ALBERT

Notes: Abstract Tissue maps, and cell characteristics and properties were recorded in a study under the optical microscope of the development of the mouse calvarium from pre-natal to 26 days. Osteocyte populations in left and right halves of the calvarium were similar, but decreased with time for a given volume. Small isolated areas of bone matrix stained for phosphate (or carbonate) in a more readily available form from that in the surrounding matrix, which could be stained after sectioning but failed to stain in bulk. Osteocyte types were defined on the basis of histochemical methods for calcium and phosphate, which were associated inside bone cells in a complex manner, varying with time and position. The calcium and phosphate were not always present within the cell in discrete regions and were not always present in the same place in a given cell type. On the basis of a study of changes in cell types with time in selected sites a sequence of “loading” and “unloading” is proposed.

Notes: Abstract Mucopolysaccharides (MPS) and collagen have been analyzed in different types of cartilage and some other tissues at various stages of prenatal and postnatal human development. The concentration of the chondroitin sulfate-type MPS increased from the 38th embryonal day, the 6-sulfate exceeding in amount the 4-sulfate. The increase in collagen concentration was accompanied by a decrease in the proportion of neutral salt-soluble collagen. There was no accumulation of chondroitin sulfates in the calvaria. A maximal concentration of MPS of a low molecular weight occurred in the skin and in the ear pinna after the 10th prenatal week. Postnatally, the MPS-concentration in the iliac crest and in the tibial articular cartilages decreased gradually, the highest values being reached in the newborn; the collagen-concentration showed an approximately reciprocal course.

Notes: Summary When the complexity of a developmental system evolves to a certain point, appreciable variation must occur in the process. The problem the biologist faces is whether this point constitutes a limit to the evolution of complexity in developmental systems. If not, what mechanisms are employed to cope with the problem ? The problem—essentially one in “custom fitting” of parts, — and the possible solution(s) to it that have evolved are discussed. The antibody producing system appears to be one that “solves” the custom-fitting problem.

Notes: Summary The nucleotide sequence ofDrosophila melanogaster 5S RNA has been determined and appears to be homogeneous both in the KC cell line and in the insect at different developmental stages. Experimental evidence on the conformation of this molecule is in agreement with a general class of 5S RNA models.

Notes: Summary Injection of α-ecdysone into the larval haemolymph of late third instar larvae ofD. virilis induces both the extrusion of secretory proteins and the inactivation of the enzyme glutamine-fructose-6-phosphate-aminotransferase (E.C. 2.6.16) in the salivary glands. In the presence of actinomycin D or cycloheximide the hormone is ineffective. If before adding these inhibitors RNA synthesis is allowed to proceed for 1.5h, or protein synthesis for 2h after ecdysone injection, however, the protein extrusion and the enzyme inactivation do occur. It is proposed that ecdysone controls these two cytoplasmic events at the transcriptional level by the activation of specific Correlations with puff activities are discussed.

Notes: Summary 1. The developmental potentials of dissociated cells of the different regions of the male foreleg disc ofDrosophila melanogaster were analysed. To this end, various amounts of foreleg disc material were dissociated together with an excess of heavily irradiated wing discs (“feeding layer”), and the reaggregates were cultured for 10 days in the abdomens of adult hosts prior to metamorphosis. 2. The foreleg disc cells were in most cases unable to regenerate missing structures in a circular direction within the leg segments. Instead they strongly tended to adopt the specifications of more distal leg segments (distal transformation), irrespective of the region of origin of the ancestor cells within the disc. 3. The distal transformation occurred mainly, if not exclusively, during an early phase (“initial phase”) in the reaggregates. 4. The extent of distal transformation was most pronounced in those series in which the foreleg cells were initially least diluted by the “feeding layer” cells. 5. Cells of the lower lateral quadrant were very poor both in proliferative activity and in the extent of distal transformation, compared to cells of the three remaining quadrants. In the experiments with a low initial dilution of the foreleg cells, cells of the lower medial quadrant underwent distal transformation much more distinctly than cells of the upper medial and the upper lateral quadrants. 6. Allotypic structures occurred exclusively in reaggregates of the upper medial and upper lateral quadrants. In these implants, however, the frequency of transdetermination was extremely high. 7. Two alternative mechanisms are discussed which could have led to the general occurrence of distal transformation. They differ in the basic assumption of whether or not the “feeding layer” cells were able to interact with the leg cells to influence their regulative behaviour. In addition, interactions among the leg cells themselves seemed to stimulate proliferation to varying degrees and may account for the observed differences in the degree of distal transformation.

