Studies on the yolk granules of the silkworm,Bombyx mori L.: The morphology of diapause and non-diapause eggs during early developmental stages

Summary The morphological features during development of diapause and non-diapause eggs of the silkworm,Bombyx mori, were investigated by means of light and electron microscopy, with special reference to eggs up to 24 h after oviposition.The blastoderm and yolk cells began to be formed about 6 and 24 h after oviposition, respectively, in both the diapause and non-diapause eggs, indicating that the diapause and non-diapause eggs develop at similar rates at least until 24 h after oviposition.Specific changes in the distribution of yolk granules were observed during early development of the diapause egg. Its yolk granules gradually aggregated into clusters from the periphery toward the inside of the egg during the period of blastoderm formation. Aggregation of yolk granules was most noticeable about 12 h after oviposition and then they dispersed again before yolk cell formation. On the other hand, yolk granules of the non-diapause eggs remained dispersed during development.     

Quantitative assessment of lysosomal size, number and enzyme activity in mouse kidney during maturational development.

Image and cytochemical analyses were undertaken to determine possible correlation between the number and size of acid phosphatase-positive granules (lysosomes), and variation in acid phosphatase (AcP) activity in the proximal tubule cells of mouse-kidney during growth and development. Eighteen ddY strain mice ages: 1 day, 1 and 2 weeks, and 1, 2 and 10 months were used. The lanthanide-based method for the ultrastructural localization of AcP-activity was employed. The number and size of AcP positive granules were quantitatively analyzed by image analysis, and AcP activity by X-ray microanalysis. Significance was evaluated by 2-tailed-Student's t-test for the difference between means. AcP activity was observed in the lysosomes and the reaction product appeared dense and heterogeneous. In some cells, it appeared apparently homogeneous. The results showed that the number and size of AcP Positive granules (lysosomes) increased significantly from the first day after birth, recorded a peak in one week time and thereafter, it gradually declined until the 10th month. The result of X-ray microanalysis demonstrated a variation in accordance with the degree of AcP activity at different ages of the animals studied. The AcP activity decreased significantly from day one and progressively until the 10th month. From the results of the present work, it could be inferred that the changes in size and number of AcP positive granules, at least, at the early stage, and/or the variation in AcP activity are related to the growth and development of the animal.     

Maturation and degeneration of the fat body in the Drosophila larva and pupa as revealed by morphometric analysis

Using morphometric and cytochemical techniques we have described changes taking place in the fat body cells during three different stages of development. The cell number remains constant at about 2200 cells during larval life and then decreases gradually and continuously throughout metamorphosis and the first 3 days of the adult stage until no more cells can be observed. Cell size increases rapidly during the larval period and decreases steadily during metamorphosis and adult stage. The size of the nuclei increases during the larval instars and decreases during the pupal interval. The change in nuclear size is correlated with the amount of DNA present throughout development implying the nuclear DNA is synthesized during the larval period and degraded gradually during metamorphosis. The cell size changes are due in large part to accumulation or loss of reserve substances: lipid droplets, glycogen deposits and protein granules. During metamorphosis the amount of lipid decreases slightly whereas glycogen experiences two loss cycles. The protein granules in the form of lysosomes continue to increase in amount during the first day of metamorphosis because of a short period of massive autophagy. Then the lysosomes decrease in amount throughout the remainder of metamorphosis. The lysosomes stain positively for lipofuscin.     

