The genetics of egg retention and fertilization success in Drosophila: One step closer to understanding the transition from facultative to obligate viviparity

Oviparous, facultative egg retention enables Drosophila females to withhold fertilized eggs in their reproductive tracts until circumstances favor oviposition. The propensity to retain fertilized eggs varies greatly between species, and is correlated with other reproductive traits, such as egg size and ovariole number. While previous studies have described the phenomenon, no study to date has characterized within‐species variation or the genetic basis of the trait. Here, we develop a novel microscope‐based method for measuring egg retention in Drosophila females and determine the range of phenotypic variation in mated female egg retention in a subset of 91 Drosophila Genetic Reference Panel (DGRP) lines. We inferred the genetic basis of egg retention using a genome‐wide association study (GWAS). Further, the scoring of more than 95,000 stained, staged eggs enabled estimates of fertilization success for each line. We found evidence that ovary‐ and spermathecae‐related genes as well as genes affecting olfactory behavior, male mating behavior, male‐female attraction and sperm motility may play a crucial role in post‐mating physiology. Based on our findings we also propose potential evolutionary routes toward obligate viviparity. In particular, we propose that the loss of fecundity incurred by viviparity could be offset by benefits arising from enhanced mate discrimination, resource specialization, or modified egg morphology.     

Progress on reproductive modes of sarcosaphagous flies

The reproduction strategies of invertebrates, oviparity, ovoviviparity and viviparity, always reflect the relationship between individuals and their surroundings. There is plasticity in the reproductive strategies of sarcosaphagous flies as they adjust to rapidly changing circumstances. The transition from oviparity to ovoviviparity or viviparity involves numerous changes in physiology, morphology and immunology. Demonstrating these processes can make the application of entomology work in forensic practice more reliable. This essay reviews means of reproduction in sarcosaphagous flies and identifies related features. It is shown that not only the reproduction traits, such as fast location of carrion, and uncommon number of ovaries and oogenesis, but also some morphological features are related to viviparity. In general, viviparous flies have larger adult bodies but smaller eggs and chorions. Moreover, the length of terminalia and the shape of the vagina also varies among those three modes. Reproductive plasticity is a bridge between the three reproductive modes, and it can greatly influence the inference of the post-mortem interval (PMImin).     

Field Assessment of Adhesion and Hatch ofChrysoperlaEggs Mechanically Applied in Liquid Carriers

A mechanical technique was evaluated for releasing green lacewing eggs in liquid suspensions. Deposited eggs were enclosed within a circle of nonpoisonous adhesive to protect them from predation and to prevent escape of hatched larvae. Released eggs were monitored daily for 5 days after release by measuring three response variables: adhesion rate of eggs to foliage, hatch rate of eggs, and “yield” of larvae from discharged eggs; “yield” was the product of egg adhesion and egg hatch. Factors tested were: egg conditioning prior to release (incubated or refrigerated), carrier (distilled water or commercial carrier solution), application technique (mechanical or hand application), and row facing (North or South). Release technique did not significantly effect egg hatch on any day. Conditioning eggs prior to release had the greatest effect on hatch of eggs and resulting yield of larvae during the 5-day monitoring period. Carrier had a significant effect on adhesion of eggs to leaves and hatch of eggs. Commercial carrier solution increased egg adhesion but decreased egg hatch compared to water. Overall mean yield of larvae from incubated eggs distributed mechanically was not significantly different for eggs suspended in water (36.4% on day 5 post-release) and for eggs suspended in commercial carrier solution (36.1% on day 5 post-release). Hand-applied eggs had a higher hatch and subsequent yield of larvae than mechanically released eggs; however, the hand technique was labor intensive.     