Notes: Summary Three-hundred and twenty fertile,pal-induced Y-chromosome mosaic males and females were obtained. Fractional analysis of the sons of 55 somatically mosaic flies that were also germinally mosaic tentatively suggests that the number of functional primordial germ cells inDrosophila melanogaster is variable and that it is seldom greater than 24. From the observed 0.17 frequency of germinal mosaicism it was estimated that the average number of pole cells at the end of blastoderm formation is 45. At present, the germ cells afford the only opportunity to compare genetic estimates of the number of blastoderm or primordial cells with available histological counts. The good agreement between them suggests that both the fractional and the mosaic frequency methods for estimating primordial or blastoderm cell numbers of various larval and imaginal anatomical structures provide reasonably close approximations of the actual values.

Notes: Summary The developmental accretion of up to nine individual gangliosides in foetal brains, peri- and postnatal cortices, postnatal cerebelli and olfactory lobes and in the liver and the spleen were investigated in mice and compared with that of glycoprotein-bound sialic acid and the activity of the acetylcholinesterase. In foetal brain and in postnatal liver and spleen more sialic acid was found bound to glycoproteins than to gangliosides. In postnatal brain structures, however, ganglioside-NeuAc predominated and increased between the 7th and 21st d about 2-fold in the olfactory lobes and cerebellum and more than 3-fold in the cortex. During foetal development the relative quantities (mol %) as well as the absolute concentrations (compared with the fresh weight) of GM1, GM2 and GM3 in the brain decreased, whereas those of GD1a, GD1b and GQ increased. This pattern change continued perinatally in the cortex up to the end of the first week. Thereafter the pattern changed little, but the concentration of all gangliosides present increased much more rapidly, especially between the 10th and 13th d. The postnatal cerebellum and olfactory lobes contained higher concentrations of GM1 and GM3 than the cortex, both gangliosides decreasing in favour of their di-, tri- and tetrasialo-homologues during the third postnatal week. In all brains structures the accretion of GD1a and GT1 was proportional to the increase in the activity of the acetylcholinesterase. Unlike the brain structures, the ganglioside pattern in the liver and spleen, characterised by a predominance of monosialogangliosides and of GD3, did not change noticeably during the first three weeks after birth. The coincidence of the changes in ganglioside accretion observed in the different brain structures with successive periods of morphological differentiation further support the suggestion that gangliosides may play an important role in control of the growth and differentiation of developing nerve cells.

