Half Of Embryonic Development Research Articles

Dynamic changes in DNA methylation during embryonic and postnatal development of an altricial wild bird.

DNA methylation could shape phenotypic responses to environmental cues and underlie developmental plasticity. Environmentally induced changes in DNA methylation during development can give rise to stable phenotypic traits and thus affect fitness. In the laboratory, it has been shown that the vertebrate methylome undergoes dynamic reprogramming during development, creating a critical window for environmentally induced epigenetic modifications. Studies of DNA methylation in the wild are lacking, yet are essential for understanding how genes and the environment interact to affect phenotypic development and ultimately fitness. Furthermore, our knowledge of the establishment of methylation patterns during development in birds is limited. We quantified genome‐wide DNA methylation at various stages of embryonic and postnatal development in an altricial passerine bird, the great tit Parus major. While, there was no change in global DNA methylation in embryonic tissue during the second half of embryonic development, a twofold increase in DNA methylation in blood occurred between 6 and 15 days posthatch. Though not directly comparable, DNA methylation levels were higher in the blood of nestlings compared with embryonic tissue at any stage of prenatal development. This provides the first evidence that DNA methylation undergoes global change during development in a wild bird, supporting the hypothesis that methylation mediates phenotypic development. Furthermore, the plasticity of DNA methylation demonstrated during late postnatal development, in the present study, suggests a wide window during which DNA methylation could be sensitive to environmental influences. This is particularly important for our understanding of the mechanisms by which early‐life conditions influence later‐life performance. While, we found no evidence for differences in genome‐wide methylation in relation to habitat of origin, environmental variation is likely to be an important driver of variation in methylation at specific loci.

Melanin-concentrating hormone (MCH) neurons in the developing chick brain

The present study was undertaken because no previous developmental studies exist on MCH neurons in any avian species. After validating a commercially-available antibody for use in chickens, immunohistochemical examinations first detected MCH neurons around embryonic day (E) 8 in the posterior hypothalamus. This population increased thereafter, reaching a numerical maximum by E20. MCH-positive cell bodies were found only in the posterior hypothalamus at all ages examined, restricted to a region showing very little overlap with the locations of hypocretin/orexin (H/O) neurons. Chickens had fewer MCH than H/O neurons, and MCH neurons also first appeared later in development than H/O neurons (the opposite of what has been found in rodents). MCH neurons appeared to originate from territories within the hypothalamic periventricular organ that partially overlap with the source of diencephalic serotonergic neurons. Chicken MCH fibers developed exuberantly during the second half of embryonic development, and they became abundant in the same brain areas as in rodents, including the hypothalamus (by E12), locus coeruleus (by E12), dorsal raphe nucleus (by E20) and septum (by E20). These observations suggest that MCH cells may play different roles during development in chickens and rodents; but once they have developed, MCH neurons exhibit similar phenotypes in birds and rodents.

Embryonic development of mushroom bodies in Pterostichus niger schall. (Coleoptera: Carabidae)

This study demonstrates that the number of stem cells in mushroom bodies of the ground beetle Pterostichus niger increases already in the first half of embryogenesis and that this timing allows for an intensive increase of the number of neurons that compose the mushroom bodies during the second half of embryonic development. The degree of development of the mushroom bodies in new-born P. niger larvae was shown to be higher than that in new-born larvae of any other holometabolous insect investigated previously.

Use of high-frequency ultrasound to study the prenatal development of cranial neural tube defects and hydrocephalus in Gldc-deficient mice.