Light and electron microscopic studies on the neurosecretory control of diapause incidence in Pieris rapae crucivora

The incidence of diapause was shown to be determined humorally during the larval-pupal ecdysis by means of brain extirpation experiments.On the basis of this observation, light and electron microscopic changes in the neurosecretory type II cells in the pars intercerebralis-corpus cardiacum system during pharate pupal and early pupal stages were examined in insects reared under long day-length (non-diapause individuals) and in insects reared under short day-length (diapause individuals). In the diapause individuals, neurosecretory granules in NS-II cells increased during the pupal instar and large aggregates of granules packed the cytoplasm. Thereafter, inclusion bodies showing cytoplasmic breakdown of the granules appeared.In the non-diapause individuals, on the contrary, electron micrographs suggesting the release of neurosecretory material from axon terminals were obtained just after the pupal ecdysis. There were very few granules, with many Golgi bodies and much rough ER 8 to 12 hr after the ecdysis.It is concluded that adult development is determined by the release of neurosecretory material from the axon terminals of NS-II cells at the larval-pupal ecdysis. If release does not occur, the pupae enter diapause. It is also thought that differences in day-length during the larval stages influence the activities of NS-II cells before pupation.     

Catecholamine-storing cells in the adrenal medulla of the pre- and postnatal rat

Summary The development of the rat adrenal medulla was studied at the ultrastructural level with particular emphasis placed on early discrimination of different catecholamine-storing cells. The first granule-containing cells, phaeochromoblasts, were seen at day 15 of gestation migrating into the anlage of the cortex. These cells were characterized by a few small granules (80–120 nm in diameter) and a high nuclear to cytoplasmic ratio. Presumably due to differentiation into chromaffin cells, they were no longer present after the eighth postnatal day. Maturation of phaeochromoblasts was indicated by an increase in number and size of their storage granules and a decrease in the nuclear to cytoplasmic ratio. Noradrenaline and adrenaline cell types were first clearly discernible at day 21 of gestation. Another cell type, a giant cell, was also recognized at this stage. In the adult animal, noradrenaline, two morphologically different types of adrenaline, and small granule-containing cells were observed.By applying acetylcholinesterase histochemistry, it was found that at day 17 of gestation a small population of granule-storing cells showed strong positive staining in the endoplasmic reticulum. In the adult animal this cell type was further characterized by small-storage granules. Other chromaffin cells began to show weak staining within the endoplasmic reticulum at day 19 of gestation. This staining appeared more frequently within adrenaline than noradrenaline cells. However, even in the adult animal many cells of both types were completely negative.It is concluded that acetylcholinesterase histochemistry is a useful method for early discrimination of small granule-containing cells in the developing rat adrenal medulla.Supported by grants from the Deutsche Forschungsgemeinschaft     

Ontogenesis of Spermatozoa Autoantigens in Guinea Pigs

The ontogenetic appearance of three independant spermatozoa autoantigens (S, P and T) has been studied in guinea pig germinal cells by immunofluorescence and comparison with cytology and histological structures during early maturation of seminiferous tubule cells. The maturation of 120 testes from 60 guinea pigs studied from day 1 to day 50 after birth has shown an evolution in 3 periods. During the first, or negative, period (day 1 to day 25), only spermatogonia (from day 1) and spermatocytes I (from day 16) are present. No significant PAS-positive formations are seen and no autoantigen is detected. During the second, or transitional, period (day 26 to 29), spermatocytes II and spermatids appear as well as paranuclear PAS-positive golgian proacrosomal and acrosomal granules. At the same time, the three autoantigens S, P and T are detected on the same PAS-positive formations with a frequency that increases from day to day. During the third, or positive, period (from day 30) all testes present cells with PAS-positive formation, progressive maturation of acrosomes in spermatids and appearance of spermatozoa (present on day 39) leading to the adult structure of seminiferous tubules. The three autoantigens are constantly present during that period. The simultaneous appearance of the 3 antigens in haploid germinal cells (spermatids and possibly spermatocytes II) as an early expression of cytodifferentiation and their total absence from diploid germinal cells (spermatogonia and spermatocytes I) seem to be of biological significance.     

Sex and strain differences in the brush border and PAS-positive granules and giant bodies of the mouse renal S3 segment cells.

