Formation of an egg envelope in the pycnogonid, Propallene longiceps (Pycnogonida, Callipallenidae)

Vitellogenic oocytes of all pycnogonids studied so far contain dilated elements of the endoplasmic reticulum, filled with characteristic electron-dense bodies. During vitellogenesis these bodies fuse and form larger, almost spherical granules that were traditionally interpreted as nascent yolk granules. Here, we present the results of ultrastructural investigations of previtellogenic and early vitellogenic oocytes of Propallene longiceps (Pycnogonida, Callipallenidae). We show that the intra-cisternal bodies/granules of pycnogonids are not involved in vitellogenesis but contain macromolecules that are released from the oocyte and contribute to the formation of an egg envelope. The obtained results are discussed in a phylogenetic context. We suggest that the presence of the intra-cisternal electron-dense bodies in the oocyte cytoplasm represents a plesiomorphic character of arthropods inherited from the arthropod ancestor.     

Postembryonic development of Tanystylum bealensis (Pycnogonida, Ammotheidae) from Barkley Sound, British Columbia, Canada

All developmental stages of Tanystylum bealensis are described. This is the first complete developmental series of a pycnogonid species collected from a natural, rather than a laboratory-reared population. Development proceeds through a series of nine instars from egg to adult, with major defining characteristics of the instars being the addition of walking legs, loss of chelifores, and modification of larval appendages into adult palps and ovigers. All stages are free-living on the hydroid Plumularia setacea, except for the first instar (protonymphon), which remains on the ovigers of the adult male for a short time after hatching. Development in T. bealensis is compared to development in both the closely related species T. orbiculare and to the more distantly related Achelia alaskensis.     

Morphogenesis of Pseudopallene sp. (Pycnogonida, Callipallenidae) II: postembryonic development

Pycnogonida (sea spiders) are bizarre marine arthropods that are nowadays most frequently considered as being the sister group to all other chelicerates. The majority of pycnogonid species develops via a protonymphon larva with only three pairs of limbs affiliated with the future head region. Deviating from this, the hatching stage of some representatives shows already an advanced degree of trunk differentiation. Using scanning electron microscopy, fluorescent nucleic staining, and bright-field stereomicroscopy, postembryonic development of Pseudopallene sp. (Callipallenidae), a pycnogonid with an advanced hatching stage, is described. Based on external morphology, six postembryonic stages plus a sub-adult stage are distinguished. The hatching larva is lecithotrophic and bears the chelifores as only functional appendage pair and unarticulated limb buds of walking leg pairs 1 and 2. Palpal and ovigeral larval limbs are absent. Differentiation of walking leg pairs 3 and 4 is sequential. Apart from the first pair, each walking leg goes through a characteristic sequence of three externally distinct stages with two intermittent molts (limb bud-seven podomeres-nine podomeres). First external signs of oviger development are detectable in postembryonic stage 3 bearing three articulated walking leg pairs. Following three more molts, the oviger has attained adult podomere composition. The advanced hatching stages of different callipallenids are compared and the inclusive term "walking leg-bearing larva" is suggested, as opposed to the behavior-based name "attaching larva". Data on temporal and structural patterns of walking leg differentiation in other pycnogonids are reviewed and discussed. To facilitate comparisons of walking leg differentiation patterns across many species, we propose a concise notation in matrix fashion. Due to deviating structural patterns of oviger differentiation in another callipallenid species as well as within other pycnogonid taxa, evolutionary conservation of characteristic stages of oviger development is not apparent even in closely related species.     

Morphogenesis of Pseudopallene sp. (Pycnogonida, Callipallenidae) I: embryonic development

