Ultrastructural aspects of the germovitellarium of two prorhynchids (Platyhelminthes,Lecithoepitheliata)

Summary

The female gonad of two fresh-water prorhynchids, Geocentrophora baltica and Prorhynchus stagnalis, has been investigated by means of conventional electron microscopy and cytochemical techniques. Both species have an unpaired germovitellarium located under the gut; accessory cells surround the germovitellarium of G. baltica. The germovitellarium consists of a restricted germinative area where early differentiating oocytes and vitellocytes are randomly associated, and an extensive growth area with follicular organization. Each follicle consists of a single alecithal oocyte surrounded by numerous vitellocytes. The main features of oocyte differentiation are the accumulation of lipid droplets and the appearance of Golgi complexes and small bodies possibly representing secondary lysosomes. Vitellocytes show features typical of secretory cells, including well-developed rough endoplasmic reticulum (RER) and Golgi complexes which are involved in the production of type A and type B inclusions, hi both species, type A inclusions appear first, have a glycoprotein content, do not contain polyphenols, and become localized in the peripheral cytoplasm of mature vitellocytes; they have been interpreted as eggshell forming granules. Type B inclusions are larger, have a proteinaceous content with a different structure in the two species examined, and remain scattered in the cytoplasm of mature vitellocytes; they are considered to be yolk. The finding of eggshell forming granules without polyphenols in prorhynchids contrasts with the condition in most platyhelminths that have a sclerotized eggshell formed through a tanning process of polyphenolic substances. The small bodies in the oocytes and the eggshell granules in the vitellocytes of Lecithoepitheliata differ from those observed in prolecithophorans, which have oocyte and vitellocyte inclusions similar to those of the Rhabdocoela.     

Tyrosine kinase and cooperative TGFbeta signaling in the reproductive organs of Schistosoma mansoni

Drug-induced suppression of female schistosome sexual maturation is an auspicious strategy to combat schistosomiasis since the eggs are the causative agent. The establishment of drug targets requires knowledge about the molecular mechanisms that regulate the development of the female reproductive organs, which include vitellarium and ovary. This review summarizes recent studies suggesting tyrosine kinases as important factors for the regulation of female gonad development. In this context, especially cytoplasmatic tyrosine kinases of the Src class seem to play dominant roles. Moreover, experimental data and theoretical concepts are provided supporting a crosstalk between tyrosine kinase and TGFbeta signaling in the production of vitellocytes. Finally, we take advantage from the schistosome genome project to propose a model for the regulation of vitelline-cell production and differentiation.     

Intake of Erythrocytes Required for Reproductive Development of Female Schistosoma japonicum

The reproductive development and maturation of female schistosomes are crucial since their released eggs are responsible for the host immunopathology and transmission of schistosomiasis. However, little is known about the nutrients required by female Schistosoma japonicum during its sexual maturation. We evaluated the promoting effect of several nutrients (calf serum, red blood cells (RBCs), ATP and hypoxanthine) on the reproductive development of pre-adult females at 18 days post infection (dpi) from mixed infections and at 50 dpi from unisexual infections of laboratory mice in basic medium RPMI-1640. We found RBCs, rather than other nutrients, promoted the female sexual maturation and egg production with significant morphological changes. In 27% of females (18 dpi) from mixed infections that paired with males in vitro on day 14, vitelline glands could be positively stained by Fast Blue B; and in 35% of females (50 dpi) from unisexual infections on day 21, mature vitelline cells were observed. Infertile eggs were detected among both groups. To analyze which component of mouse RBCs possesses the stimulating effect, RBCs were fractionated and included in media. However, the RBC fractions failed to stimulate development of the female reproductive organs. In addition, bovine hemoglobin hydrolysate, digested by neutral protease, was found to exhibit the promoting activity instead of untreated bovine hemoglobin. The other protein hydrolysate, lactalbumin hydrolysate, exhibited a similar effect with bovine hemoglobin hydrolysate. Using quantitative RT-PCR, we found the expression levels of four reproduction-related genes were significantly stimulated by RBCs. These data indicate that RBCs provide essential nutrients for the sexual maturation of female S. japonicum and that the protein component of RBCs appeared to constitute the key nutrient. These findings would improve laboratory culture of pre-adult schistosomes to adult worms in medium with well-defined components, which is important to investigate the function of genes related to female sexual maturation.     