Placental calcium provision in a lizard with prolonged oviductal egg retention

A prominent scenario for the evolution of viviparity and placentation in reptiles predicts a step-wise pattern with an initial phase of prolonged oviductal egg retention accompanied by progressive reduction in eggshell thickness culminating in viviparity; calcium placentotrophy evolves secondarily to viviparity. Saiphos equalis is an Australian scincid lizard with a reproductive mode that is uncommon for squamates because eggs are retained in the oviduct until late developmental stages, and the embryonic stage at oviposition varies geographically. We studied calcium mobilization by embryos in two populations with different oviductal egg retention patterns to test the hypothesis that the pattern of nutritional provision of calcium is independent of the embryonic stage at oviposition. Females from one population are viviparous and oviposit eggs containing fully formed embryos, whereas embryos in oviposited eggs of the second population are morphologically less mature, and these eggs hatch several days later. The reproductive mode of this population is denoted as prolonged oviductal egg retention. Yolk provided the highest proportion of calcium to hatchlings in both populations. Eggs of both populations were enclosed in calcified eggshells, but shells of the population with prolonged egg retention had twice the calcium content of the viviparous population and embryos recovered calcium from these eggshells. Placental transfer accounted for a substantial amount of calcium in hatchlings in both populations. Hatchling calcium concentration was higher in the population with prolonged egg retention because these embryos mobilized calcium from yolk, the eggshell and the placenta. This pattern of embryonic calcium provision in which both a calcified eggshell and placentotrophy contribute to embryonic nutrition is novel. The reproductive pattern of S. equalis illustrates that calcified eggshells are compatible with prolonged oviductal egg retention and that viviparity is not requisite to calcium placentotrophy.     

The effect of temperature on the egg incubation period of Capnia bifrons (Plecoptera: Capniidae) from Windermere (English Lake District)

Gravid females of Capnia bifrons (Newman) from Windermere (English Lake District) were almost completely ovoviviparous, the eggs hatching within 15 min after oviposition in the water. When kept in the laboratory at constant temperatures between 3.8 and 19.8°C, few females survived to lay eggs at temperatures above 12.1°C. The relationship between air temperature (T°C) and the egg incubation period (Y days between fertilisation and oviposition) was given by the regression equation: Y = 316.4 T^−0.9996 (r²= 0.957, p < 0.001). This equation successfully predicted egg incubation periods for gravid females kept in cages in the field.
Comparisons with similar studies on four non-ovoviviparous species of Plecoptera showed that egg development was rarely more rapid in C. bifrons . It was also shown that the hypothesis of ovoviviparity being an adaptation to combat low water temperatures could be rejected for C. bifrons from Windermere.     

Plasticity and limitations of extended egg retention in oviparous Zootoca vivipara (Reptilia: Lacertidae)

The transition between oviparity and viviparity in reptiles is generally accepted to be a gradual process, the result of selection for increasingly prolonged egg retention within the oviduct. We examined egg retention plasticity in an oviparous strain of the lacertid lizard Zootoca vivipara, a species having both oviparous and viviparous populations. We forced a group of female Z. vivipara to retain their clutch in utero by keeping them in dry substrata, and assessed the effect on embryonic development and hatching success, along with offspring phenotype and locomotor performance. Forced egg retention for one additional week affected the developmental stage of embryos at oviposition, as well as hatchling robustness and locomotor performance. However, embryos from forced clutch retention treatment reached one stage unit more than control embryos at oviposition time. Embryos from control eggs were more developed than embryos from experimental eggs after approximately the same period of external incubation, showing that embryonic development is retarded during the period of extended egg retention, despite the high temperature inside the mother's body. Significant differences in external incubation time were only found in one of the two years of study. Hatching success was much lower in the experimental group with forced egg retention (21.1%) than in the control group (95.4%). Therefore, we conclude that there are limitations that hinder the advance of intrauterine embryonic development beyond the normal time of oviposition, and that extended egg retention does not represent clear advantages in this population of Z. vivipara. Nevertheless, the fact that some eggs are successful after forced egg retention could be advantageous for the females that are able to retain their clutch under unfavourable climatic conditions. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 75–82.     