Notes: Summary 1. Histological analyses were made of imaginal discs and histoblasts during the larval development ofDrosophila melanogaster to determine the number of cells, the patterns of cell division and the growth dynamics in these adult primordia. Histological studies were also made of the imaginal rings which are the primordia of the adult salivary gland, fore-and hindgut, the anlage cells of the midgut and several larval and embryonic tissues. 2. In the newly-hatched larva, the immature eye-antenna, wing, haltere, leg and genital discs contain about 70, 38, 20, 36–45 and 64 cells respectively. These numbers include cells destined to form cuticular elements as well as peripodial, tracheal and nerve cells and probably the progenitors of adepithelial cells. The number of cells counted in the various imaginal disc anlagen is 1.5 to 4 times higher than the numbers deduced from genetic mosaic analyses by other investigators and reasons for these differences are given. 3. About 12 h after fertilization, mitosis ceases in all tissues of the embryo except the nervous system. After the larva hatches, mitosis resumes in most of the imaginal anlagen and in some larval tissues. The time of resumption of mitosis in the imaginal anlagen was determined after treating the larvae with colchicine for 2 h. 4. Among the imaginal discs, the eye disc is the first to begin cell division, at about 13–15 h after the hatching of the larva (first instar) followed by the wing (15–17 h), the haltere (18–20 h), the antenna, leg, and genitalia (24–26 h, early second instar), and finally the labial and dorsal prothoracic discs (52–54 h, early third instar). The cell doubling time for various discs was calculated from cell counts and the times agree closely with the doubling times deduced from clonal analyses by other workers: e.g., 7.5 h for the cells of the wing disc. 5. The imaginal ring of the hindgut first shows cell division early in the second instar. The imaginal rings of the foregut and salivary glands, the anlage cells of the midgut and the cells of the segmental lateral tracheal branches begin to divide early in the third instar. 6. The histoblasts which are the anlagen of the integument of the adult abdomen do not increase in number from the time of larval hatching until about 5 h after pupation when they begin to divide. Their behaviour contrasts with that of the histoblasts of the other dipterans such asCalliphora, Musca andDacus, which begin to divide during the second instar. 7. The histoblasts are an integral part of the larval abdominal epidermis and, unlike imaginal disc cells, secrete cuticle during larval life. Each hemisegment consists of an anterior dorsal, a posterior dorsal, and a ventral histoblast nest containing about 13, 6 and 12 cells respectively. The 62 histoblasts in each larval segment represent about 7–8% of the total number of cells that form the integument of that segment. 8. The number of cells in a particular type of histoblast nest was constant for both male and female larvae and among the different abdominal segments, except that the anterior dorsal group of the first and the seventh segments contains fewer cells than those of the other segments. Although the male and female adultDrosophila lack the first abdominal sternite and the male lacks the seventh abdominal tergite and sternite, the ventral histoblast nests of the first and the dorsal and ventral nests of the seventh abdominal segments are present in the larval stages as well as in the prepupa and have the same morphology and cell number as similar nests in the rest of the abdominal segments. 9. The cells of the imaginal discs increase in volume about six-fold and their nuclei increase in volume three-fold between the time of hatching and the initiation of mitosis. The histoblasts increase in volume about 60-fold and their nuclei increase in volume about 25-fold between larval hatching and pupariation. 10. Prior to each cell division, the nuclei of the columnar cells of the disc epithelium and of the histoblasts appear to migrate toward the apical surface of the epithelium. The cells round up and shift toward the apical region where mitosis occurs. After cytokinesis, the daughter cells move back to deeper positions in the epithelium. Because the nuclei of the non-dividing cells continue to lie deep in the epithelium, this intermitotic migration of nuclei gives these epithelia a pseudostratified appearance. 11. Analyses of the growth of larval cells and of organs confirmed the observations of earlier investigators that cell division occurs only in a few larval tissues, whereas growth in the rest of the larval tissues is by cell enlargement and polyteny. During larval life, cell division was detected only in the central nervous system, gonads, prothoracic glands, lymph glands and haemocytes. Each tissue began mitosis at a characteristic stage in larval life. The larval cells that did not divide, grew enormously, e.g., epidermal cells increased in volume 150-fold and their nuclei increased in volume 80-fold. 12. The adepithelial cells, which give rise to some of the imaginal muscles, were first identified between the thick side of the imaginal dise epithelium and the basement membrane at the beginning of the third larval instar (50–52 h). The origin of these precursors of mesodermal structures was analysed and evidence is presented that the adepithelial cells come from the disc epithelium. The question of the origin of the mesoderm of cyclorrhaphan Diptera is reviewed and it is suggested that the imaginal disc ectoderm may become segregated from the rest of the embryo before gastrulation has occurred, that is before the mesoderm has been established.