Ultrastructural and cytochemical aspects of the germarium and the vitellarium in Syndesmis patagonica (Platyhelminthes,Rhabdocoela, Umagillidae)

The cytoarchitecture of the female gonad of the endosymbiont umagillid Syndesmis patagonica has been investigated using electron microscopy and cytochemical techniques. The female gonad consists of paired germaria and vitellaria located behind the pharynx in the mid‐posterior region of the body. Both the germaria and the vitellaria are enveloped by an outer extracellular lamina and an inner sheath of accessory cells which contribute to the extracellular lamina. Oocyte maturation occurs completely during the prophase of the first meiotic division. Oocyte differentiation is characterized by the appearance of chromatoid bodies and the development of endoplasmic reticulum and Golgi complexes. These organelles appear to be involved in the production of round granules, about 2–2.5 μm in diameter, with a homogeneous electron‐dense core surrounded by a granular component and a translucent halo delimited by a membrane. These egg granules migrate to the periphery of mature oocytes, are positive to the cytochemical test for polyphenol detection, are unaffected by protease and have been interpreted as eggshell granules. The mature oocytes also contain a small number of yolk granules, lipid droplets, and glycogen particles scattered throughout the ooplasm. The vitellaria are branched organs composed of vitelline follicles with vitellocytes at different stages of maturation. Developing vitellocytes contain well‐developed rough endoplasmic reticulum and small Golgi complexes involved in the production of eggshell and yolk globules. Eggshell globules are round, measure 4–5 μm in diameter, and have a mosaic‐like patterned content which contains polyphenols. The yolk globules, 2–3 μm in diameter, show a homogeneous protein content of medium electron density, devoid of polyphenols, and completely digested by protease. The mature vitellocytes also contain glycogen as further reserve material. The presence of polyphenolic eggshell granules in the oocytes and of polyphenolic eggshell globules with a mosaic‐like pattern in the vitellocytes have been considered apomorphic features of the Rhabdocoela + Prolecithophora. J. Morphol. 275:703–719, 2014. © 2014 Wiley Periodicals, Inc.     

Seasonal characteristics and tetrodotoxin-producing ability of bacteria in gastropod Oliva hirasei

Lycoteuthis lorigera is among the most abundant mesopelagic squid on the continental slope of the south Atlantic. It is the dominant prey in some commercially harvested groundfish. Despite its abundance and importance in the mesopelagic foodweb, its general biology is poorly known. In this study, the spawning pattern and fecundity of L. lorigera from southern African waters were investigated. Both histological examination and length frequency analysis of ovaries in various stages of development revealed that the ovulation pattern of L. lorigera is asynchronous, which indicates a spawning strategy where batches of eggs are spawned repeatedly over time. More specifically, ovulation is group synchronous, where distinct batches of 1,200–2,400 eggs ripen in the ovary and accumulate in the oviducts. The potential fecundity was estimated to be 20,000–50,000 in immature females (n = 6) and 8,000–25,000 in mature females (n = 21). The number of ripe eggs in the oviducts suggests that batches of spawned egg masses contain between 1,000 and 4,000 eggs. The reproductive strategy of L. lorigera is discussed and compared to the reproductive strategies of oceanic squid inhabiting the continental slope of southern Africa.     

Failure of in vitro-cultured schistosomes to produce eggs: how does the parasite meet its needs for host-derived cytokines such as TGF-β?

When adult schistosome worm pairs are transferred from experimental hosts to in vitro culture they cease producing viable eggs within a few days. Female worms in unisexual infections fail to mature, and when mature adult females are separated from male partners they regress sexually. Worms cultured from the larval stage are also permanently reproductively defective. The cytokine transforming growth factor beta derived from the mammalian host is considered important in stimulating schistosome female worm maturation and maintenance of fecundity. The means by which schistosomes acquire TGF-β have not been elucidated, but direct uptake in vivo seems unlikely as the concentration of free, biologically active cytokine in host blood is very low. Here we review the complexities of schistosome development and male–female interactions, and we speculate about two possibilities on how worms obtain the TGF-β they are assumed to need: (i) worms may have mechanisms to free active cytokine from the latency-inducing complex of proteins in which it is associated, and/or (ii) they may obtain the cytokine from alpha 2-macroglobulin, a blood-borne protease inhibitor to which TGF-β can bind. These ideas are experimentally testable.     

The metabolic control of schistosome egg production

Schistosomiasis is a neglected tropical disease caused by infection with trematode parasites of the genus Schistosoma. Despite ongoing treatment programmes, the prevalence of schistosomiasis has failed to decline and the disease remains a cause of severe morbidity in millions of people. Understanding the biology of egg production by schistosomes is critical since eggs allow transmission of the infection, and when trapped in host tissues induce the immune responses that are responsible for the pathologic changes that underlie disease development. Unusually among trematodes, adult schistosomes exhibit sexual dimorphism and display a fascinating codependency in that the female is dependent on the male to grow and sexually mature. Thus, virgin females are developmentally stunted compared with females from mixed‐sex infections and are unable to lay eggs. Moreover, fecund female schistosomes rapidly lose the ability to produce eggs when placed in tissue culture. Here we discuss the metabolic regulation of egg production in schistosomes, and in particular the critical role played by fatty acid oxidation in this process.     