ObjectiveWe used non‐invasive high‐frequency ultrasound (HFUS) imaging to investigate embryonic brain development in a mouse model for neural tube defects (NTDs) and non‐ketotic hyperglycinemia (NKH).MethodUsing HFUS, we imaged embryos carrying loss of function alleles of Gldc encoding glycine decarboxylase, a component of the glycine cleavage system in mitochondrial folate metabolism, which is known to be associated with cranial NTDs and NKH in humans. We serially examined the same litter during the second half of embryonic development and quantified cerebral structures. Genotype was confirmed using PCR. Histology was used to confirm ultrasound findings.ResultsHigh‐frequency ultrasound allowed in utero detection of two major brain abnormalities in Gldc‐deficient mouse embryos, cranial NTDs (exencephaly) and ventriculomegaly (corresponding with the previous finding of post‐natal hydrocephalus). Serial ultrasound allowed individual embryos to be analysed at successive gestational time points. From embryonic day 16.5 to 18.5, the lateral ventricle volume reduced in wild‐type and heterozygous embryos but increased in homozygous Gldc‐deficient embryos.ConclusionExencephaly and ventriculomegaly were detectable by HFUS in homozygous Gldc‐deficient mouse embryos indicating this to be an effective tool to study CNS development. Longitudinal analysis of the same embryo allowed the prenatal onset and progression of ventricle enlargement in Gldc‐deficient mice to be determined. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Developmental stages of the climbing gecko Tarentola annularis with special reference to the claws, pad lamellae, and subdigital setae.

Studying the in ovo mode of development of squamates has the advantage of allowing easy access to embryos without surgically compromising gravid females. Despite the non-ophidian squamates being a very diverse lineage of reptiles, embryonic tables for individuals of this group are very few. Here, I present the first in ovo embryonic table for a basal multi-scansored, pad-bearing gecko, Tarentola annularis. In this gecko, only the III and IV digits bear claws. Eleven embryonic stages are described based on chronological development of morphological characteristics. In contrast to other previously studied geckos, this species exhibits a longer incubation period. Comparison with other squamates, embryonic development of T. annularis is an indicative of a conserved developmental strategy. Interestingly, the clawless digits of this gecko do exhibit claws during the first half of embryonic development. Thus, regression of claws in these digits could be an advantage of studying this particular taxon, as it raises the question, to be answered in future study, of which mechanisms could be responsible for such claw regression. Before hatching, the outer periderm layer sloughs revealing the functional setae. The present study provides not only a model for pentadactyl limbs and digit development, but also an example of a unique developmental phenomenon, as represented by claw regression.

Serial block face-scanning electron microscopy: a tool for studying embryonic development at the cell-matrix interface.

Studies of gene regulation, signaling pathways, and stem cell biology are contributing greatly to our understanding of early embryonic vertebrate development. However, much less is known about the events during the latter half of embryonic development, when tissues comprising mostly extracellular matrix (ECM) are formed. The matrix extends far beyond the boundaries of individual cells and is refractory to study by conventional biochemical and molecular techniques; thus major gaps exist in our knowledge of the formation and three-dimensional (3D) organization of the dense tissues that form the bulk of adult vertebrates. Serial block face-scanning electron microscopy (SBF-SEM) has the ability to image volumes of tissue containing numerous cells at a resolution sufficient to study the organization of the ECM. Furthermore, whereas light microscopy was once relatively straightforward and electron microscopy was performed in specialist laboratories, the tables are turned; SBF-SEM is relatively straightforward and is becoming routine in high-end resolution studies of embryonic structures in vivo. In this review, we discuss the emergence of SBF-SEM as a tool for studying embryonic vertebrate development.

Photosynthetic carbon from algal symbionts peaks during the latter stages of embryonic development in the salamander Ambystoma maculatum.

BackgroundIt was recently discovered that symbiotic algae in the eggs of the salamander Ambystoma maculatum translocate fixed carbon from photosynthesis to developing embryos. Fixed carbon translocation was shown in embryos at one time point during development, however, it was unknown if fixed carbon translocation occurs throughout all developmental stages.FindingsIn this study, fixed carbon translocation was measured in salamander eggs at six time points over the latter half of development. Fixed carbon translocation did not occur until the middle tailbud portion of development (stages 26–30), and translocation was measured in 20% or less of eggs sampled. Peak carbon translocation occurred during the late tailbud phase of development (stages 31–35), where as much as 87% of eggs sampled showed translocation, and average percent translocation was 6.5%. During the final stages of development, fixed carbon translocation declined, and translocation was not detected in embryos five days prior to hatching.ConclusionsThe onset of fixed carbon translocation from Oophila to A. maculatum embryos during the second half of embryonic development is likely due to the corresponding settlement and concentration of Oophila in the inner egg envelope. In addition, carbon translocation ceases in late stage embryos as the inner egg envelope thins and ruptures in preparation for hatching.