Notes: Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis. The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos. Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development. All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae. Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.

Notes: Summary The lineages of cells on the second-leg basitarsus ofDrosophila melanogaster were analyzed by examining gynandromorphs andMinute mosaics. Bracts lie proximal to bristles on the adult basitarsus, yet bract precursor cells were found to originate lateral to bristle precursor cells. In 6 of the 8 longitudinal rows of bristles on this segment, the bract cells arise ventral to the bristle cells; in the others they arise dorsally. The lateral cell origins are interpreted as reflecting a pattern of lateral cell movements associated with evagination of the leg disc. An unusual discrepancy was observed in the relative frequencies of male vs. female bracts and bristles in gynandromorphs. The discrepancy suggests that there is a cell-autonomous sexual difference in either the time at which cells begin moving during evagination or the speed with which they move. On the basis of the results, it is reasoned that the bristle pattern of the basitarsus does not originate in its final form. Prior to evagination, the bristle cells of each row are apparently closer together than in the final pattern, and the rows are farther apart. Evidence is presented which suggests that the bristle cells of each row may originally be arranged in a jagged line which is later straightened by cell movements. The two locations where the anterior/posterior compartment boundary of the second leg passes through the basitarsus were found to vary relative to the bristle pattern. If this boundary is assumed to be a fixed line of positional values, then the extent of the observed variability — which is estimated to be ± 1 or 2 cell diameters — provides a measure of the precision of patterning around the circumference.

Notes: Summary The differentiation of muscles in primary cultures of cells fromDrosophila melanogaster embryos was investigated. In early cultures, and in the absence of exogenous ecdysone, two main classes of muscle were found. Comparison, by light and electron microscopy, of one of these classes (the “myotube” class) with muscles from third instar larvae shows that this class corresponds to the muscles of the body wall of the larva. When α- or β-ecdysone is added to the cultures, these undergo a number of metamorphic changes. Most of the larval muscles disappear, and two new types of muscle form. Ultrastructural and light microscopic examination of these two types indicates that they correspond to the two classes of skeletal muscle (fibrillar and tubular) found in adult flies.

Notes: Summary The pathway of adult sensory nerves has been analysed in three experimental situations: (i) in flies with grossly abnormal thoracic morphology resulting from X-irradiation early during development, (ii) in flies which had been subjected to surgical operations late in the larval period, (iii) in homoeotic mutants. The results provide experimental support for a simple mechanism in which developing adult axons join the nearest larval nerve and are guided by it up to the central nervous system. In particular, experimental interference with normal development can result in nerves from different segments, or from dorsal and ventral appendages, joining each other and entering the central nervous system together.

Notes: Summary Since to earlier results indicated a dependence of the optic lobe on the compound eye during post-embryonic development, it appeared essential to specify the part played by the post-retinal fibres connecting these two organs. Especially, we wondered if the mitotic activity in the outer optic anlage common to the two outer optic ganglia (lamina and medulla) was controlled by the number of newly-neoformed post-retinal fibres, or if the regulating influence from the post-retinal fibres takes place later, during the differentiation of the ganglion cells. In order to answer these questions, three kinds of operation were performed: (1) removal, in young larvae, of the zone producing new ommatidia. This operation deprives the optic of the arrival of new post-retinal fibres below the operated level. (2) overloading of post-retinal fibres, by inducing zones that produced supernumerary ommatidia. (3) removal of an ocular volet, followed by its immediate reinsertion, to provide a “surgery-control”. The following results were obtained: (1) A preliminary growth regulation controlled the total number of neuroblasts in the outer optic anlage. However, the permanent mitotic activity of these cells was not controlled by post-retinal fibres. (2) A second regulation, much more precise, occurring in the lamina, consisted in the differentiation of the ganglion cells being affected by the new post-retinal fibres. The supernumerary cells then rapidly degenerated. (3) A last regulatory process, implying the integrity of post-retinal fibres, maintained the ganglion cells.