Embryonic development of Pycnogonida (sea spiders) is poorly understood in comparison to other euarthropod lineages with well-established model organisms. However, given that pycnogonids potentially represent the sister group to chelicerates or even to all other euarthropods, their development might yield important data for the reconstruction of arthropod evolution. Using scanning electron microscopy, fluorescent nucleic staining and immunohistochemistry, the general course of embryonic morphogenesis in Pseudopallene sp. (Callipallenidae), a pycnogonid with prolonged embryonic development, is described. A staging system comprising ten stages is presented, which can be used in future studies addressing specific developmental processes. The initially slit-like stomodeum anlage forms at the anterior end of an eight-shaped germ band and predates proboscis outgrowth. The latter process is characterized by the protrusion of three cell populations that are subsequently involved in pharynx formation. In later stages, the proboscis assumes distally a horseshoe-like shape. At no time, a structure corresponding to the euarthropod labrum is detectable. Based on the complete lack of palpal and ovigeral embryonic limbs and the early differentiation of walking leg segments 1 and 2, the existence of an embryonized protonymphon stage during callipallenid development is rejected. The evolution of pycnogonid hatching stages, especially within Callipallenidae and Nymphonidae, is re-evaluated in the light of recent phylogenetic analyses. Specifically, the re-emergence of the ancestral protonymphon larva (including re-development of palpal and ovigeral larval limbs) and a possible re-appearance of adult palps in the nymphonid lineage are discussed. This challenges the perception of pycnogonid head appendage evolution as being driven by reduction events alone.     

Postembryonic development of Nymphon unguiculatum Hodgson 1915 (Pycnogonida, Nymphonidae) from the South Shetland Islands (Antarctica)

Male specimens of the sea spider species Nymphon unguiculatum, carrying eggs, larvae and postlarvae in various stages of development, were collected off the South Shetland Island at water depths between 112 and 472 m in austral summer 2006/2007. Here, we describe the external morphology of four postembryonic stages (protonymphon, instar 1, instar 2, and instar 3) carried by these specimens. We found that (1) protonymphon larvae hatch from the eggs; (2) larvae and postlarval stages have yolk reserves and are characterized by a relatively large size (average body lengths of 0.46, 0.55, 0.65 and 0.73 mm in the successive stages); (3) postlarvae remain on the ovigerous legs of males during several moults; (4) a spinning apparatus is present; (5) the development of walking legs is sequential. The larval and postlarval development of N. unguiculatum is compared with that known from other pycnogonid species.     

Development of the female reproductive system in the freshwater crayfish Cherax quadricarinatus (Decapoda, Parastacidae)

Abstract. The differentiation of the female reproductive system from a macroscopic and microscopic point of view was studied in Cherax quadricarinatus. For this characterization, 184 females were dissected and processed for the histological analysis. From the differentiation of the ovary up to its maturity, three ovarian morphotypes could be distinguished macroscopically: parallel strands without any contact between them, an H-shaped ovary, and a Y-shaped ovary. These morphotypes were compared within the Astacida. Four ovarian developmental stages were recognized based on ovary color, and the histological structure and relative proportion of cellular types. The post-spawning ovary was also characterized. The components of the female reproductive system sheath were described and its modifications in the ovary and oviducts were determined and compared. Theoretical aspects of the study of sexual differentiation in C. quadricarinatus were discussed within a phylogenetic framework.     

Morphology of female reproductive system of Mediterranean flatheaded peachborer,Capnodis tenebrionis (Linnaeus, 1761) (Coleoptera: Buprestidae)

Capnodis tenebrionis causes damage in many species of Rosaceae. The present study investigates on the morphology of the female reproductive system of C. tenebrionis. The female reproductive system of C. tenebrionis has a pair of ovaries, lateral oviducts, a common oviduct, spermatheca, and bursa copulatrix. Each ovary in C. tenebrionis consists of approximately 24 telotrophic meroistic type ovarioles. The ovarioles of C. tenebrionis have four regions (terminal filament, tropharium, vitellarium, and pedicel). Tropharium have trophocytes, young oocytes, and prefollicular cells. Vitellarium consists of previtellogenic, vitellogenic, and choriogenic oocytes. Previtellogenic oocyte is surrounded by cylindrical epithelial cells. Its ooplasm is homogeneous and basophilic. In vitellogenic oocyte, there are intercellular spaces between monolayered follicle cells. Its ooplasm has yolk granules and lipid droplets. Choriogenic oocyte are surrounded by chorion and single-layered cylindrical cells. There are yolk granules and lipid droplets in its ooplasm which is asidophilic. In C. tenebrionis female, spermatheca and bursa copulatrix wall is surrounded by thin cuticular intima, monolayer epithelial, glandular cells, and muscle layer. Spermatheca lumen contains a large number of spermatozoa. Bursa copulatrix lumen is filled with secretory material. This study may be useful in terms of the morphology of mature female reproductive organs of Buprestidae and other coleopteran species.     

