Developmental potencies of nuclei from cleavage,preblastoderm, and syncytial blastoderm transplanted into unfertilized eggs ofDrosophila melanogaster

Summary Wild-type nuclei from eggs ofDrosophila melanogaster at various developmental stages and from different regions of the egg—cleavage nuclei, pole nuclei from preblastoderm, and lateral nuclei from syncytial blastoderm—were singly implanted into unfertilizedy w sn ³ lz _50e eggs to determine their developmental potencies.All three types of transplanted nuclei were almost equally effective in initiating development of unfertilized eggs. Development was arrested in one of five critieal embryonic stages or in one of the three larval instars. The frequency of individuals reaching a distinct stage was approximately the same for all three types of donor nuclei. The stage-specific pattern of defects was independent of the type of nucleus transplanted.The deviations from normal development were broadly similar to those seen in controls developing from fertilized eggs which had only been punctured or into which cytoplasm had been injected. Many defective embryos also occurred in these control experiments. These and other observations indicate that a large proportion of irregularly developed individuals found after nuclear transfer can be ascribed to loss of egg material, disturbances in the internal organization of the egg during nuclear implantation, and the difficulty the implanted nucleus has in adjusting to the autonomous processes within the egg, such as the formation and migration of cytoplasmic islands.Some of the defective embryos and larvae originating from nuclear transfer were implanted into adult hosts. After culture for 14 days the early embryonic stages had formed several larval structures, and the late embryonic and larval stages had developed all larval organs. The proliferated imaginal primordia of thesein vivo cultured embryos and larvae, as well as the imaginal disks of the third instar larva, were then implanted into larval hosts with which they passed through metamorphosis and differentiated into imaginal structures. All three types of donor nuclei were capable of producing all adult structures derivedin situ from imaginal disks. The phenotype of these structures waswild-type, thus demonstrating their origin from the transplanted nuclei.The problem as to why not all transplanted nuclei initiated development, and why development after nuclear transplantation was arrested at the third larval instar, at the latest, is discussed.This article is dedicated to Professor Friedrich Seidel on the occasion of his 75th birthday.     

The acquisition of egg cytoplasmic non-histone proteins by nuclei during nuclear reprogramming

Inseminated eggs and nuclear transplants of Rana pipiens were labeled with [³H]tryptophan. Both the pronuclei of fertilized eggs and the late gastrula endodermal nuclei of transplants concentrated label during the first cell cycle of the egg, and this label was resistant to boiling TCA. These studies demonstrate that nuclear reprogramming is accompanied by the nuclear acquisition of cytoplasmic non-histone proteins from the egg.     

Die Zytologie der bisexuellen und parthenogenetischen Fortpflanzung von Heteropeza pygmaea Winnertz,einer Gallmücke mit pädogenetischer Vermehrung