Notes: Summary The mature labial disc, when implanted into a larva of the same age, undergoes metamorphosis along with the host and produces one lateral half of the medi- and distiproboscis. On the basis of results obtained from transplanted disc halves (including the separate peripodial membrane) a tentative fate map of the labial disc was constructed, which shows most of the presumptive mediproboscis to be located in the dorsal, and most of the presumptive distiproboscis in the ventral part of the disc. The distal protion of the peripodial membrane also contains imaginal anlagen, viz. part of the mediproboscis, prementum, and labellar cap anlagen. The involvement of this part of the peripodial membrane was checked by a careful histological analysis of labial disc development during the first ten hours after prepupation. The results were compared with the situation described forCalliphora imaginal discs. In addition, a detailed morphological analysis was made of the proboscis of the homoeotic mutantproboscipedia (pb). At 27°C,pb changes the distiproboscis into a “telopodite” (leg segments distal to the coxa); the (unchanged) prementum may therefore correspond to the coxa. At 15° C, the tarsus of this homoeotic “telopodite” is replaced to a greater or lesser extent by an arista. The present analysis thus confirms (a) the fundamental morphological correspondence of the medi- and distiproboscis with the labium of other insects, and (b) the fundamental developmental correspondence of the labial, antennal, and leg discs.

Notes: Summary A temperature-sensitive period during early embryogenesis for three stocks carrying thetuh-3 gene suggests that it is a homoeotic mutation involved in the initial determination of the eye-antennal disc rather in maintenance of the determination.

Notes: Summary The development of the adult abdomen ofDrosophila melanogaster was analyzed by histology, microcautery, and genetic strategies. Eight nests of diploid histoblasts were identified in the newly hatched larva among the polytene epidermal cells of each abdominal segment: pairs of anterior dorsal, posterior dorsal, and ventral histoblast nests and a pair of spiracular anlagen. The histoblasts do not divide during larval life but begin dividing rapidly 3 h after pupariation, doubling every 3.6 h. Initially they remain confined to their original area, but 15 h after pupariation the nests enlarge, and histoblasts replace adjacent epidermis cell by cell. The histoblasts cover half the abdomen by 28 h after pupariation and the rest by 36 h. Polytene epidermal cells of the intersegmental margin are replaced last. Cautery of the anterior dorsal nest caused deletion of the whole corresponding hemitergite, whereas cautery of the posterior dorsal nest caused the deletion of the macrochaetae of the posterior of the hemitergite. Cautery of the ventral nest deleted the hemisternite and the pleura, whereas cautery of the spiracular anlagen deleted the spiracle. Results of cautery also revealed that no macrochaetae formed on the tergite in the absence of adjacent microchaetae. Clonal analysis revealed that there were no clonal restrictions within a hemitergite at pupariation. Cautery of polytene epidermal cells other than those of the intersegmental margin failed to affect tergite development. However, cautery of polytene epidermal cells of the intersegmental margin adjacent to either dorsal histoblast nest caused mirror-image duplications of the anterior or posterior of the hemitergite in 10% of the hemitergites. Forty percent of the damaged presumptive hemitergites formed complete hemitergites, indicating extensive pattern regulation and regeneration. Pattern duplication and regeneration were accounted for in terms of intercalation and a model of epimorphic pattern regulation (French et al., 1976). Histoblasts in adjacent segments normally develop independently, but if they are enabled to interact by deleting the polytene epidermal cells of the intersegmental margin, they undergo intercalation which results in duplication or regeneration. The possible role of the intersegmental margin cells of insects in development was analyzed.