Hypoxic level and duration differentially affect embryonic organ system development of the chicken (Gallus gallus)

Hypoxia inhibits avian embryonic development, as well as increases embryonic mortality. However, the key organ systems affected by hypoxia, and their critical windows for development, are poorly understood. Consequently, chicken embryos were continuously exposed to 3 levels of oxygen (21, 15, or 13% O2) throughout d 0 to 10, d 11 to 18, or d 0 to 18 of incubation, followed by morphometric and blood physiological measurements. Hypoxia occurring early during incubation (d 0 to 10) had larger effects on embryonic mortality and organ growth than hypoxia occurring at later stages (d 10 to 18). Growth of the heart and chorioallantoic membrane was stimulated by chronic hypoxia, whereas the lung, brain, eye, liver, stomach, beak, and toes showed no disruption. Sustained hypoxia from the beginning of incubation decreased blood hemoglobin, hematocrit, and red blood cell concentration of embryos at d 10, but the values among hypoxic and normoxic groups were not significantly different at d 18. Blood partial pressure of O2 and partial pressure of CO2 were dependent upon incubation O2 level at a given day of development. These results indicated that either modest hypoxia (15% O2) throughout development, or hypoxia at any level during the late stages (d 11 to 18), increased the heart and chorioallantoic membrane weight, which partly compensated for the detrimental effects of hypoxia on embryonic development. We conclude that the first half of embryonic development contained the critical windows for the detrimental effects of hypoxia, and the second half contained the critical windows for the compensatory response of hypoxia in key organs.

Expression and regulation of glucocorticoid-induced leucine zipper in the developing anterior pituitary gland

The expression profile of glucocorticoid-induced leucine zipper (GILZ) in the anterior pituitary during the second half of embryonic development in the chick is consistent with in vivo regulation by circulating corticosteroids. However, nothing else has been reported about the presence of GILZ in the neuroendocrine system. We sought to characterize expression and regulation of GILZ in the chicken embryonic pituitary gland and determine the effect of GILZ overexpression on anterior pituitary hormone levels. Pituitary GILZ mRNA levels increased during embryogenesis to a maximum on the day of hatch, and decreased through the first week after hatch. GILZ expression was rapidly upregulated by corticosterone in embryonic pituitary cells. To determine whether GILZ regulates hormone gene expression in the developing anterior pituitary, we overexpressed GILZ in embryonic pituitary cells and measured mRNA for the major pituitary hormones. Exogenous GILZ increased prolactin mRNA above basal levels, but not as high as that in corticosterone-treated cells, indicating that GILZ may play a small role in lactotroph differentiation. The largest effect we observed was a twofold increase in FSH beta subunit in cells transfected with GILZ but not treated with corticosterone, suggesting that GILZ may positively regulate gonadotroph development in a manner not involving glucocorticoids. In conclusion, this is the first report to characterize avian GILZ and examine its regulation in the developing neuroendocrine system. We have shown that GILZ is upregulated by glucocorticoids in the embryonic pituitary gland and may regulate expression of several pituitary hormones.