Heteropeza pygmaea (syn. Oligarces paradoxus) can reproduce as larvae by paedogenesis or as imagines (Fig. 1). The eggs of imagines may develop after fertilization or parthenogenetically. The fertilized eggs give rise to female larvae, which develop into mother-larvae with female offspring (Weibchenmütter). Only a few of the larvae which hatch from unfertilized eggs become motherlarvae with female offspring; the others die. Spermatogenesis is aberrant, as it is in all gall midges studied to date. The primary spermatocyte contains 53 or 63 chromosomes. The meiotic divisions give rise to two sperms each of which contains only 7 chromosomes (Figs. 5–11). The eggs of the imago are composed of the oocyte and the nurse-cell chamber. In addition to the oocyte nucleus and the nurse-cell nuclei there are three other nuclei in the eggs (Figs. 15–17). They are called small nuclei (kleine Kerne). In prometaphase stages of the first cleavage division it could be seen that these nuclei contain about 10 chromosomes. Therefore it is assumed that these nuclei originate from the soma of the mother-larva. The chromosome number of the primary oocyte is approximately 66. The oocyte completes two meiotic divisions. The reduced egg nucleus contains approximately 33 chromosomes. The polar body-nuclei degenerate during the first cleavage divisions. The fertilized egg contains 2–3 sperms. The primary cleavage nucleus is formed by the egg nucleus and usually all of the sperm nuclei and the small nuclei (Figs. 21–29). The most frequent chromosome numbers in the primary cleavage nuclei are about 77 and 67. The first and the second cleavage divisions are normal. A first elimination occurs in the 3rd, 4th, and 5th cleavage division (Fig. 30). All except 6 chromosomes are eliminated from the future somatic nuclei. Following a second elimination (Figs. 33, 34), the future somatic nuclei contain 5 chromosomes. No elimination occurs in the divisions of the germ line nucleus. In eggs which develop parthenogenetically the primary cleavage nucleus is formed by the egg nucleus and 2–3 small nuclei. It's chromosome number is therefore about 53 or 63. After two eliminations, which are similar to the ones which occur in fertilized eggs, the soma contains 5 chromosomes. The somatic nuclei of male larvae which arrise by paedogenesis contain 5 chromosomes; while the somatic nuclei of female larvae of paedogenetic origin contain 10 chromosomes. It was therefore assumed earlier that sex was determined by haploidy or diploidy. But the above results show that larvae from fertilized as well as from unfertilized eggs of imagines have 5 chromosomes in the soma, but are females, and the female paedogenetic offspring of larvae from unfertilized eggs have either 5 or 10 chromosomes in their somatic cells. Therefore sex determination is not by haploidy-diploidy but by some other, unknown, mechanism. The cytological events associated with paedogenetic, bisexual, and parthenogenetic reproduction in Heteropeza pygmaea are compared (Fig. 37). The occurrence and meaning of the small nuclei which are found in the eggs of most gall midges are discussed. It has been shown here that these nuclei function to restore the chromosome number in fertilized eggs; it is suggested that they function similarity in certain other gall midges. Consideration of the mode of restoration of the germ-line chromosome number leads to the conclusion that in Heteropeza few, if any, of the chromosomes are limited to the germ-line, i.e. can never occur in somatic cells (p. 124).     

The cytology of paedogenesis in the gall midgeMycophila speyeri

In paedogenetically developing female eggs of the gall midgeMycophila speyeri only one equational meiotic division occurs. The primary cleavage nucleus contains 29 chromosomes. In the fourth cleavage division 23 chromosomes are eliminated from the future somatic nuclei while the primordial germ-line nucleus keeps the high chromosome number.—The paedogenetic development of male eggs begins with two meiotic divisions. The egg nucleus with 14 or 15 chromosomes fuses with two, sometimes only one, somatic nuclei (2n=6) of maternal origin (regulation). Thus the primary cleavage nucleus contains 26 or 27 chromosomes, sometimes only 20 or 21. Elimination in cleavage divisions V and VI leeds to somatic nuclei with 3 chromosomes while the primordial germ-line nucleus keeps the high chromosome number.—Differences between male and female eggs and the evolution of regulation in gall midges are discussed.     

Lebendbeobachtungen der Chromosomen-Aufregulation bei der Gallmücke Heteropeza pygmaea (Cecidomyiidae,Diptera)

In male-determined, paedogenetically developing eggs of Heteropeza pygmaea a restitutive fertilization takes place after meiosis. Two small nuclei of maternal origin (somatic nuclei) and the egg nucleus migrate to the center of the egg chamber. Their chromosomes then form the metaphase plate of the primary cleavage nucleus. The in vitro observations and the analysis of photomicrographs and time lapse films revealed that the metaphase stage can be reached in three different ways: 1. The egg nucleus and the two somatic nuclei form one common spindle. 2. The egg nucleus forms a spindle and the two somatic nuclei together form another one. The two spindles then fuse in late prometaphase and form a single spindle. 3. The egg nucleus alone forms a spindle. The chromosomes of the somatic nuclei migrate to the equator of this spindle. This variation in the restitutive fertilization is explained by an increasing asynchrony between the development of the egg nucleus and the slower somatic nuclei from the first to the third type.     