Notes: Summary The early development ofPhallusia mammillata eggs, dechorionated with trypsin and treated with Concanavalin A, was studied. Vital staining with a very dilute solution of acridine orange (0.01 μg/ml) helped to visualize the mitochondrial ‘crescent’ by fluorescence. At high concentrations of Concanavalin A (20–200 μg/ml) fertilized eggs did not cleave, but went through early ooplasmic segregation movements (formation of the crescent) and multinuclear syncytia were formed. At lower concentrations of Concanavalin A (less than 10 μg/ml), cleavage occurred, but the blastomeres remained rounded, leading to a grapelike embryo. Eggs attached to Concanavalin A treated nylon surfaces either did not cleave or produced grapelike embryos. Attachment of the eggs did not affect ooplasmic segregation. Considering modern theories of membrane structure it was concluded that Concanavalin A prevented cleavage either by immobilizing surface structures connected with microfilaments or by indirectly modifying other membrane structures. These structures could not have been involved in ooplasmic segregation, but their mobility was necessary for further morphogenesis.

Notes: Summary The development of the rhabdomeric pattern in the compound eye ofDrosophila has been studied using combined transplantation and electron microscope techniques. In a first series of experiments eye imaginal discs of increasing age were implanted into larvae ready to pupate, thus losing variable amounts of the normal time for development. A sequence of differentiative abilities was found in the metamorphosed test pieces. As far as the photoreceptor cells are concerned, the most prominent steps of this sequence are: ability to form groups with other similar elements, anatomical polarization of microvilli, establishment of the rhabdomeric pattern and formation of an equator line. The stability of determination of the equator line was tested in a second experimental series. Fragment of different topographical origin within the mature eye anlage were brought to metamorphosis by implantation into larvae ready to pupate. It was found that an equator line differentiates only in those pieces which according to the published anlage maps contain the prospective equator region prior to metamorphosis. The mitotic abilities of implanted eye imaginal discs were investigated by means of “in vitro”3H-thymidine pulse-labelling and light microscope autoradiography of the differentiated test pieces. During the third larval stage the eye anlage is traversed by two consecutive mitotic waves, each one of them producing different categories of receptor cells. The first, anterior wave predominantly produces cells oriented toward the poles of the eye within the ommatidia, while the second, posterior wave gives rise to elements exclusively in an equatorial position. The dynamics of this proliferation are discussed in relation to the findings in the implantation experiments. Silver-grain counts support the possibility that at least two successive cell divisions occur in the eye anlage between labeling with tritiated thymidine and beginning of morphological differentiation. The relevance of this finding for the understanding of the concept of acquisition of competence is discussed.

Notes: Summary The derivatives of 110 mosaic genital discs of gynandromorphs have been analysed microscopically. It has been found that theanalia of both sexes are homologous and derive from a single primordium (see Fig. 1a). Whether male or female anal plates are formed depends on the genetic constitution of the cells. This is analogous to the development of male sex combs versus female transversal rows on the forelegs of gynandromorphs. In contrast, the data for thegenitalia (see Fig. 1 b) are best explained if it is assumed that there are two genital primordia in everyDrosophila embryo: a male primordium that will only develop into genitalia if populated by XY (or XO) nuclei, and a female primordium that will only do so if populated by XX nuclei. This model, as depicted in Figure 2, is compatible with all our gynandromorph data and also with observations onMusca andCalliphora where in fact two separate genital primordia are found.

Notes: Summary Five regions of the compound eye have been found to be preferential boundaries for clones of labelledMinute + cells, and to act restrictively on the growth of cell clones after a given developmental stage. One of these regions is topographically related to the line of pattern inversion existing at the level of the equator. The results of experiments showing independency of origin of restriction lines and line of pattern inversion are reported.