Maternal serum and yolk hormone concentrations in the placental viviparous bonnethead shark, Sphyrna tiburo

Among vertebrates, maternal transfer of hormones to offspring has been studied extensively in mammals (placental transfer) and more recently in oviparous birds and reptiles (yolk transfer). The placental viviparous bonnethead shark, Sphyrna tiburo, allows the investigation of both yolk and placental hormone transfers in a single organism. In this species, yolk provides nutrition for the first half of embryonic development and placental transfer provides the second half. As sex determination is complete prior to development of placental connections, it was postulated that yolk hormones would have a prominent role in embryonic regulation. The goal of the current study was to determine serum and yolk hormone concentrations during five reproductive stages, from pre-ovulatory through pre-implantation (pre-placental) stages. Radioimmunoassay was used to determine 17β-estradiol, progesterone, and testosterone concentrations in both serum and yolk. When yolk and serum concentrations were compared, the yolk had significantly higher concentrations of both estradiol and progesterone during post-ovulation and early pregnancy. Yolk concentrations of testosterone were significantly less than serum at pre-ovulation, but there were no differences after that stage. When yolk concentrations were compared between stages, significantly higher concentrations of estradiol were present in ovulatory, post-ovulatory, and pre-implantation stages, while progesterone was significantly higher in post-ovulatory, early pregnancy, and pre-implantation stages and testosterone was higher in pre-ovulation. Most of these results are consistent with the published findings in birds and reptiles. Further, in the bonnethead shark, they suggest that yolk transfer of hormones is adequate for sexual differentiation in embryonic development and that estradiol probably has a significant developmental role.

Hypothalamic indolamines during embryonic development and effects of steroid exposure

The serotonin system has been implicated in the modulation of endocrine and behavioral components of reproduction. In this study, we examined endogenous hypothalamic indolamines during sexual differentiation and long-term effects of exogenous steroids during this time. In Experiment 1, Japanese quail were studied during the last half of embryonic development and early post-hatch. Samples were taken at embryonic day 10 (E10), E12, E14, E16, hatch (day 0), and days 3 and 5, post-hatch. Hypothalamic indolamines, including serotonin (5-HT) and its metabolite, 5-hydroxy indole acetic acid (5-HIAA) were measured by HPLC-EC detection. Females had relatively higher hypothalamic 5-HT at E14 than males, with both sexes showing increasing levels thereafter. By day 5, post-hatch, hypothalamic 5-HT content was higher in males than in females. When turnover was estimated by comparing relative concentrations of 5-HT to 5-HIAA, males were significantly higher at E12 and E14 than females. These data suggest that there are stage specific changes in the serotonin system, as well as sexually dimorphic patterns in the ontogeny and activity of this system. In Experiment 2, we investigated the effects of embryonic steroid hormone treatment on the serotonin system and on male sexual behavior. Birds were treated with either estradiol benzoate (EB), testosterone propionate (TP) or sesame oil (vehicle control) at selected embryonic days (E10, E12, E14, E16, 0, D3, and D5). At 4 weeks post-hatch, birds were transferred to short photoperiod (16D:8L) for 3 weeks to prevent photostimulated reproductive development. At 7 weeks of age, males were implanted with a 20 mm silastic capsule filled with testosterone and sexual behavior was tested 1 week later. Brains were collected from both males and females, and preoptic area (POA) indolamines were measured. Steroid treatment at E10 or E12 resulted in the loss of male sexual behavior. Moreover, males treated with EB or TP on E12 also had increased POA 5-HT content as adults, compared to control males. Females treated with EB on either E10 or E 12 also had higher POA 5-HT content than control or TP treated females. These data provide evidence for sexual dimorphism in the hypothalamic 5-HT system at specific stages during embryonic development. Moreover, males were sensitive to exogenous EB and TP on E12, whereas females appeared to be affected by EB only and appeared to be sensitive to steroid effects over a longer period of time in development. Moreover, exogenous steroids at E12 in males also correlated with impaired sexual behavioral. These data suggest that long-term effects of embryonic steroid exposure may be mediated in part through effects on the serotonin neurotransmitter system.

Gender-related changes in the avian vasotocin system during ontogeny.