Egg size,embryonic development time and ovoviviparity in Drosophila species

Lengths, widths and volumes of eggs from 11 species of Drosophila whose genomes have been fully sequenced exhibit significant variation that is not explained by their phylogenetic relationships. Furthermore, egg size differences are unrelated to embryonic development time in these species. In addition, two of the species, Drosophila sechellia and, to a lesser degree, D. yakuba, both ecological specialists, exhibit ovoviviparity, suggesting that female control over oviposition in these species differs from what is observed in D. melanogaster. The interspecific differences in these reproductive characters, coupled with the availability of whole genome sequences for each, provide an unprecedented opportunity to examine their evolution.     

Effect of host deprivation on egg quality,egg load,and oviposition in a solitary parasitoid,Chetogena edwardsii (Diptera: Tachinidae)

Solitary parasitoids are limited to laying one egg per host because larvae compete within hosts. If host encounter rate is low, females should not increase the number of eggs/host in response. The tachinid fly, Chetogena edwardsii,was used to evaluate the effect of host deprivation on egg accumulation, oviposition behavior, and egg quality in a solitary parasitoid. Females deprived of hosts for 2– 7 days accumulate about 1 day's supply of eggs. Egg output of deprived females once hosts are restored does not differ from that of control females. Deprived females retain one egg in the uterus where it undergoes embryogenesis. Maggots emerging from retained eggs are more likely to survive in hosts molting in 40 h or less after receipt of an egg than are maggots emerging from eggs fertilized shortly before oviposition. Egg retention is a consequence of host deprivation that permits females to broaden the range of hosts they can exploit to include soon-to-molt hosts and possibly multiply parasitized hosts.     

Vitellocytes and vitellogenesis in cestodes in relation to embryonic development, egg production and life cycle

Vitellocytes have two important functions in cestode embryogenesis: (1) formation of hard egg-shell (e.g. Pseudophyllidea) or a delicate capsule (e.g. Cyclophyllidea), and (2) supplying nutritive reserves for the developing embryos. During evolution any of these two functions can be reduced or intensified in different taxa depending on the type of their embryonic development, degree of ovoviviparity and life cycles. Within the Cestoda, there are three monozoic taxa with only one set of genital organs: Amphilinidea, Gyrocotylidea and Caryophyllidea. In these monozoic taxa and some polyzoic groups with well developed vitellaria (e.g. Pseudophyllidea, Trypanorhyncha) a single oocyte [=germocyte] and a large number of vitellocytes (up to 30) are enclosed within a thick, hardened egg-shell, forming a type of eggs typical for the basic pattern of Neodermata. Only one type of egg-shell enclosures, the so-called 'heterogeneous shell-globule vesicle' is common for the above mentioned cestode taxa. Each membrane-bounded vesicle of mature vitellocytes contains numerous electron-dense shell globules embedded in a translucent matrix. In free-living Neoophora and Monogenea there are two types of vesicles with dense granules; the second is considered to be proteinaceous reserve material. Within the Cestoda, the numbers of vitellocytes per germocyte are reduced in those taxa forming eggs of the 'Cyclophyllidean-type' (e.g. Cyclophyllidea, Tetraphyllidea, Pseudophyllidea). This is particularly evident in Cyclophyllidea; for example, in vitellocytes of Hymenolepis diminuta (Hymenolepididae) there are numerous vitelline granules of homogeneously electron-dense material; in Catenotaenia pusilla (Catenotaeniidae) there are three large, homogenous vitelline vesicles, while in Inermicapsifer madagascariensis (Anoplocephalidae) there is only one large vitelline vesicle, containing homogeneously electron-dense material, which occupies most of the vitelline cell volume. In this respect the Tetraphyllidea and Proteocephalidea, in forming eggs that lack a hard egg-shell, hold an intermediate position. A comparison of interrelationships which exist among types of vitellocytes, vitellogenesis, types of embryonic development, ovoviviparity and life cycles indicates parallelisms and analogies in adaptation to the parasitic way of life in different groups of cestodes. Knowledge on cestode vitellogenesis may also have an important applied aspect. Vitellocytes, due to their high metabolic rate, represent a very sensitive target for analysing effect of anthelminthic drugs upon the egg formation (ovicidal effects); rapid degeneration of vitellocytes is usually accompanied by a cessation of egg production.     