We recently demonstrated sexual dimorphism in the S3 segment of the ICR mouse kidney, as differences in periodic acid Schiff (PAS) staining on the brush border and the number and size of PAS-positive granules. However, whether these sex dependent features in the S3 segment of the mouse kidney occur only in the ICR strain or are a general feature also observed in other strains is unclear. In the present study, we examined the renal S3 segment of the ICR, BALB/c, C57BL/6, C3H/He and DBA/2 mice strains, which are commonly used in laboratory experiments. PAS staining of the brush border in females of all strains was more intense than that of males, and PAS-positive granules were detected in all females. In male groups, PAS-positive granules were detected in the DBA/2 strain only, but their number was very few. In addition, PAS-positive giant bodies, larger than the nuclear size, were detected in females except those of the C57BL/6 strain. Histometrical investigation demonstrated apparent strain differences in a number of PAS-positive granules and PAS-positive giant bodies. The ultrastructural and cytochemical investigations suggest that the PAS-positive granules and PAS-positive giant bodies were multilamellar lysosomes. We propose that the present findings are significant for comparative morphology in laboratory animal science.     

Seasonal regulation of dormancy in Chrysopa carnea (Neuroptera)

In Ithaca, New York, diapause incidence in Chrysopa carnea exceeds 50 per cent among adults emerging between 31 August and 3 September. Apparently, decreasing late summer day lengths, which exceed the stationary critical photoperiod in the laboratory, act on the sensitive stages and induce diapause. Full diapause intensity occurs about 2 weeks after adult emergence; complete winter coloration appears approximately 2 weeks later. Photoperiodic stimili in combination with the geographic strain largely determine diapause depth.Decreasing autumn day lengths decelerate diapause development and maintain diapause until the winter solstice. Subsequently, short day lengths and cold temperatures slow diapause development. In nature, neither long days, increasing day lengths, nor chilling hasten diapause termination. Diapause duration is largely determined by the interaction between inducing photoperiod, maintaining photoperiodic and thermal stimuli, and geographic strain. In Ithaca, diapause ends (all response to photoperiod ceases) between 22 January and 12 March.After diapause ends, the animals retain their diapause characteristics until temperatures exceed 4°C. Post-diapause reproductive development in females requires approximately 100 heat units above 4°C; however, below 8°C mating and oviposition are absent. Although the winter coloration remains, mating occurs within 1 day after transfer from 4 to 24°C. Temperature determines the rate at which the green summer colour returns.     

A fine structural survey of the development of the adult fat body of Leptinotarsa decemlineata

The fat body of a female Colorado potato beetle, Leptinotarsa decemlineata, at adult ecdysis, contains a large number of protein granules which are composed of light and dark zones. Part of the light zone in some of these granules is believed to be urate. During the first two days after adult ecdysis, fat body development is not essentially different in females reared either under long- or short-day conditions. Protein granules and large vacuoles disappear and the first cell organelles are regenerated. The effect of the photoperiod on the histological structure of the fat is expressed after these events. In females reared under long-day conditions, the fat body becomes specialized for vitellogenin synthesis. Under short-day conditions, the fat body stores massive amounts of lipid until day 6 after adult ecdysis. Then the first electron-dense protein granules develop near the nucleus, and on day 10 the first autophagic vacuoles are seen. These structure changes are discussed in connection with the known biochemical properties of the adult faty body of Leptinotarsa.     

Photoperiodic control of larval development in the semivoltine cockroachPeriplaneta japonica (Blattidae: Dictyoptera)

Periplaneta japonica is semivoltine, entering early diapause in any (except the first) larval instar before the last, and late diapause in the last instar. Early diapause was induced under a short day of 13 h or less at 28°C, and under both short and long daylength (12–16 h) at 20°C. The shorter the daylength and the lower the temperature, the younger the instar was entering early diapause. Early diapause was terminated by a long day (16 h) or a high temperature (28°C), after which larvae grew faster in short days than in long days until the last instar, when they again entered diapause, always in short days and frequently in long days. This late diapause was terminated also by an increase in daylength and was always followed by adult emergence. In this case, 13 and 14 h daylengths after exposure to 12 h daylength were as effective as 16 h daylength. Ourdoor samples collected in late autumn, winter and early spring at Hirosaki (40.5°N) comprised two distinct size groups, corresponding with the early and late diapause instars. Based on these results, the seasonal development strategy and intriguing aspects of the photoperiodic response in this cockroach are discussed.     

