração seria mecânica e resultado da ação conjunta do aumento gradual do diâmetro dos anéis do filamento espiral e da distância entre os mesmos, bem como do poder de ancoragem proporcionado pelas respectivas espículas. Finalmente, o quinto capítulo apresenta uma revisão da literatura acerca do fenômeno de implante de espermatóforos em Decapodiformes, e reúne evidências que corroboram o modelo teórico proposto. Neste capítulo, é apresentada também uma reinterpretação da função da reação espermatofórica em Octopodiformes. Com base no levantamento de diversos caracteres reprodutivos, foi possível testar hipóteses de evolução da estrutura do espermatóforo e do sistema de implante dos espermatângios, bem como hipóteses de co-evolução de estruturas envolvidas no processo de transferência e armazenamento de espermatozóides. Duas hipóteses principais acerca da evolução do sistema de implante dos espermatângios são propostas. / Male coleoid cephalopods produce elaborate spermatophores, which are transferred to the female during mating. These spermatophores are capable of functioning autonomously and extracorporeally, undergoing complicated changes during the so-called spermatophoric reaction, i.e., a complex process of evagination of the spermatophoric tunics and membranes that, ultimately, leads to the extrusion and attachment of the sperm mass on the females body. Few detailed morphological studies regarding this structure have yet been conducted, and much of the knowledge on the coleoid spermatophore was generated by classical studies of the 19th and early 20th centuries; furthermore, investigations on the functioning of this structure are even rarer. Since the extracorporeal functioning of coleoid spermatophores must rely entirely on the intricate structure and organization of the tunics, membranes, and other structures composing the spermatophore, only detailed investigations of these components would provide the basis for comprehending its mechanics. On these grounds, the present five-chapter Thesis aimed to provide solid evidence that could allow for postulating hypothesis on the functioning and evolution of this unique structure. In the first chapter, an investigation of the morphology of the spermatophore of Doryteuthis plei (Blainville, 1823) applying several microscopy techniques was carried on. A much more complex structural arrangement was revealed for the loliginid spermatophore, the most striking findings being: 1) the complex, layered structure of the middle membrane, which bears an additional, chemically distinct segment surrounding part of the cement body; 2) the presence of a space between the inner tunic and middle membrane filled with a fine reticulated material, presumably a viscous fluid in the fresh state; 3) the presence of stellate particles not only embedded in the spiral filament, but also closely applied to the inner membrane at the level of the cement body; 4) the presence of a pre-oral chamber in the cap region; and 5) the complex organization of the cement body, formed by two distinct layers encompassing contents of different chemical and textural properties. Careful literature reassessment suggests several of these features are common to loliginids, and to some extent to other squids. Their possible functional implications are discussed in light of our knowledge on the spermatophoric reaction mechanics. As part of the investigation on the spermatophoric reaction, and the mechanisms involved in the attachment of the sperm mass on the females body, it was found that the everting spermatophore, when directed towards the incised region of an experimental tissue sample, was able to readily penetrate the artificially exposed musculature, almost resembling natural deep implantation observed in some oceanic and deep-sea squids; this finding is reported in the second chapter, where it was hypothesized that the mechanism involved in deep implantation could be inherent to the 215 spermatophore structure of all squids. The third chapter investigated the functional morphology of the spermatophore of the squid D. plei applying in vitro analysis of the reaction, as well as light and electron microscopy investigation of spermatangia (everted spermatophores containing the sperm mass) obtained either in vitro or naturally attached on the female. Hitherto unnoticed functional features of the loliginid spermatophore revealed herein required a reappraisal of some important processes involved in the spermatophoric reaction, as well as the proposal of new hypotheses to explain their mechanics. The most striking findings concern to the attachment mechanism, which is not carried out solely by cement adhesive material, as previously believed, but rather by a complex process performed by multiple structures that lead to the implantation of the base of the spermatangium into the female body. Firstly, the everting ejaculatory apparatus is presumably able to superficially puncture the female tissue. Subsequently to this process, the cement body passes through a complex structural rearrangement, which leads to the injection of both its viscid cement contents and pointed oral region through the puncture into the female tissue. When the inner membrane at the oral region of the cement body is everted, its sharp stellate particles are exposed, presumptively adhering to the scarified tissue and augmenting attachment by assuring the injection of the cement material inside the superficial hole. The