A factor necessary for normal morphogenetic function of anuran endoderm.

Cytoplasmic preparations obtained from late blastulae and early gastrulae of normal R. pipiens eggs contain factors which induce delayed lethal effects when injected into normal eggs at the beginning of cleavage. The same preparations can rescue some defective eggs which occur spontaneously (NS eggs), or are produced experimentally (ES eggs), by relieving the specific developmental syndrome displayed by either type of egg. The syndrome involves abnormal endoderm in which cytolysis is time-programmed. The lethal factors as well as the rescue factors are absent in the normal egg cytoplasm prior to the late blastula stage.     

Methods for quantitating sea urchin sperm-egg binding

Two simple photometric methods are described for determining the average number of bound sea urchin spermatozoa per egg between 0 and 60 sec after insemination. One method is based on stopping gamete interaction with formaldehyde and then, after the eggs settle with sperm bound to their surfaces, measuring the turbidity of sperm remaining in suspension. An alternative method involves removal of the dead, unbound sperm from the formaldehyde fixed eggs by repeated washing in sea water. The bound sperm are then released from the egg surface by pronase digestion and the turbidity of the sperm suspension measured and related to sperm concentration by direct cell counts. Two phases of gamete interaction exist (1) the binding phase, from 0 to 20 or 25 sec, during which time sperm continuously bind to the egg surfaces; (2) the unbinding phase, from 20 or 25 to 50 sec, during which time the sperm are unbound from the eggs and return to the suspension. The results of experiments in which the method is used to assess sperm binding at different pH values and at different calcium concentrations are presented. Data are presented suggesting that a definite number of sperm binding sites may exist on the vitelline layer.     

Culture studies of Chorda tomentosa (Phaeophyta,Laminariales)

The complete life history of Chorda tomentosa Lyngbye from northern Norway has been followed in culture. Under relatively high temperatures (10–15°C) or low irradiance, zoospores develop into filamentous monoecious gametophytes with unlimited vegetative growth. Formation of oogonia and antheridia was induced by transfer to strong white fluorescent light and low temperatures (1–5°C). By variation of these environmental factors the degree of fertility can be controlled. In a light-dark regime, egg release occurs exclusively during the dark periods. Freshly released eggs secrete a sexual hormone which effects explosive discharge of spermatozoids from the antheridia and subsequent chemotaxis towards the egg. Plasmogamy occurs immediately. Chromosome staining reveals interesting nuclear activities during karyogamy. Frequently, unfertilized eggs develop parthenogenetically. The resulting sporophytes are haploid and show the same developmental pattern as those originating from fertilized eggs. All sporophytes produce sporangia and release zoospores within 60 to 90 days after egg discharge.     

Extensive cell movements accompany formation of the otic placode

A centrally important factor in initiating egg activation at fertilization is a rise in free Ca(2+) in the egg cytosol. In echinoderm, ascidian, and vertebrate eggs, the Ca(2+) rise occurs as a result of inositol trisphosphate-mediated release of Ca(2+) from the endoplasmic reticulum. The release of Ca(2+) at fertilization in echinoderm and ascidian eggs requires SH2 domain-mediated activation of a Src family kinase (SFK) and phospholipase C (PLC)gamma. Though some evidence indicates that a SFK and PLC may also function at fertilization in vertebrate eggs, SH2 domain-mediated activation of PLC gamma appears not to be required. Much work has focused on identifying factors from sperm that initiate egg activation at fertilization, either as a result of sperm-egg contact or sperm-egg fusion. Current evidence from studies of ascidian and mammalian fertilization favors a fusion-mediated mechanism; this is supported by experiments indicating that injection of sperm extracts into eggs causes Ca(2+) release by the same pathway as fertilization.     

