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Scanning electron microscopy (SEM) enables a close-up investigation of topographical features at the cellular level down to a resolution of about 3 nm. In terms of gametes this allows for examination of minute surface details and changes that occur during fertilization. Sometimes these features are hidden from view and must be carefully exposed during preparation in order to be made visible in the SEM. This chapter describes methods that can reveal surface details of gametes for observation, as well as fix them permanently, while keeping fixation artifacts to a minimum.

Early events of fertilization are described in Chaetopleura apiculata and other selected Chitonida. C. apiculata egg hulls are elaborated into multi-branched spines with interlocking polygonal bases. Around the perimeter of each base are a series of open pores, ranging in size from 0.1-0.5 microm, which permit sperm direct access to the vitelline layer. In Callochitonidae (Chitonida) even larger pores occur in egg jelly coats, but this is considered to be the plesiomorphic condition, found also in Lepidopleurida such as Deshayesiella curvata. Other Chitonina, such as Rhyssoplax tulipa and Acanthopleura granulata, have a continuous outer dense layer that lacks pores and must be digested by penetrating sperm. Fertilization in Chitonida is unique and involves injection of chromatin into the egg via a narrow tubular nuclear extension that appears to exclude other sperm organelles, including mitochondria, centrioles, and flagellum. New evidence from studies of fertilization in Mopalia muscosa (Chitonida: Acanthochitonina) supports this hypothesis. This type of fertilization implies maternal inheritance of both mitochondria and centrioles, which is highly unusual, because in most animals one sperm centriole assists movements of pronuclei and regulates organization of the mitotic spindle. This mechanism of fertilization is defined by a series of apomorphic characters that unify the order Chitonida.

Fifty years ago, it was reported that individuals of the Pacific boreal limpet Problacmaea (= Erginus) bear a penis for copulation and brood their young in the pallial cavity. These observations were based on light microscopy, but now we reveal new details of the reproductive biology of Erginus (Problacmaea) puniceus with electron microscopy. Gametogenesis is fundamentally similar to other Patellogastropoda, but there are some key differences. Oocytes develop in the ovary, dorsal to the foot in the posterior half of the body, before passing through the oviduct to the pallial cavity, where they are fertilized and brooded. Development is direct with embryos being brooded to the crawl-away juvenile stage. However, sometimes fertilization occurs internally with embryos beginning development in or near the gonad. Free sperm, which were likely autosperm (self) but could have been allosperm (cross), were found near developing oocytes in the gonad. Spermiogenesis results in the formation of an enta-quasperm with a cap-like acrosome, a bullet-shaped nucleus and spherical mitochondria in the midpiece. Copulation was not observed, but we confirm the presence of a penis with a dorsal penial groove that lies below the right cephalic tentacle in males, which is typical of functional penises of some molluscs. Males were always smaller, but intermediate-sized individuals were hermaphroditic to different extents. The largest individuals were entirely female, and their penises were smaller and often lacked the penial groove. Thus, E. (Problacmaea) puniceus appears to be a protandrous hermaphrodite, passing from male to female phase. Remnants of the testis persist into the female phase, suggesting that they are protandrous hermaphrodites with overlap, possibly allowing for self-fertilization to occur on occasion. However, the largest females lacked any testis remnants, suggesting instead that the species may be protandrous sequential hermaphrodites. [ABSTRACT FROM AUTHOR]

Egg hull formation during oogenesis in the chiton Callochiton dentatus does not follow the typical model of merocrine secretion involving Golgi vesicle exocytosis. Instead, microapocrine secretions are primarily responsible for egg hull formation, although merocrine secretions contribute 'areolae' and the vitelline layer. Microapocrine secretion mechanisms are poorly understood, involving a different cellular pathway than is typical. Egg hull formation in C. dentatus involves two types of microapocrine secretions released by the oocyte, one of which is described here for the first time. The plesiomorphic jelly-like egg hull of chitons, as exemplified by the eggs of members of the basal order Lepidopleurida and present also in eggs of C. dentatus (Chitonida: Callichitonidae), may have evolved solely as an oocyte secretion, whereas members of some other families in the order Chitonida form their egg hulls with considerable secretory input from the follicle cells as well. [ABSTRACT FROM AUTHOR]