Adaptations for matrotrophy in the female reproductive system in the pseudoscorpion Chelifer cancroides (Chelicerata: Pseudoscorpiones,Cheliferidae)

Pseudoscorpiones (pseudoscorpions, false scorpions) is an order of small terrestrial chelicerates. While most chelicerates are lecithotrophic, that is, embryos develop due to nutrients (mostly yolk) deposited in the oocyte cytoplasm, pseudoscorpions are matrotrophic, that is, embryos are nourished by the female. Pseudoscorpion oocytes contain only a small amount of yolk. The embryos develop within a brood sac carried on the abdominal site of the female and absorb nutrients by a pumping organ. It is believed that in pseudoscorpions nutrients for developing embryos are produced in the ovary during a postovulatory (secretory) phase of the ovarian cycle. The goal of our study was to analyze the structure of the female reproductive system during the secretory phase in the pseudoscorpion Chelifer cancroides, a representative of the family Cheliferidae, considered to be one of the most advanced pseudoscorpion taxa. We use diverse microscopic techniques to document that the nutritive fluid is produced not only in the ovaries but also by the epithelial cells in the oviducts. The secretory active epithelial cells are hypertrophic and polyploid and release their content by fragmentation of apical parts. Our observations also indicate that fertilization occurs in the oviducts. Moreover, in contrast to previous findings, we show that secretion of the nutritive material starts when the fertilized oocytes reach the brood sac and thus precedes formation of the pumping organ. Summing up, we show that C. cancroides exhibits traits of advanced adaptations for matrotrophy due to coordinated secretion of the nutritive fluid by the ovarian and oviductal epithelial cells, which substantially increases the efficiency of nutritive fluid formation. Since the secretion of nutrients starts before formation of the pumping organ, we suggest that the embryos are able to absorb the nutritive fluid also in the early embryonic stages.     

Postembryonic development of Antygomonas incomitata (Kinorhyncha: Cyclorhagida)

Postembryonic development in the kinorhynch species Antygomonas incomitata was examined using scanning electron microscopy. The morphology of the six juvenile stages, J‐1 to J‐6, varies at numerous details, but they can also be distinguished by a few key characters. Juvenile stage 1 by its composition of only nine trunk segments; J‐2 by the combination of possessing 10 trunk segments, but no cuspidate spines on segment 9; J‐3 by the presence of cuspidate spines on segment 9, but only one pair of cuspidate spines on segment 8; J‐4 by the combination of 10 trunk segments only, but having two pairs of cuspidate spines on segment 8; J‐5 by possessing 11 trunk segments and same spine compositions as adults but is still maintaining postmarginal spiculae; J‐6 specimens closely resemble adults and are most easily identified by their reduced trunk lengths. New segments are formed in a growth zone in the anterior part of the terminal segment. The complete number of segments is reached in J‐5. Development of cuticular head and trunk structures are described through all postembryonic stages and following developmental patterns could be outlined: the mouth cone possesses outer oral styles from J‐1, but in J‐1 to J‐3, the styles alternate in size. Scalids of the introvert are added after each molt, and scalids appear earliest in the anterior rings, whereas scalids in more posterior rings are added in older postembryonic stages. The early J‐1 stage is poor in spines and sensory spots and both structures increase in number after each molt. The complete spine composition is reached in J‐4, whereas new sensory spots appear after all molts, inclusive the final one from J‐6 to adult. Sensory spots in the paraventral positions often appear as Type 3 sensory spots but are through development transformed to Type 2. This transformation happens earliest on the anterior segments. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Microscopic structures of the ovary and female genital ducts of Supachai's caecilian,Ichthyophis supachaii Taylor, 1960 (Amphibia: Gymnophiona)