The arginine vasotocin (AVT) system of the avian brain includes a sexually dimorphic part that extends from the caudal part of preoptic region through the medial part of the bed nucleus of stria terminalis (BSTm) to the lateral septum. It is composed of the parvocellular neurons located in the BSTm and the dense innervation of the medial preoptic region and lateral septum. In this part of the brain, AVT expression is stronger in males than in females in a few bird species investigated to date. This review focuses on the ontogeny of sexual differences in the vasotocinergic system of two gallinaceous species, domestic chicken and Japanese quail, and on the role of gonadal hormones in organizing during development and maintaining in adulthood these differences. Parvocellular AVT neurons become discernible in the BSTm of males and females during the second half of embryonic development. These cells undergo a profound and irreversible sexual differentiation during ontogenetic development. Recent findings demonstrate a dual role of estrogens in the organization and activation of sex differences in the AVT system. During the embryonic period of ontogeny, estrogens differentiate the AVT system in a sexually dimorphic manner in parallel with the differentiation of sexual behavior, while in adulthood estrogens, locally produced from testosterone in the male brain, activate AVT synthesis in the BSTm. The sexually dimorphic part of the AVT system is sensitive to a number of abiotic factors such as light, temperature, and water availability. It is suggested that sex dimorphic vasotocinergic systems could be implicated in processes of social recognition in various behavioral contexts.

Mechanism of the fluorescent light induced suppression of Curly phenotype in Drosophila melanogaster.

A dominant mutation Curly (Cy), frequently used as a marker on the second chromosome in Drosophila melanogaster, was previously shown to be suppressed by several factors, including larval crowding, low temperature, and fluorescent light. While the first two factors affect this mutation only partially, fluorescent tube exposed flies exhibit an almost completely suppressed (wild type) phenotype. This suppressive effect is the result of a combination of the electric field and light, both factors being produced by common fluorescent tubes. In this study, experiments were carried out to clarify the basic mechanism of this unique phenomenon. Two fluorescent tube sensitive stages of Drosophila development were found in the second half of embryonic development and first half of the pupal stage. Riboflavin, which is administered to Drosophila larvae with yeast, and decomposed by light, seems to play a key role in this phenomenon. In a medium lacking riboflavin caused by light exposure, Cy expression is inhibited by the action of electric field. Positive results of experiments with lithium ions, which block the opening of Ca(2+) channels, support the hypothesis that electromagnetic fields may alter ion currents during ontogenic development of Drosophila, and thus influence, expression of the Cy gene. Also, fluorescent light induces an overexpression of a specific protein in the imaginal wing disc of Cy pupae.

I(f) current and spontaneous activity in mouse embryonic ventricular myocytes.

Knowledge of the initiation of electrical and contractile activity in the embryonic heart relies to a large extent on data obtained in chicken. In recent years, molecular biological techniques have raised an interest in mouse physiology, including early embryonic development. We studied action potentials and the occurrence of one of the pacemaker currents, I(f), by the whole-cell voltage and current-clamp technique at the earliest stage at which a regular heartbeat is established (9.5 days postcoitum) and at 1 day before birth. We show, first, that at the early stage there is a prominent I(f) in mouse embryonic ventricles, which decreases by 82% before birth in concert with the loss of regular spontaneous activity of ventricular cells. Second, the decrease in I(f) current is associated with a slight change in channel gating kinetics and a decrease in total mRNA expression of the genes encoding for I(f) current. Third, the most prevalent mRNA subtype is switched from HCN4 to HCN2 during the second half of embryonic development. Fourth, the I(f) current may be modulated by the beta-adrenergic cascade, although the coupling to the beta-adrenoceptor in the sarcolemma itself is not yet mature. We conclude that I(f) current of the sinus node type is present in early embryonic mouse ventricular cells. In association with a loss of I(f) current, the ventricle tends to lose pacemaker potency during the second half of embryonic development.

Tet-system for the regulation of gene expression during embryonic development.

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTACMV(M1) and rTACMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and beta-galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTACMV(M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTACMV-3/NZL-2 embryos at E13.5. Doxycycline abolished beta-gal expression in tTACMV(M1)/NZL-2 but induced it in rTACMV-3/NZL-2 embryos including late stages of embryo-genesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that 'double reporter' animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.