Schistosoma mansoni: sex-specific modulation of parasite growth by host immune signals

Development of female schistosomes from infectious cercariae to mature egg-producing adults requires both male schistosomes and an intact adaptive immune system. By examining single sex infections in immunodeficient mice, we provide evidence that female schistosome development is not directly influenced by the adaptive immune system, whereas male development is. Our data are consistent with a sequential model of schistosome development, where the adaptive immune system signals development of mature males, which subsequently stimulate development of mature females. The male schistosome therefore appears to play a central role both in transducing signals from the adaptive immune system and in facilitating female development.     

Ovoviviparity and morphology of the female reproductive system in Palumbina (Lepidoptera,Gelechiidae, Thiotrichinae)

Ovoviviparity is reported for the genus Palumbina of Thiotrichinae. The females of eight species were found to retain a hatched first instar larva or a fully developed embryo in the oviduct (vestibulum), which suggests that the females may deposit larvae or mature eggs that are ready to hatch. In the mature embryos, the mandibles and a head capsule were visible and enclosed in chorion. Taking this into consideration, the embryos may contain sufficient nutrition for development within the eggshell, which indicates an ovoviviparous reproduction. The embryos in the oviduct were not dissolved while the early-stage embryos in the anterior portion of the abdomen were destroyed by the treatment of KOH. Therefore, the formation of chorion likely takes place in the oviduct. The female reproductive system of Palumbina displayed typical morphology of ditrysian Lepidoptera although it showed minor variations among the species. The paired reservoirs of the colleterial gland were rather reduced in the species that carried larvae. We assume that this might be related to the ovoviviparous reproductive physiology. The lagena of the spermatheca was present or absent, and if present, the shape and the size were various even in the same species. The species without lagena had a numerous thorn-like structures on the utriculus. The function is possibly related to the maintenance of sperm, but the correlation between the structure and the absence of the lagena remains unclear. Further detailed studies are required to reveal the comprehensive mechanism of the ovoviviparous reproduction in Palumbina.     

Ultrastructural study of vitellogenesis and oogenesis of Metadena depressa (Stossich, 1883) Linton, 1910 (Digenea,Cryptogonimidae), intestinal parasite of Dentex dentex (Pisces,Teleostei)

The ultrastructural organization of the female reproductive system of Metadena depressa, digenean intestinal parasite of Sparidae (Dentex dentex), was investigated by electron microscopy. The vitellogenesis is divided into four stages: stage I, vitellocytes have a cytoplasm mainly filled with ribosomes and few mitochondria; stage II, beginning of the synthetic activity; stage III, active shell globule clusters synthesis; stage IV, mature vitellocytes are filled with shell globule clusters and generally contain several large lipid droplets. Glycogen granules are grouped at the periphery of the cell. The three stages of the oogenesis process take place in the ovary: stage I, oogonia are undifferentiated small cells located at the periphery of the organ; stage II, primary oocytes possess a higher nucleo-cytoplasmic ratio and a nucleus with a nucleolus and synaptonemal complexes indicating the zygotene-pachytene stage of the first meiotic division; stage III, mature oocytes are located in the proximal region of the organ and possess a cytoplasmic chromatoid body and cortical granules in a monolayer close to the periphery of the cell.     

A comparative proteomic study of the undeveloped and developed Schistosoma mansoni egg and its contents: The miracidium, hatch fluid and secretions