The structures of the female reproductive system (ovary, oviduct and cloaca) of Ichthyophis supachaii were investigated by dissection, histology and light microscopy. Paired, elongated, sac‐like ovaries are parallel to the gut and fat bodies. Follicle stages include germinal nests of oogonia and primary oocytes, early and late previtellogenic follicles, early and late vitellogenic follicles and atretic follicles. Germinal nests of oogonia comprise oogonia and prefollicular cells. Nests of primary oocytes contain clusters of synchronously developing primary oocytes enclosed by connective tissue. Primary oocytes are associated with follicular cells. Previtellogenic follicles initially form the vitelline envelope, theca cell layers and patches of ooplasmic glycoproteins. Vitellogenic follicles contain heterogeneously sized spherical yolk granules. Atresia is present in several stages of developing follicles. The oviduct is divided into the anterior, middle and posterior parts. All oviductal parts are lined by non‐ciliated epithelium. A small number of mucous cells are present in the middle part. The cloaca of female I. supachaii is divided into the anterior and posterior chambers. The anterior chamber is lined by glandular stratified columnar epithelium, while the posterior chamber has stratified cuboidal epithelium with less mucus production. Our results contribute to useful information on the reproductive biology of caecilians.     

Localization of transient receptor potential ion channels in primary and motile cilia of the female murine reproductive organs

We have examined the subcellular localization of transient receptor potential (TRP) ion channels and the potential sensory role of cilia in murine female reproductive organs using confocal laser scanning microscopy analysis on ovary and oviduct tissue sections as well as on primary cultures of follicular granulosa cells. We show that the Ca2+ permeable cation channel, polycystin-2, as well as polycystin-1, a receptor that forms a functional protein complex with polycystin 2, distinctively localize to primary cilia emerging from granulosa cells of antral follicles in vivo and in vitro. Both polycystins are localized to motile oviduct cilia and this localization is greatly increased upon ovulatory gonadotropic stimulation. Further, the Ca2+ permeable cation channel, TRP vaniloid 4 (TRPV4), localizes to a sub-population of motile cilia on the epithelial cells of the ampulla and isthmus with high intensity in proximal invaginations of the epithelial folds. These observations are the first to demonstrate ciliary localization of TRP ion channels and their possible receptor function in the female reproductive organs. We suggest that polycystins 1 and 2 play an important role in granulosa cell differentiation and in development and maturation of ovarian follicles. In the oviduct both TRPV4 and polycystins could be important in relaying physiochemical changes in the oviduct upon ovulation.     

A scanning electron microscopy study of the embryonic development of Pycnogonum litorale (Arthropoda,Pycnogonida)

The phylogenetic position of the enigmatic Pycnogonida (sea spiders) is still controversial. This is in part due to a lack of detailed data about the morphology and ontogenesis of this, in many aspects, aberrant group. In particular, studies on the embryonic development of pycnogonids are rare and in part contradictory. Here, we present the first embryological study of a pycnogonid species using scanning electron microscopy (SEM). We describe the late embryogenesis of Pycnogonum litorale from the first visible appendage anlagen to the hatchling in 11 embryonic stages. The three pairs of appendage anlagen gain in length by growth, as well as by extension of furrows into the embryo. The opening of the stomodaeum is located far in front of the anlagen of the chelifores and has a Y‐shaped lumen from the onset. During further embryogenesis, the position of the mouth shifts ventrally, until it is located between the chelifores. The proboscis anlage grows out as a circumoral wall‐like structure, which is initially more pronounced ventrally. Hypotheses about the evolution of the proboscis by fusion of originally separated components are critically discussed, because the proboscis anlage of P. litorale shows no indications of a composite nature. In particular, a participation of post‐cheliforal elements in proboscis formation is rejected by our data. Further, no preoral structure and no stage in proboscis formation was found, which could plausibly be homologized with the labrum of othereuarthropods. Thus, our study supports the assumption of a complete lack of a labrum in Pycnogonida. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Lysosomal hydrolases in reproductive organs during estrous cycle of the hamster

Changes in the total protein content and the activities of lysosomal hydrolases (arylsulfatase, acid phosphatases, β-glucuronidase, β-N-acetylhexosaminidase, α-L-fucosidase, and β-galactosidase) of the hamster genital tract during the 4 days of estrous cycle and in hormonally superovulated hamsters were measured. Levels of lysosomal hydrolases in uteri and uterine fluid changed significantly during the cycle. Similar changes were observed in uterine wet weight and uterine proteins. The pattern of enzyme activities in both the ovary and the oviduct were different from those in uteri. In the ovary, most enzyme activities and the total protein concentration remained elevated after ovulation. Protein concentration and enzyme activities were significantly higher in the ovary, oviduct, and uteri of superovulated hamsters as compared to controls.     