Some aspects of the breeding biology of the equatorial land snail Limicolaria martensiana (Achatinidae: Pulmonata)

A study of the breeding season of the polymorphic equatorial land snail Limicolaria martensiana Smith (Achatinidae: Pulmonata) lasting six years and six months in populations in the Kampala District (lat. 0 20') and the Equator line shows that the snail breeds continuously throughout the year but has bimodal peaks in its breeding which occurs during the dry seasons, while during the rainy seasons breeding is low. It is the decrease in the amount of rainfall which induces peak breeding and as a result there is an inverse relationship between the amount of rainfall and the breeding intensity. The evolution of ovoviviparity with the ability to retain eggs and/or young in utero during aestivation is the phenomenon that enables L. martensiana to have peak breeding during the dry seasons. Moisture which enables young to feed and thus grow is an ultimate factor in determining breeding in L. martensiana . The gestation period is 10 to 14 days during the rainy seasons but the snail retains its eggs and/or young in utero during the dry seasons and only deposits them when rain returns. The eggs take 18.6±5.43 days ( N= 130) to hatch after they are laid. Hatching in utero and outside occurs by the young eating the egg shells. Oogenesis in the snails occurs at all times. L. martensiana stores viable allosperms which it can use for further reproduction.     

Evolution of viviparity: variation between two sceloporine lizards in the ability to extend egg retention

The evolutionary transition between oviparity and viviparity in squamate reptiles presumably occurs via a gradual increase in the duration of egg retention, the production of thinner eggshells, and increases in the vascularity of maternal and embryonic tissues. The 'ease' of this transition may differ among taxa. For example, in the genus Sceloporus , the scalaris species group contains both oviparous and viviparous species, and female Sceloporus scalaris can extend egg retention facultatively in response to the absence of a suitable site for oviposition without impairing embryonic development. In contrast, the undulatus species group contains only oviparous species, and, while female Sceloporus virgatus can extend egg retention, doing so retards embryonic development. I tested several hypotheses that would explain the greater ability of 5. scalaris than S. virgatus to extend egg retention. In this study, female S. scalaris retained eggs for 19 d without affecting the mortality of embryos, total developmental time, or dry mass of hatchlings. In contrast, when female S. virgatus retained eggs for 18 d, embryos had very high mortality and eggs took significantly longer to hatch than control (non-retained) eggs, although the dry mass of hatchlings was not affected. The ability of S. scalaris females to retain eggs with little negative effect on embryonic development was associated with relatively large chorioallantois, relatively thin eggshells, and relatively small clutch masses. These observations suggest that phylogenetic differences in the ability to extend egg retention may facilitate or constrain the evolution of viviparity in some lineages.     

Signal transduction pathways leading to Ca2+ release in a vertebrate model system: lessons from Xenopus eggs

At fertilization, eggs unite with sperm to initiate developmental programs that give rise to development of the embryo. Defining the molecular mechanism of this fundamental process at the beginning of life has been a key question in cell and developmental biology. In this review, we examine sperm-induced signal transduction events that lead to release of intracellular Ca(2+), a pivotal trigger of developmental activation, during fertilization in Xenopus laevis. Recent data demonstrate that metabolism of inositol 1,4,5-trisphosphate (IP(3)), a second messenger for Ca(2+) release, is carefully regulated and involves phospholipase C (PLC) and the tyrosine kinase Src. Roles of other potential regulators in this pathway, such as phosphatidylinositol 3-kinase, heterotrimeric GTP-binding protein, phospholipase D (PLD) and phosphatidic acid (PA) are also discussed. Finally, we address roles of egg lipid/membrane microdomains or 'rafts' as a platform for the sperm-egg membrane interaction and subsequent signaling events of egg activation.     

Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.     