The schistosome egg is the key agent responsible both for transmission of the parasite from human to molluscan host, and is the primary cause of pathogenesis in schistosomiasis. Characterisation of its proteome is a crucial step in understanding the egg’s interactions with the mammalian host. We devised a scheme to isolate undeveloped eggs from mature schistosome eggs by Percoll gradient and then fractionate the mature egg into miracidial, hatch fluid and secreted protein preparations. The soluble proteins contained within the five preparations were separated by two-dimensional electrophoresis and their spot patterns compared by image analysis. Large numbers of representative spots were then excised and subjected to tandem mass spectrometry to obtain identities. In this way, the principal components of each sub-proteome were established. Chaperones were the most abundant category, with heat shock protein 70 (HSP70) dominant in the undeveloped egg and Schistosoma mansoni protein 40 (Smp-40) in the miracidium. Cytoskeletal proteins were expressed at similar levels in the undeveloped egg and miracidium, with tubulins the most abundant. The proteins of energy metabolism reflected the change from anaerobic to aerobic metabolism as the miracidium developed. None of the above categories was abundant in the hatch fluid but this peri-miracidial compartment was highly enriched for defence proteins such as thioredoxin. Hatch fluid also contained several host proteins and schistosome proteins of unknown function, highlighting its distinct nature and potentially its role. The egg secretions could not be compared with the other preparations due to their unique composition featuring the previously characterised IL-4-inducing principal of S. mansoni eggs (IPSE), Omega-1, egg secreted protein 15 (ESP15), a micro-exon gene 2 (MEG-2) protein and two members of the recently described MEG-3 family. This last preparation contains the subset of egg proteins that probably enables eggs to escape from host tissues and may also initiate granuloma formation, emphasising the need to establish fully the roles of its components in schistosome biology.     

Effect of multiple mating on the reproductive performance of the rice leaffolder moth,Cnaphalocrocis medinalis (Lepidoptera: Crambidae)

To clarify whether multiple mating of females and males affects the reproductive performance of the rice leaffolder moth, Cnaphalocrocis medinalis (Guenée), we examined the effect of the number of matings (once, twice, or three times) for females (female treatment) and males (male treatment) on the incidence of moth mating, number of eggs laid, egg hatchability, and adult longevity. We also compared the effect of multiple mating imposed on males or females separately with the effect of that imposed on both sexes simultaneously (both sexes treatment). The incidence of mating of females and males that mated three times (3-mated females and males) was significantly lower than for females and males that mated twice or once (2-mated or 1-mated females and males). The incidence of mating of 1-mated moths (both sexes) was significantly higher than for 2-mated or 3-mated moths (both sexes). Two-mated or 3-mated females laid significantly more eggs with significantly higher hatchability than 1-mated females. Females that mated with 1-mated males (second male mating) or 2-mated males (third male mating) laid significantly fewer eggs than those that mated with virgin males (first male mating). Females laid significantly more eggs after the second and third matings for moths of both sexes than after the first mating for moths of both sexes. The mechanisms of improvement and decline of female reproductive performance when multiple mating was imposed on males or females are also discussed in relation to the reproductive biology of C. medinalis.     

Ultrastructure of vitellogenesis and vitellocytes in the trypanorhynch cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax

This is the first TEM examination of vitellogenesis in the cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax and a member of a little-studied trypanorhynch family, the Aporhynchidae. The synthetic activity of vitellocytes plays two important functions in the developmental biology of cestodes: (1) their shell-globules serve in eggshell formation; and (2) their accumulated reserves of glycogen and lipids represent a food source for the developing embryo. In A. menezesi, vitelline follicles consist of cells at various stages of development, from peripheral, immature cells of the gonial type to mature cells towards the centre of the follicle. These stages are: (I) immature; (II) early differentiation; (III) advanced maturation; and (IV) mature. Gradual changes involved in this process occur within each stage. Vitellogenesis involves: (1) an increase in cell volume; (2) the development of a smooth endoplasmic reticulum and an accelerated formation and accumulation of both unsaturated and saturated lipid droplets, along with their continuous enlargement and fusion; (3) the formation of individual β-glycogen particles and their accumulation in the form of glycogen islands scattered among lipid droplets in the cytoplasm of maturing and mature vitellocytes; (4) the rapid accumulation of large, moderately saturated lipid droplets accompanied by dense accumulations of β-glycogen along with proteinaceous shell-globules or shell-globule clusters in the peripheral layer during the advanced stage of maturation; (5) the development of cisternae of granular endoplasmic reticulum that produce dense, proteinaceous shell-globules; (6) the development of Golgi complexes engaged in the packaging of this material; and (7) the progressive and continuous enlargement of shell-globules into very large clusters in the peripheral layer during the advanced stage of maturation. Vitellogenesis in A. menezesi, only to some extent, resembles that previously described for four other trypanorhynchs. It differs in: (i) the reversed order of secretory activities in the differentiating vitellocytes, namely the accumulation of large lipid droplets accompanied by glycogenesis or β-glycogen formation during early differentiation (stage II), i.e. before the secretory activity, which is predominantly protein synthesis for shell-globule formation (stage III); (ii) the very heavy accumulation of large lipid droplets during the final stage of cytodifferentiation (stage IV); and (iii) the small number of β-glycogen particles present in mature vitellocytes. Ultracytochemical staining with PA-TCH-SP for glycogen proved positive for a small number of β-glycogen particles in differentiating and mature vitellocytes. Hypotheses, concerning the interrelationships of patterns of vitellogenesis, possible modes of egg formation, embryonic development and life-cycles, are commented upon.     