Changes in amino acid pools in the silkworm,Bombyx mori during embryonic life: Alanine accumulation and its conversion to proline during diapause

Alanine accumulated in silkworm eggs at the onset of diapause. When the eggs were kept at 4°C during diapause, this alanine was converted to glutamate, glutamine and especially proline. On resumption of development at 25°C after diapause, proline was used as an energy source for protein synthesis. In HCl-treated diapause eggs, which develop like non-diapause eggs, most amino acids showed similar developmental changes to those in eggs in resumption of embryogenesis after diapause. However, the proline level increased until the middle of embryonic development and then decreased. Continuous incubation of diapause eggs at 25°C after day 10 of oviposition caused a decrease in alanine with increases in glutamine and proline, while the levels of most other amino acids either decreased slightly or remained unchanged until day 80, when most eggs died. These results show that diapause eggs have a metabolic complex coupled with carbohydrate and amino acid metabolism inclusive of the 2-oxoglutarate-glutamate shuttle. Under conditions when embryogenesis proceeded, the level of phosphoethanolamine decreased rapidly.     

Egfr signaling controls the size of the stem cell precursor pool in the Drosophila ovary

In many animals, germline progenitors are kept undifferentiated to give rise to germline stem cells (GSCs), enabling continuous production of gametes throughout animal life. In the Drosophila ovary, GSCs arise from a subset of primordial germ cells (PGCs) that stay undifferentiated even after gametogenesis has started. How a certain population of PGCs is protected against differentiation, and the significance of its regulatory mechanisms on GSC establishment remain elusive. Here we show that epidermal growth factor receptor (Egfr) signaling in somatic stromal intermingled cells (ICs), activated by its ligand produced in germ cells, controls the size of the PGC pool at the onset of gametogenesis. Egfr signaling in ICs limits the number of cells that express the heparan sulfate proteoglycan Dally, which is required for the movement and stability of the locally-produced stromal morphogen, Decapentaplegic (Dpp, a BMP2/4 homologue). Dpp is received by PGCs and maintains them in an undifferentiated state. Altering Egfr signaling levels changes the size of the PGC pool and affects the number of GSCs established during development. While excess GSC formation is compensated by the adult stage, insufficient GSC formation can lead to adult ovarioles that completely lack GSCs, suggesting that ensuring an absolute size of the PGC pool is crucial for the GSC system.     

Development of ATPase-positive,immature Langerhans cells in the fetal mouse epidermis and their maturation during the early postnatal period

Summary The development and maturation of Langerhans cells during the differentiation of skin was studied in mice from fetal day 13 to adult using 3 indices: (1) ATPase activity; (2) ultrastructure; and (3) quantitative evaluation of the cell population.ATPase-positive Langerhans cells appeared in the epidermis at first at fetal day 16, and they increased in number in the differentiating epidermis during the late fetal period. The earliest appearance of Birbeck granules was at postnatal day 4. Cored tubules were also formed in the Langerhans cells in the dermis at around the same age. The cells containing Birbeck granules or cored tubules are considered to be mature Langerhans cells. In the Langerhans-cell lineage, those cells in the epidermis at stages earlier than postnatal day 4 and not yet containing specific organelles are considered to be immature Langerhans cells. These immature Langerhans cells can be identified ultrastructurally in the epidermis at fetal day 16, coinciding with the appearance of ATPase-positive cells. The increase in the number of immature Langerhans cells during the perinatal period was shown by quantitative analysis of nuclear density and relative Langerhans-cell area on the electron micrographs.It is concluded that ATPase is a marker of the Langerhans-cell lineage from the early development stages, while Birbeck granules and cored tubules are markers that identify mature Langerhans cells in electron micrographs.     