Expression of insulin-like growth factor-I (IGF-I) and IGF-II in the avian brain: relationship of in situ hybridization patterns with IGF type 1 receptor expression

Insulin-like growth factors (IGFs) are expressed in defined spatiotemporal patterns during the development of the mammalian central nervous system (CNS). Since IGF expression in avian species is less well documented, we studied here the expression of IGF-I and IGF-II during chicken CNS development, using in situ hybridization and reverse transcriptase-PCR, and compared the results with the expression of the IGF type 1 receptor (IGF-1R). IGF-II expression started early in embryonic life, shortly after the onset of IGF-1R expression. During organogenesis, IGF-II was strongly expressed in kidney, liver and gut primordia, in contrast with IGF-1R mRNA, which is highly enriched in proliferating neuroepithelia. During the second half of embryonic development, IGF-I and IGF-II had distinct expression patterns, suggesting specific roles for each ligand during brain maturation. IGF-II mRNA was found in numerous brainstem nuclei and in the optic tectum, whereas IGF-I mRNA was found predominantly in telencephalic regions. Both ligands were expressed in the cerebellum, but each by different cell layers. Some brain regions (olfactory bulb and olivo-cerebellar system) did not exhibit the postnatal downregulation typical of extrahepatic IGF-I expression, but continued to express IGF-I into adulthood. Purkinje cells expressed IGF-II in the embryo, but switched to IGF-I expression in the adult. The conservation of embryonic and postnatal IGF expression patterns in the CNS between avians and mammals suggests that the involvement of the IGF system in neurogenesis and differentiation, and possibly in neural plasticity and learning, may have arisen early during tetrapode/vertebrate evolution.

Prenatal Development of Hematopoietic and Hormone-Producing Cells in the Chicken Adenohypophysis

The developmental sequence of markers for hematopoietic, hormone-producing, and folliculo-stellate cells in the chicken adenohypophysis is described using immunohistochemical staining techniques. Hematopoietic cells are detected in cryosections of the adenohypophysis starting from 10- or 12-day embryos, using chicken-specific monoclonal antibodies against the leukocyte common antigen (CD45) and the macrophage antigen CVI-ChNL-68.1, respectively. During the second half of embryonic development, CVI-ChNL-68.1-positive macrophages, which are also found in several lymphoid and nonlymphoid tissues of the embryo, progressively populate the adenohypophysis, simultaneously with the maturation of the different hormone-producing cell types in their characteristic topographical location. In cryosections of embryonic chicken adenohypophyses, from day 10, distinct cell populations gradually become immunoreactive to chicken-specific monoclonal antibodies against proopiomelanocortin, the β-subunit of luteinizing hormone, growth hormone, and prolactin. At hatching, these pituitary hormones are immunohistochemically detectable in a topographical pattern corresponding to the known distribution of hormone-producing cells in the adult chicken adenohypophysis. However, in the hatchling, there is no immunoreactivity to the S100 protein, a marker characteristic for the non-hormone-producing folliculo-stellate (FS) cells in the adult adenohypophysis, although FS cells in the 4-week-old chicken show a strong immunoreactivity to a polyclonal antiserum against bovine S100. Immunoreactivity to the major histocompatibility complex class II (MHC-class II of the chicken) is also absent in the embryonic adenohypophysis, thereby corroborating the absence of these characteristic markers of dendritic cells (MHC class II) and FS cells (S100) in the perinatal adenohypophysis, as in the rat. It is concluded that, whereas early macrophages populate the adenohypophysis simultaneously with the maturation of hormone-producing cells (i.e., during the second half of embryonic development), the FS cell-specific expression of S100 protein does not take place before hatching, and neither does the expression of MHC-class II antigens in the embryonic chicken adenohypophysis.