Postembryonic development of the sea spider Ammothella biunguiculata (Pycnogonida,Ammotheidae) endoparasitic to an actinian Entacmaea quadricolor (Anthozoa,Stichodactylidae) in Izu Peninsula,Japan

Postembryonic developmental stages of an endoparasitic pycnogonid, Ammothella biunguiculata in Izu Peninsula, Japan are described. Eleven stages were identified beginning with a protonymphon larva attached to the male oviger. We found endoparasitic individuals in the host actinian from the second to tenth instar, and forms in the ninth stage to adult were found free-living. This indicates a transition from being endoparasitic to free-living during the ninth to tenth instar stages. The first instar protonymphon attached to the adult male oviger has a gland duct on the anterior margin of each chelifore scape which completely disappears with the second instar. The disappearance of the chelifore gland duct coincides with the beginning of an endoparasitic stage in the development of this species. Although the larval morphology and the postembryonic development of pycnogonids have been summarized by several authors, the present study concludes that much remains to be learnt.     

A histological and ultrastructural investigation of the female reproductive system of the water snake (Erythrolamprus miliaris): Oviductal cycle and sperm storage

We studied the structural and cellular organisation of the oviduct of Erythrolamprus miliaris including its morphological variation during the reproductive cycle using light microscopy, scanning electron microscopy and transmission electron microscopy. Four anatomically distinct regions compose the oviduct of E. miliaris including the anterior and posterior infundibulum, glandular uterus, non-glandular uterus and pouch. The cells of the oviductal epithelium secrete material by apocrine and merocrine processes, which vary between the anatomical regions and according to each phase of the reproductive cycle. The infundibular epithelium secretes electron dense vacuoles, which suggests the production of lipids, whereas the epithelial secretion of the glandular uterus, non-glandular uterus and pouch creates lucent and slightly electron dense vacuoles, indicating the production of glycoproteins. The timing of mating, vitellogenesis and sperm storage directly influences the morphofunctional alterations in the oviducts of E. miliaris. Sperm storage occurs only in the infundibular receptacles with increased production of the neutral carbohydrates in the presence of male gametes. Sperm storage happens in vitellogenic, non-vitellogenic and pregnant females of E. miliaris. Thus, females may be able to produce multiple clutches at different seasons of the year regardless of mating during autumn.     

Histological and ultrastructural investigation of the female reproductive system of Argulus bengalensis Ramakrishna, 1951 (Crustacea: Branchiura)

In order to understand branchiuran reproductive biology, it is imperative to know the sites of oogenesis and oocyte maturation, locate the accessory reproductive glands, and identify the fertilization site with the present knowledge of the sperm transfer mechanism of the genus Argulus. With these objectives, we attempted to describe the female reproductive system of Argulus bengalensis using serial histological sections through the ovaries and associated ducts in the transverse, longitudinal, and sagittal planes. The reproductive organs include a median ovary, one pair of ovarian lumina, a median oviduct, and a pair of collateral accessory glands. A duct from each of the collateral accessory glands leads into the proximal part of the median oviduct, which opens to the exterior through a genital opening at the distal end. The glandular secretion presumably contributes to the jelly coat of the egg. The ovary is bound with a tunica propria which extends further diametrically inside the ovary forming the paired lumina. The lumina are confluent into the median oviduct. Two distinct areas, the germarium and differentiating zones, are clearly distinguishable within the ovary. The tunica propria itself houses the oogonia within a matrix, serving as the germarium. Transmission electron micrograph reveals that the matrix is made of collagen. The collagen matrix confers elasticity to the tunica propria to accommodate the postvitellogenic oocytes within the ovarian lumen. The differentiating zone is situated in between the germarium: dorsally it is covered with a chromatophore layer. The ovary is ensheathed by a circum ovarian striated muscle. The presence of spermatophores in the ovarian lumen indicates the fertilization site. J. Morphol. 277:707–716, 2016. © 2016 Wiley Periodicals, Inc.     