Insemination of immature sea urchin (Arbacia punctulata) eggs

Nuclei from osmotically opened erythrocytes and erythroblasts were injected into nucleated or enucleated Xenopus laevis eggs. Although the cleavage pattern of the recipient eggs which started to divide was normal in about half of the cases, nuclei from erythrocytes injected into nucleated or enucleated eggs never promoted development beyond the early gastrula stage. In contrast, nuclei from osmotically opened erythroblasts injected into enucleated eggs promoted development to early tadpole stages (stages 29–36). Frequently, injection of osmotically broken erythroblasts injected into nonenucleated eggs gave rise to triploid larvae which all died at roughly the same early tadpole stages (29–36). Surprisingly, development did not proceed to the stage of advanced organogenesis (stages 44–47), which is easily reached by gynogenetic haploids: The presence of the haploid genome derived from the egg pronucleus did not significantly improve the developmental capacity. Embryos obtained by single injection of erythrocyte nuclei into nucleated eggs were unable to pass the gastrula stage. To invalidate the interpretation that the observed arrest in development was related to nuclear damage during injection of the recipient eggs, single unbroken erythrocytes and unbroken erythroblasts were transferred into nucleated and enucleated eggs. No cleavage was observed in both classes of eggs injected with unbroken erythrocytes. In contrast, erythroblasts were found to induce cleavage in the recipient eggs at a frequency of about 11%. To ascertain that the nucleus of unbroken erythroblasts participated in development, the 1-nucleolar marker was used. Diploid embryos with only one nucleolus present were found following injection of unbroken erythroblasts into enucleated eggs from 2nu females. Triploid 2nu embryos were detected following injection of (diploid) 1nu erythroblasts into nonenucleated eggs from 2nu females. The most advanced development stages reached by these embryos did not, however, differ from the best results found in the first class of experiments: Nuclei from erythroblasts injected undamaged into nucleated or enucleated eggs never developed into a normal tadpole. Serial transfer experiments were performed using normally gastrulating embryos which had developed, following the injection of 1nu unbroken erythroblasts into recipient eggs. These donors for serial transfer experiments were checked for the presence of the 1nu marker. In addition they had passed through a normally cleaving eight-cell stage. No improvement in developmental capacity as compared to first transfer experiments could be found.     

A comparative study of the egg-laying behaviour and larval development of Pieris rapae L. and P. brassicae L. on the same host plants

Summary Pieris rapae and P. brassicae feed on the same host plants and have synchronized seasons. P. brassicae, whose larvae are twice the size of P. rapae, lays eggs in clusters of 40–100 eggs whereas P. rapae lays single eggs. In this paper we examine how egg clustering may be advantageous for P. brassicae. The larval development of each species was studied, and found not to differ significantly. P. brassicae larvae were observed to migrate from their host plant after defoliating it. A comparison of the efficiency of host plant utilization by the two pierid species was undertaken by measuring the effect of larval feeding on the growth of their host plants (kale and brussel sprouts). The results show that egg clustering is advantageous for larval fitness in terms of host resource exploitation, and we suggest that P. brassicae is adapted for ovipositing on clumped vegetation, while P. rapae is selected for exploiting isolated plants.     

Chicken erythrocyte membranes: comparison of nuclear and plasma membranes from adults and embryos