Egg-release behaviour in Antarctic krill

The process of egg release is a complex and crucial step in the life cycle of euphausiids, especially with regards mortality and recruitment success. We examined this process in Antarctic krill (Euphausia superba) in terms of the functioning of the female genital apparatus and associated swimming behaviour. A tethering technique combined with video analysis was used to make observations of three females during the release process. We found eggs were steadily extruded over a period of up to 10 h, during which time the krill released between 1,600 and 4,000 eggs and the ovary reduced by a half in length and a third in height. Eggs were mainly released individually or, less commonly, in batches of between 2 and 4 eggs. Release rates were between 5 and 30 eggs min⁻¹. The steady release of eggs fits well with histological evidence that mature oocytes must pass individually through tight genital ducts with narrow apertures, before coming in to close contact with the sperm plug for fertilisation, and then expulsion from the thelycum as fertilised eggs. During spawning, the female alternated between slow and rapid rates of pleopod beating with egg release occurring at the moment of beat acceleration. At the point of release, the descent of the egg was accelerated through downward beats of the 7th thoracic leg. The cyclic pattern in pleopod beat-rate during spawning may alter swimming performance and contribute to the widely reported sex- and maturity-based biases within krill swarms.     

Capillaria hepatica: Relation of structure and composition of egg shell to antigen release

Clean, nonembryonated Capillaria hepatica eggs recovered from infected liver tissue by physical methods as well as eggs obtained after passage through the mouse gastrointestinal tract were examined with the electron microscope. In eggs collected by physical methods the outer membrane showed defects where it was lacking at various points or disrupted. The pillars of the outer shell were frequently broken or fractured resulting in virtual collapse or separation of the outer shell from the inner shell. No shell matrix was observed in any eggs examined. Even more dramatic effects were observed in eggs recovered following passage through the mouse gastrointestinal tract. Clean, nonembryonated eggs collected by physical methods were suspended at 5 C in phosphate-buffered saline. A large amount of protein was released initially into the medium; the amount released then fell to a low level at which it remained for several weeks. Gel diffusion tests with concentrated protein supernatant and C. hepatica egg-derived antigen were compared using appropriate antisera. Bands of identity were present in both; however, egg antigen contained other proteins not present in egg supernatant. These studies indicate that during physical collection of C. hepatica eggs, sufficient damage occurs to allow for the release of materials into host tissue during experimental egg granuloma formation. An hypothesis is presented concerning the viability of C. hepatica eggs in host liver tissue following host cellular response and the possible modes of action which trigger development of eggs after release from infected liver.     

Ascidian follicle cells: Multifunctional adjuncts to maturation and development

Ascidians are primitive chordates, subphylum Tunicata, that are sessile filter‐feeding hermaphrodites as adults. Released oocytes are enclosed within a monolayer of follicle cells, a non‐cellular vitelline coat and a monolayer of test cells that cover the egg membrane. Follicle cell structure is distinctive in different groups. They originate from circulating hemoblasts with functional nuclei. They are necessary for germinal vesicle breakdown in several species and may secrete a meiosis‐inducing substance to the oocyte. In some families the follicle cells are necessary for fertilization. Although all ascidians are hermaphrodites, many are not capable of self fertilization. The follicle cells seem to be involved in self, non‐self discrimination. Attachment of sperm to egg involves a sperm surface glycosidase binding to an egg surface glycoside. The primary block to polyspermy involves a glycosidase released by the follicle cells. In one species with direct development, the follicle cells secrete a sticky substance that anchors the embryos in a wave‐swept rocky area; a brooding solitary ascidian with a tadpole larva uses a sticky substance secreted by follicle cells to attach the brood to the atrial chamber. Several species have floating eggs due to buoyancy of their follicle cells, a result of ammonia sequestration in at least one species. Many other marine invertebrates release eggs with attached follicle cells, and all vertebrates ovulate oocytes covered with follicle cells. Comparisons are discussed between these groups and ascidians.