functioning of the loliginid spermatophore is revisited in light of these findings. The forth chapter, building upon evidence from the literature along with evidence from these experiments, proposes a theoretical model to explain how the everting ejaculatory apparatus would be able to mechanically perforate, and concomitantly implant the spermatophore into the female body during the spermatophoric reaction. It is proposed that this process is achieved chiefly through the combination of 1) an \"evaginating-helix\" mechanism performed by the everting ejaculatory apparatus\' spiral filament, and 2) the anchorage provided by its numerous, minute sharp stellate particles. Finally, the fifth chapter reviews the literature concerning the phenomenon of implantation of spermatophores in decapodiforms, and presents evidence corroborating the proposed theoretical model ascribing the role of implantation to the mechanical perforation performed by the spiral filament. The mechanisms of spermatophore transfer are also reviewed for octopodiforms, and a reinterpretation of the function of the spermatophoric reaction in this case is provided. In light of parsimonious character optimizations performed onto recently published phylogenetic trees, a complete ejaculatory apparatus with a spiral filament, as well as the spermatophoric reaction, apparently emerged once and early in the evolution of the Coleoidea. This novelty possibly provided an efficient attachment mechanism and presumably countered the changes associated with the adoption of an active mode of life by coleoids, augmenting fertilization success. Two main hypotheses for the evolution of the complex spermatophore within Coleoidea and Decapodiformes are proposed.

Several studies suggest the environment influences chokka squid catches which are mostly based on the successful formation of inshore spawning aggregations. None of the evidence, however, is direct observation. Acoustic telemetry offers a means to determine the response of spawners to changes in the environment and insight into the behaviour of spawning squid. A hexagonal array of VR2 receivers deployed 500 m apart was deemed to be ideal to monitor the movement patterns of squid on the spawning sites. In isothermic conditions, an area up to 1.28 km2 could be monitored as there was an approximate 50 m overlap in individual VR2 receiver range. In thermocline conditions however, “acoustic dead zones” as wide as 350 m may have existed between VR2 receivers, limiting the performance of this configuration. Similarly benthic turbidity events would also decrease detection range and limit performance. A hexagonal array of VR2 receivers was moored in Kromme Bay on and around active spawning aggregations during the squid fishery closed seasons of November 2003, 2004, 2005 and 2006. Squid were caught on jigs and tagged with V9 acoustic pressure telemetry transmitters. A total of 45 animals were tagged. Presence-absence analysis identified three general behaviours: (1) arrival at dawn and departure after dusk, (2) a continuous and uninterrupted presence for a number of days and (3) presence interrupted by frequent but short periods of absence. Overall, the data suggests frequent migrations between spawning aggregations and offshore feeding grounds. The pressure sensor data showed both males and females stayed persistently near the seabed during the day, but at night, this pattern was broken with common activity higher up in the water column. The squid did not remain exclusively in the water column and regularly made excursions to the seabed. CTD and temperature data indicated the intrusion of a cold bottom layer due to upwelling at the monitored spawning sites on a number of occasions. The formation of spawning aggregations appears to be triggered by upwelling events and spawning behaviour, once initiated, disrupted by upwelling events with a rapid onset, possibly due to an inability to adapt physiologically over such a short time period.

It is currently hypothesised that the chokka squid (Loligo reynaudi) consist of a single stock. This was tested through a spatial comparison of the morphology of this species. Forty three morphometric characters were measured from 1079 chokka squid collected from three regions: the south coast of South Africa, the west coast of South Africa, and southern Angola. While no significant differences were found for the hard body parts, results from discriminant function analyses showed the soft body morphometric characters from each of the three regions differed, with an overall correct classification rate of 100% for males and 99% for females in all three regions. Due to the existing model being used to assess the resource currently being updated it was not feasible to apply this model to the area-disaggregated data from this study. Rather, the CPUE trends and catches from the area-disaggregated data were compared against those of the area-aggregated data, as a first attempt to discern any appreciable differences which would suggest the use of disaggregated data in future assessments. Both the trawl and jig CPUE trends from the area-disaggregated analysis differed only slightly from those of the area-aggregated data. Similarly, the spring and autumn biomass trends for the main spawning area (east of 22°E) followed the same trends as for the full area. It is therefore concluded that there is currently no need to assess the resource on an area-disaggregated basis.