These experiments were designed to determine whether the migration of RNA molecules from an implanted nucleus to the host cytoplasm and from there into the host cell nucleus against a concentration gradient might reflect an artefact induced by the process of nuclear transplantation. That is, are RNA molecules, as previously shown for certain nuclear proteins, caused to artefactually leave a manipulated nucleus and then move into the host cell nucleus (as well as return to the grafted nucleus) during the recovery period?A variety of experiments involving different kinds of manipulative sequences and different numbers of nuclear transplantations suggest—but do not prove—that no artefact is involved in the migration of RNA from one nucleus to another but two experiments strongly support the view that the shuttling activity is a normal physiological process. One of the latter involved a determination of the rate of egress of ³H-RNA from an implanted nucleus and reveals that that rate, in contrast with the equivalent rate of egress for labeled proteins which is clearly abnormal after micromanipulation, is totally consonant with the rate of movement of RNA from nucleus to cytoplasm established from experiments that do not involve micromanipulation. The other experiment involves comparison of (1) the amount of radioactivity acquired by an unlabeled nucleus present in the cell at the time a labeled nucleus is implanted with (2) the amount of radioactivity acquired by an unlabeled nucleus implanted after a labeled nucleus had been implanted and had time to recover from any possible operation-induced trauma. With ³H-protein nuclei the host nuclei of (1) acquired much more label than the host nuclei of (2) because in (1) the host nuclei were able to acquire much of the artefactually-released ³H-protein. For the ³H-RNA experiments, however, little difference was found between (1) and (2) in the amount of label acquired by the host cell nuclei. It can be concluded that little, if any, of the non-random shuttling activity of RNA molecules can be a reflection of an artefact.     

BEHAVIOR OF INTERPHASE EMBRYONIC NUCLEI TRANSPLANTED IN NUCLEAR MULTIPLICATION STAGE EMBRYOS OF DROSOPHILA MELANOGASTER

Interphase nuclei were transplanted from syncytial blastoderm into early cleavage embryos of Drosophila melanogaster. The transplanted nuclei, when exposed to host cytoplasm, were initiated to mitosis. During the period from 10 to 50 min after transplantation, the implanted nuclei and host nuclei were found not synchronous in their mitotic cycles. Synchrony was restored usually by the blastoderm stage.
About 5% of eggs with transplanted nuclei developed significantly faster than control eggs, resulting in premature blastoderm formation. This finding is discussed in relation to chimera formation and to embryonic development of grandchildless mutants.     

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997. © 1997 Wiley-Liss, Inc.     

Host plant, oviposition behavior and larval ecology of a sawfly leafminer, Profenusa japonica (Hymenoptera: Tenthredinidae)

The host plant, oviposition behavior and larval ecology of Profenusa japonica Togashi are reported for the first time. Adults of P. japonica mated and oviposited on a polyantha rose, Rosa multiflora (Rosaceae), in April. Each female adult laid an egg on the edge of a leaflet. Hatched larvae consumed the parenchymatous layer of leaflets and in so doing created a mine. The larval stage comprised five instars. On average, 70.6% of the total area of a leaflet was consumed by one larva. Female adults of P. japonica laid eggs singly, probably to avoid larval competition for food.     

Larval host plant affects fitness consequences of egg size variation in the seed beetle Stator limbatus

Egg size variation often has large effects on the fitness of progeny in insects. However, many studies have been unable to detect an advantage of developing from large eggs, suggesting that egg size variation has implications for offspring performance only under adverse conditions, such as during larval competition, periods of starvation, desiccation, or when larvae feed on low-quality resources. We test this hypothesis by examining the consequences of egg size variation for survivorship and development of a seed-feeding insect, Stator limbatus, on both a low-quality (Cercidium floridum) and a high-quality (Acacia greggii) host plant. Our results are consistent with the hypothesis. S. limbatus larval performance was affected by egg size only when developing on the poor-quality host (C. floridum); larvae from large eggs survived better on C. floridum than those from small eggs, while there was no evidence of an effect of egg size on progeny development time, body weight, or survivorship when larvae developed on A. greggii. These results indicate intense selection for large eggs within C. floridum-associated populations, but not in A. greggii-associated populations, so that egg size is predicted to vary among populations associated with different hosts. Our results also support this hypothesis; females from a C. floridum-associated population (Scottsdale) laid larger eggs than females from an A. greggii-associated population (Black Canyon City).     