Identification of two types of melanocyte within the stria vascularis of the mouse inner ear

We have distinguished two types of melanocyte within the intermediate layer of the stria vascularis in the cochlea of normally pigmented mice: light and dark intermediate cells. The light intermediate cells are present in the stria from birth and have the typical appearance of a melanocyte. They are large and dendritic with electron-lucent cytoplasm containing numerous vesicles that show tyrosinase activity, and pigment granules in various stages of development. These granules have the ultrastructural and histochemical characteristics of premelanosomes and melanosomes. The light intermediate cells persist throughout life, but less frequently contain pigment in older animals. The dark intermediate cells, present only in adult mice, vary considerably in number and distribution between animals. Pigment granules, bound within an electron-dense acid phosphatase-rich matrix, form the main component of the dark intermediate cells. The intermediate cells may comprise either two distinct cell populations or different developmental stages of the same cell type; ultrastructural observations suggest the latter. In young mice, light intermediate cells contain the electron-dense matrices, which at later stages of development are found almost exclusively in dark cells. The dark intermediate cells contain few cell organelles other than pigment granules accumulated within lysosomal bodies and they often have pycnotic nuclei. These observations suggest that the dark intermediate cells are a degenerate form of the light intermediate cells. Clusters of melanosomes also occur in the basal cells, and to a much lesser extent in the marginal cells. These cells do not stain after incubation in DOPA, suggesting that they are not capable of melanin synthesis, and therefore probably acquire melanin by donation from adjacent melanocytes. Pigment clusters are also found within the spiral ligament at all stages of development.     

Induction and development of winter larval diapause in a drosophilid fly,Chymomyza costata

Photoperiodic response during induction of larval hibernal diapause of Chymomyza costata was characterized and the course of diapause development was analyzed in the laboratory. C. costata becomes sensitive to photoperiodic stimuli during an unspecified stage of its early development (embryo, 1st larval instar); the sensitivity gradually increases during the 2nd and early 3rd larval instars and reaches its maximum just before the moment when it abruptly ceases at the age of 15-19 days after oviposition. Diapause intensifies during a period of 2-3 weeks after induction and, later, is maintained without apparent development until death (between 150 and 250 days) under 18 degrees C and a short-day photoperiod (L10:D14, SD). Diapause may be terminated in a horotelic process by exposure to a low temperature (2 degrees C) during which larvae subsequently (1) synchronize their post-diapause development (requires up to 14 days of chilling), (2) lose photoperiodic sensitivity (2 months), and finally (3) terminate diapause (5 months). Alternatively, diapause may be terminated in a tachytelic process by exposure to a high temperature (18 degrees C) and long-day photoperiod (L16:D8, LD) during which no synchronization occurs and pupariation takes place after a mean of 25.2 days (with a broad range from 8 to more than 50 days). Larvae that are transferred from LD to SD during their sensitive period switch their developmental programming from pupariation to diapause. Proliferation of adult primordial structures (imaginal discs, neuroblasts) slows down within 1 day after transfer. In contrast, whole body growth continues for at least 3 days before its rate slows down and matches the rate characteristic for SD conditions.     

Diapause development in the chestnut weevilCurculio elephas

Larval diapause development in the chestnut weevilCurculio elephas (Coleoptera, Curculionidae) was studied in the laboratory at different temperatures. The results proved that exposure to low temperatures (3–6°C) in the period December–February is not required to complete diapause. The diapause is terminated in December and from January on, the larvae can initiate post-diapause morphogenesis in the laboratory, if temperatures allow it. In the field developmental rates are negligible during winter cold (4–6°C) and only after March morphogenesis can proceed with no interruption until adult emergence. Diapause and post-diapause quiescence contribute to individual synchronization for initiation of development. The observed spread of adult emergence was 30 days in the laboratory. This variability produced during post-diapause development may be a response to annual variation in the phenology of the chestnuts.     