Feeding and reproductive biology of the currito,Hoplosternum littorale,in the Venezuelan llanos with comments on the possible function of the enlarged male pectoral spines

Synopsis The reproductive biology of roach, Rutilus rutilus, was investigated during 1980–1982 in a small tributary of the eutrophic Lake rungen, southeastern Norway. The upstream migration started in early May with medium or falling water levels and water temperatures of 6–10° C, and lasted until late May or early June. Roach (both males and females) migrating early in the season were larger than roach migrating late in the season. Males matured on average one year younger than females. The first males were mature at age 2 years, the first females were mature at age 3 years. Males were smaller than females in all age groups. There was no significant year to year variation in mean length of male and female roach in the various age-groups. Mean population fecundity during 1980–1982 was estimated to be 19 × 10⁶ eggs or 63300 eggs m^–2 of spawning area. The survival rate of eggs and small roach varied considerably due to rapid and unpredictable changes in water and silt levels. Mean annual survival rates for mature male and female roach were 0.30 and 0.52, respectively. The survival rates did not vary with age.     

体外培养日本血吸虫成虫生殖器官超微结构的观察

将日本血吸虫成虫于851培养基中培养23天后,对其生殖器官进行透射电镜观察。观察结果显示,雌虫卵巢内卵母细胞出现不同程度的变性、坏死;成熟卵黄细胞的卵黄小滴融合,脂质小滴数量增多、体积增大;培养后期卵壳形成发生障碍,最终导致无活性、无卵壳的畸形卵形成。超微结构观察首次显示,体外初产期虫卵卵壳中有条带状低电子密度区和高电子密度区相间排列。     

Reproductive Biology of <Emphasis Type="Italic">Leptocybe invasa</Emphasis> Fisher &amp; La Salle (Hymenoptera: Eulophidae)

Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae) is an invasive pest in Eucalyptus plantations around the world. The successful colonization of L. invasa is possibly related to its reproductive biology. The objective of this study was to examine the reproductive biology of L. invasa. In Guangxi Province, the sex ratio (proportion of female, 0.99) of L. invasa was female-dominant throughout the year based on natural and artificial infestation. This result was similar to the ratios observed for other geographic populations in China, including those in Fujian (0.99), Guangdong (0.98), Hainan (0.95), Jiangxi (0.96), and Sichuan (0.99). The offspring sex ratio favored females. A large number of females emerged from the galls produced by females, with few males found. Galls on the petioles and midribs of Eucalyptus plants could be caused by newly emerged females with mature eggs. The lengths of the ovariole, spermatheca, common oviduct, and reproductive glands did not differ among L. invasa females, but their lateral oviducts showed differences from 0 to 42 h after emergence, indicating that this insect is proovigenic. These results could explain why L. invasa populations can rapidly increase in invaded areas.     

Eggshell Appearance Does Not Signal Maternal Corticosterone Exposure in Japanese Quail: An Experimental Study with Brown-Spotted Eggs

Reproduction is a critical period for birds as they have to cope with many stressful events. One consequence of an acute exposure to stress is the release of corticosterone, the avian stress hormone. Prolonged stress can have negative impacts on the immune system, resulting in, for example, increased oxidative stress. Through maternal effects, females are known to modulate their investment in eggs content according to their own physiological condition. Less is known about maternal investment in eggshells, especially in pigments. The two main eggshell pigments may possess opposite antioxidant properties: protoporphyrin (brown) is a pro-oxidant, whereas biliverdin (blue-green) is an antioxidant. In Japanese quail, we know that the deposition of both pigments is related to female body condition. Thus, a chronic stress response may be reflected in eggshell coloration. Using female Japanese quails that lay brown-spotted eggs, we explored whether physiological exposure to corticosterone induces a change in female basal stress and antioxidant factors, and eggshell pigment concentration, spectrophotometric reflectance, and maculation coverage. We supplemented adult females over a 2 week period with either peanut oil (control) or corticosterone (treatment). We collected pre- and post-supplementation eggs and analysed the effect of corticosterone treatment on female physiology and eggshell appearance parameters. Except for corticosterone-fed birds which laid eggs with brighter spots, supplementation had no significant effect on female physiology or eggshell pigment concentration, reflectance and maculation. The change in eggshell spot brightness was not detected by a photoreceptor noise-limited color opponent model of avian visual perception. Our data confirms that eggshell reflectance in spotted eggs varies over the laying sequence, and spot reflectance may be a key factor that is affected by females CORT exposure, even if the changes are not detected by an avian visual model.