Parabiotic development of fused eggs from the Hymenopteron,Pimpla turionellae,and of eggs injected with energids

Explanted oocytes and eggs of different developmental stages from the Hymenopteron Pimpla were fused in pairs as parabiotic tandems. Interactions within the tandem were analysed by time lapse films. Except for the exchange of nuclei, no joint development was observed, and each partner followed its own time pattern. The rapid cell cycles of the cleavage energids switched over to longer cycles according to the local developmental stage of the different egg regions, although all of the nuclei were still contained in a single plasmodium. In a second experimental series, nuclei in newly deposited eggs were X-rayed and replaced by cleavage energids from later stages injected into the wrong (=posterior) egg pole. Even injected blastoderm nuclei immediately took up mitotic activities and underwent rapid cell cycles characteristic of early cleavage. Normal embryos could be formed, although the nuclei had populated the egg in a reverse direction.     

Autoradiographic study of protein and RNA formation during early development of Drosophila eggs

Permeabilized eggs of Drosophila melanogaster were incubated in tritiated uridine, valine, and phenylalanine. The uptake and incorporation into TCA-insoluble material were measured by scintillation counting. There was very little incorporation of uridine before the blastoderm stage. At the blastoderm stage, the egg took up 2.4 pmoles/hr of uridine and incorporated 0.13 pmoles into RNA (assuming no dilution of specific activity of the precursor). The uptake of amino acids varied with the age of the embryo; virgin eggs synthesized about as much protein as fertilized eggs. Autoradiography of eggs incubated in uridine showed a lack of RNA synthesis in nuclei until the start of the blastoderm formation. The small amount of uridine incorporation before this stage was due to mitochondria. Incorporation of amino acids was uniform in the cytoplasm until the blastoderm; there was no incorporation by yolk granules. Regional difference in labeling appeared during gastrulation. The pole cells did not form RNA during the blastoderm stage, formation started during gastrulation. Protein labeling of the pole cells, on the contrary, was very strong in the blastoderm and early gastrula. These results indicate that the expression of zygotic genome before the blastoderm stage is unlikely.     

Life Cycle of Culicospora magna (Kudo, 1920) (Microsporida: Culicosporidae) in Culex restuans Theobald with Special Reference to Sexuality1

The life cycle of Culicospora magna (Kudo, 1920) Weiser, 1977, consists of two major developmental sequences that alternate in host individuals of successive generations, each of the sequences starting with a sporoplasm and ending with spores. The first sequence occurs in larval, pupal, and adult stages of a parental generation of the host mosquito, Culex restuans Theobald; it begins with a sporoplasm from an ingested uninucleate spore and progresses through stages in gametogony, plasmogamy, nuclear association, merogony, karyogamy, and disporous sporulation with production of binucleate spores that discharge sporoplasms into the oocytes. The second sequence occurs in egg and larval stages of a filial generation of the same host species; it begins with the binucleate sporoplasm that entered the egg, includes stages in merogony, nuclear dissociation, and mictosporous sporulation, and ends with uninucleate spores. These spores are released into the environment following death of the host and are capable of infecting new parental generation host individuals. The life cycle is conceived as an alternation of generations related to haploidy and diploidy in the nuclei, the transition from haploidy to diploidy occurring with nuclear association and the transition from diploidy to haploidy occurring with nuclear dissociation.     

Mediterranean flour moth Ephestia kuehniella eggs and larvae exposed to a static magnetic field and preference by Trichogramma embryophagum