Specialized basophilic cells in the ventral lobe of the pituitary of the smooth dogfish,Mustelus canis

The ventral lobe of the adenohypophysis of the smooth dogfish, Mustelus canis, a viviparous elasmobranch, has been found to possess distinctive cells identified as basophils on the basis of staining properties. At maximum size, such a cell consists of a distended vesicle containing PAS-positive, AF-negative material surrounded by a thin envelope of cytoplasm and an eccentric nucleus. In earlier stages of these cells, vesicles are small or absent and granules in the more abundant cytoplasms are AF or Alcian-positive. Basophil numbers are high in pre-ovulation and mid-ovulation females, decrease markedly after the end of ovulation until embryos are about 1 cm long then increase greatly during August and September while embryos grow to 8 cm in length. Early high counts, if these basophils are gonadotropes, may be correlated with stimulation of the ovary and ovulation; reduced numbers suggest inhibition, possibly by ovarian hormones for a period, while subsequent increase may indicate indirect involvement in uterine conditions in this viviparous species. Conclusion are, admittedly, tentative as specimens were available during only a fraction of the ten month gestation period.     

Differential effects of post-natal development, animal strain and long term recovery on the restoration of neuromuscular function after neuromyotoxic injury in rat

We have analysed the effect of long term recovery, post-natal development and animal strain on the extent of restoration of neuromuscular function after neuromyotoxic injury in the rat (Rattus norvegicus). Muscle isometric contractile properties of soleus muscle in response to nerve stimulation were measured in situ in snake venom injured muscles and compared to contralateral uninjured muscles. We show here that neuromuscular function was not fully recovered until 24 weeks after injury in young adult (2-3 month old) Wistar rats. Moreover, the level of functional recovery 3 weeks after injury induced in juvenile rats (1 month old) was not globally different from that in younger adult, adult (10 month old) and older adult (24 month old) Wistar rats. Furthermore, the level of recovery of some contractile parameters differed between Wistar and Sprague-Dawley strains 3 weeks after injury. In conclusion, a very long time (>12 weeks) is required for full neuromuscular recovery following neuromyotoxic injury of young adult rats. Moreover, neuromuscular recovery during post-natal development is not markedly different from that during adult stage in the Wistar rat strain. Finally, some rat strain differences are observed in the recovery after injury of young adult rats.     

Diapause decision in the small tortoiseshell butterfly,Aglais urticae

Insects in temperate areas spend the inhospitable winter conditions in a resting stage known as diapause. In species that diapause in the larval or pupal stage, the decision whether to diapause or develop directly is customarily taken during the late instars, with long days (i.e., long light phases) and high temperatures promoting direct development. Among butterflies that overwinter as adults, data are rare and variable, but imply that the larval daylength conditions can affect the pathway decision. We studied the small tortoiseshell, Aglais urticae L. (Lepidoptera: Nymphalidae, Nymphalini), which is partially bivoltine from Central Scandinavia and southwards, and tested whether the pathway decision is taken in the larval or adult stage. We reared larvae under long‐day (L22:D2) or short‐day (L12:D12) photoperiods, and recorded the pathway taken by the eclosing adults by scoring their propensity to mate and produce eggs. We also tested whether the larval photoperiod influenced adult ability to diapause by assessing adult survival. The results clearly indicate that (1) there is no detectable effect of larval photoperiod treatment on the pathway decision taken by adults whether to enter diapause or to develop directly, (2) some individuals are obligately univoltine and insensitive to photoperiod during adulthood, whereas (3) other individuals can facultatively enter diapause or direct development, depending on the photoperiod experienced after adult eclosion.