Primary homology assessment of structures in the female atrial system among species of the Polycystididae (Rhabditophora, Eukalyptorhynchia)

Abstract. The female atrial system of members of the Polycystididae has been studied at the light microscopical level and compared among the constituent taxa. Based on the criteria of position and conjunction, hypotheses of homology are put forward and compared with the assessments of homology found in earlier literature. Contradictory terminology is synonymized and adapted to fit new findings that recognize homology among structures in different taxa. Based on differences in position and structure, 2 types of female duct are recognized: female duct type I and female duct type II. The term “female bursa” is restricted to a sperm resorbing organ at the proximal end of the female duct type I and/or at the end of a common oviduct. Some species have bundles of glands at the place where the oviduct(s) enter the female duct type I, which are considered homologous among these species. Different types of seminal receptacles are recognized. The term “insemination duct” is restricted to the ducts which in some species, in addition to the oviducts, connect the ovaries with the female duct type I. A single muscular duct, the common oviduct, connects the joined oviducts with the female duct type I in species of Duplacrorhynchus and is considered homologous with the similar duct present in some other species. A uterus is always present, entering the common genital atrium through its frontal wall, or entering a female duct type II. This morphological review of the female atrial system reveals a mosaic distribution of characters if applied to previous phylogenies that are based primarily on the male reproductive system.     

Ultrastructural investigations of the female genital system of Epiperipatus biolleyi ( Bouvier 1902) (Onychophora,Peripatidae)

Brockmann, C., Mummert, R., Ruhberg, H. and Storch, V. 1999. Ultrastructural investigations of the female genital system of Epiperipatus biolleyi (Bouvier 1902) (Onychophora, Peripatidae). — Acta Zoologica (Stockholm) 80: 339–349. The female genital system of the neotropical peripatid Epiperipatus biolleyi was examined using transmission and scanning electron microscopy. Special attention is given to the two accessory organs of the paired oviducts: the receptacula seminis and the ovarian funnels (Ovarialtrichter). The latter occur only in the Peripatidae, whereas receptacula seminis may also be present in the Peripatopsidae, the only other family in the Onychophora. The ovarian funnels of E. biolleyi are thin-walled and closed from the haemocoel. This trait has also been reported from Epiperipatus trinidadensis, Macroperipatus torquatus, and Eoperipatus weldoni, whereas other peripatids have ovarian funnels which have been reported to open into the haemocoel. The occurrence of artificially opened ovarian funnels, caused by tissue rupture during specimen preparation, is discussed. The presence of spermatozoa both in the receptaculum seminis and in parts of the uterus of the female examined in this study supports the hypothesis that in E. biolleyi insemination of juvenile females occurs directly via the genital opening. The female contained one unstalked cleavage embryo in each uterus horn. Two features of the uterus were found to be unique to E. biolleyi: (1) a second cell layer overlying the uterine epithelium with a pronounced secretory activity (2) embryos are enclosed in a noncellular coat interspersed with numerous transport vesicles.     

Ultrastructural investigations of the ovary and oogenesis in the pycnogonids Cilunculus armatus and Ammothella biunguiculata (Pycnogonida, Ammotheidae)

Abstract. Ovarian ultrastructure and oogenesis in two pycnogonid species, Cilunculus armatus and Ammothella biunguiculata , were investigated. The ovary is morphologically and functionally divided into trunk and pedal parts. The former represents the germarium and contains very young germ cells in a pachytene or postpachytene phase, whereas the latter houses developing previtellogenic and vitellogenic oocytes and represents the vitellarium. Intercellular bridges were occasionally found between young (trunk) germ cells. This indicates that in pycnogonids, as in other animal groups, at the onset of oogenesis clusters of germ cells are generated. As nurse cells are absent in the ovaries of investigated species, the clusters must secondarily split into individual oocytes. In the vitellarium, the oocytes are located outside the ovary. Each oocyte is connected to the ovarian tissue by a stalk composed of several somatic cells. The stalk cells directly associated with the oocyte are equipped with irregular projections that reach the oocyte plasma membrane. This observation suggests that the stalk cells may play a nutritive role. The ooplasm of vitellogenic oocytes comprises mitochondria, free ribosomes, stacks of annulate lamellae, active Golgi complexes, and vesicles derived from these complexes. Within the latter, numerous electron-dense bodies are present. We suggest that these bodies contribute to yolk formation.