Titres of juvenile hormone I, II and III in Spodoptera littoralis (Noctuidae) from the egg to the pupal moult and their modification by the egg–larval parasitoid Chelonus inanitus (Braconidae)

Physico-chemical analysis of juvenile hormones (JHs) of Spodoptera littoralis revealed highest quantities in the second half of embryonic development and in newly hatched 1st instar larvae. At these stages, mostly JH II, JH I and little JH III were found, while in later stages only JH II and JH III were found. Titres fluctuated in a similar manner in all larval instars, being lowest during the moults. In last (=6th) instar larvae, JHs disappeared in the late feeding–digging stage and again increased in the early prepupal stage. Parasitisation with Chelonus inanitus, a solitary egg–larval parasitoid which induces in its host the precocious onset of metamorphosis in the 5th instar, did not alter JH homologue composition but led to a disappearance of JHs in the 5th instar. Implantation of a parasitoid larva into early 5th instar larvae containing polydnavirus/venom caused a drop in the JH titre which indicates that the parasitoid larva plays an important role in the manipulation of the host's JH titre. In the parasitoid larva, only JH III was found; titres were highest in the 2nd larval instar, a stage when the host is in the 5th instar and contains almost no JHs. Thus, JHs of the parasitoid and the host fluctuate in an independent manner.

Ecdysteroids during ovarian development and embryogenesis in solitary and gregarious schistocerca gregaria

Maternal ecdysteroids identified in the vitellogenic oocytes of Schistocerca gregaria included more than 80% polar conjugates, up to 5% free ecdysteroids, and up to 15% non-hydrolyzable polar metabolites. The representations of ecdysone (E), 20-hydroxyecdysone (20E), and 2-deoxyecdysone (2dE) in the conjugates was about 16:3:1, and in the free ecdysteroids about 3:1:1. The quantity of ecdysteroids in the ovaries before egg-laying reached 2.3 ng 20E equiv. per mg tissue in the solitary, and 8.9 ng/mg in the gregarious females. Newly laid eggs contained 14 ng and 89 ng, respectively, of 20E equiv. per egg. Nearly all egg ecdysteroids were in form of conjugates and their content declined during the first half of embryonic development. The amount of ecdysteroids sharply increased to over 70 ng 20E equiv./egg in the solitary, and to nearly 400 ng/egg in the gregarious phase. In the second half of embryonic development, the representation of conjugates in total ecdysteroids was reduced to 45-55%, whereas that of free E + 20E rose to 30-40%. Free 2dE remained low but, in the gregarious embryos, free 26E increased to 10% of all ecdysteroids. The conjugates of solitary embryos contained nearly exclusively E and 20E (in ratio 2:1), whereas those of the gregarious embryos included E, 20E, 2dE, and 26E (in ratio 12:7:4:1). Towards the end of embryonic development, the amounts of conjugates and of free ecdysteroids decreased, while that of polar metabolites rose. A sharp drop in ecdysteroid content was associated with hatching but the more than five times higher ecdysteroid level in the gregarious than in the solitary phase was maintained in the newly hatched larvae. Arch. Copyright 1999 Wiley-Liss, Inc.

Staging of middle and late embryonic development in the medicinal leech,Hirudo medicinalis

We present a description of the last half of embryonic development in the European medicinal leech, Hirudo medicinalis, based entirely on externally visible morphological features, and establish reliably observable stages during that development. Embryogenesis, from the time fertilized eggs are deposited in an eggcase (called a cocoon) to the emergence of juveniles from the cocoon, takes approximately 4 weeks at room temperature. The stages described in this paper extend from the completion of segmentation to the appearance of the final bands of pigmentation. Developmental stages are expressed as percentages of total embryonic developmental time. This staging table was constructed for embryos kept at 20 degrees C. In addition, the development of animals kept at 17 degrees C or at 24 degrees C was compared with those held at 20 degrees C. Development proceeds more quickly at higher temperatures. Because development in embryos held at higher or lower temperatures was linearly related to the stages determined for embryos held at 20 degrees C, the rate of development at any intermediate temperature can be predicted from the staging table at 20 degrees C by simple multiplication.