This study investigated the effect of strong magnetic fields as insecticidal activity on Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) larvae and eggs at different stages of development and their preference by the egg parasitoid, Trichogramma embryophagum Hartig (Hymenoptera: Trichogrammatidae). Eggs ranging in age from 24-h to 48-h and 72-h-old and larvae (1 to 2 days) were exposed to 1.4 Tesla (T) magnetic fields from a DC power supply at 50 Hz for different time periods (3, 6, 12, 24, 48 and 72 h). Twelve hours of exposure at 1.4 T was toxic to 24-h-old eggs of E. kuehniella. The 72-h-old host eggs treated with 1.4 T for 6–72 h were not significantly preferred by T. embryophagum. The magnetic field was toxic to 24-h-old eggs of E. kuehniella exposed to 1.4 T for 12. The treatment of magnetic fields on the 72-h-old host egg with 1.4 T at 6–72 h was not significantly preferred by T. embryophagum. Magnetization of 24-h-old eggs of E. kuehniella for 3 h could be effectively used with T. embryophagum as sterilised host eggs. These eggs were markedly preferred by T. embryophagum. The LT₅₀ and LT₉₉ values of magnetic fields at different egg stages of E. kuehniella, and larvae were measured. A level of 1.4 T at 72 h completely prevented the development of the larvae. There was no significant effect on larval survival at 1.4 T at 48 and 72 h. Increasing magnetic fields exposure times for eggs that were 24-h, 48-h and 72-h-old prevented larval emergence and increased their mortality rate. Consequently, magnetic fields could be used in controlling stored-product pest eggs and larvae of E. kuehniella.     

Developmental defects of female-sterile mutants of Drosophila melanogaster

Gans et al. (1975) isolated female-sterile mutants, viable and normal in the homozygous state, producing apparently normal eggs which were unable to develop or developed into defective embryos or adults, even when fertilized with wild-type sperm. Developmental abnormalities of these mutants were surveyed by observing living and fixed material. The following types of mutants were distinguished according to the predominant developmental defect: (i) Eggs not developing at all, mostly remaining unfertilized. (ii) Eggs stopping development after a few cleavages, some with polyploid nuclei. (iii) Eggs stopping development at various embryonic stages, with haploid nuclei. (iv) Eggs with abnormal blastoderm, not developing further or giving abnormal embryos. (v) Eggs with abnormal gastrula, in one case with excessive invaginations, in another case with germ band failing to elongate. (vi) Eggs with embryos dying at different stages, without easily visible effects. Several of the mutants were temperature sensitive. In all the mutants there were eggs that died without developing. Most of the developmental defects appear to be due to general metabolic disturbances of the egg, not directly related to morphogenesis. There were no mutants affecting determination of a particular adult organ. The closest to a morphological type of mutation were those with abnormal blastoderm having successive bands of nuclei of different sizes. Those mutants were thermosensitive; at permissive termperatures they developed into agametic adults or adults with various defects of abdomens, wings or eyes. The nature of maternal genetic control of early morphogensis was discussed.     

Heterotopic transplantation in the syncytial blastoderm ofDrosophila: Evidence for anterior and posterior nuclear commitments

Summary The interaction ofDrosophila syncytial blastoderm nuclei and cortical cytoplasm in the control of somatic developmental commitments was studied by transplanting genetically marked nuclei and surrounding cytoplasm between anterior and posterior flanks. After completion of cellularization the host egg was cut. Host anterior or posterior partial embryos were cultured in adult abdomens for 8–10 days, then the larval tissue removed and injected into larval hosts for metamorphosis. Differentiated ectodermal implants were recovered from emerged adults and characterized. One hundred sixteen clearly interpretable control and experimental implants were found. Of the 73 experimental implants 15 were derived from donor nuclei.Among the 15 donor implants, 14 autonomously formed donor site anterior (head and thoracic) or posterior (abdomen and genital) structures. This donor autonomy is interpreted to mean that nuclear and cytoplasmic factors necessary for anterior and posterior somatic commitments are present and transplantable prior to the completion of cellularization. Since donor nuclei injected directly into host flanks, or premixed with host cytoplasm, would have been well exposed to any host cytoplasmic factors, donor nuclei appear to have adopted anterior or posterior somatic commitments which are stable to significant cytoplasmic alterations.In 14 implants, host nuclei exposed to donor material altered somatic fate and formed donor type structures. These conversions are interpreted to imply that cytoplasmic factors controlling anterior or posterior somatic fates are present in the syncytial balstoderm embryo.