Notes: Summary sev LY3,the only existing allele at thesev locus (1–33,2±0,2), behaves as strongly hypomorph or even as amorph. Ommatidia in asev compound eye have only seven receptor cells, the position of the R7 pattern element being vacant. Various criteria showing that the missing cell is R7 have been verified. These include (i) anatomical characteristics ofsev ommatidia; (ii) behaviour of central R cells insev rdgB double mutants; (iii) medullary projection of central R cell axons; and (iv) mitotic pattern ofsev imaginal discs. The analysis of morphogeneticsev-sev + mosaics has shown thatsev is expressed autonomously by R7 cells, indicating that thesev phenotype is not due to asev genotype of ommatidial pattern elements other than R7. The study of third instarsev imaginal discs has not brought any direct evidence for death of clustered presumptive R7 cells; however, clonal analysis of the developingsev compound eye has given evidence of developmental parameters comparable to those ofsev +, therefore favouring the hypothesis that R7 cells die insev mutants. On the other hand,sev + seems to be required for the determination of the R7 cells, since thesev phenotype cannot be uncovered during the last mitoses of heterozygous mutant cells.

Notes: Summary These experiments examined whether inDrosophila immature imaginal disc tissue and tissues from embryonic stages can influence pattern regulation in a disc fragment in the same way as can mature imaginal discs. Immature imaginal discs, or the cells of whole embryos, were mixed with a test fragment (presumptive notum) from a mature wing disc. The immature tissues in each mixture were genetically marked and had been heavily irradiated (25 Kr gamma) prior to mixing to prevent growth and maturation during subsequent culture in vivo. Alteration of the regulative behavior of the test fragment (that is, regeneration of wing) thus provided an assay for the communication of positional information by the immature tissues. The results suggest that this capacity arises well before competence to metamorphose, as early as the 16th hour of embryonic development, whereas prior to 16 h, essentially no stimulation of regeneration occurred. It is suggested that the imaginal disc (or presumptive disc) cells of the embryo may have been responsible for this early stimulatory capacity.

Notes: Summary The ontogeny of allozyme patterns has been studied in embryos ofDrosophilamelanogaster, which are doubly heterozygous for alleles specifying the slow and fast forms of alcohol dehydrogenase (ADH) and α-glycerophosphate dehydrogenase (GPDH). The ontogeny of esterase-2 was studied in embryos and young larvae of the flour mothEphestia kühniella, which are heterozygous for two of the three existing esterase-2 alleles. In freshly laidDrosophila eggs only the maternal enzyme forms are present and during the first 15 hours of development the staining of these forms becomes progressively fainter. After 16 and 17 h, the paternal and hybrid bands of ADH and GPDH respectively become obvious. Before hatching, the intensity distribution in the three-banded pattern of reciprocal hybrids is asymmetric in favour of the persisting maternal enzyme form. InEphestia embryos, however, there is no persistence of the maternal esterases. In all reciprocal heterozygotes a three-banded pattern suddenly appears 96 h after egg deposition, indicating synchronous activation of both parental alleles. The relative intensity distribution in the hybrid patterns approaches that of the mature larvae stepwise and in an allele-specific manner. This result and the fact that the various heterozygous types exhibit unequal total activities suggest that the Esterase-2 alleles have different activities, which are fixed late in embryogenesis.

Notes: Summary The females produced in the crossesD. melanogaster×D. simulans andD. melanogaster×D. mauritiana are sterile and have reduced ovaries. Normal and fertile ovaries were produced when genetically marked pole cells ofD. melanogaster were transplanted into eggs which gave rise to the hybrid females. These results eliminate the possibility that the sterility of these hybrids is due to the somatic component, i.e. the follicular cells of the ovaries, or to other physiological causes. The results also suggest that the control of gonadal morphogenesis is dependent mainly on the germ line.

Notes: Summary The dormant shelled gemmulae of the fresh water spongeEphydatia fluviatilis contain uniform, totipotent statocytes (thésocytes), which can differentiate either into archaeocytes (mono- and binucleated) or into histoblasts. The histoblasts accumulate at the villus near the micropyle. The other cell types orientate in a three-dimensional pattern at the micropyle, according to a developing gradient. After the primary pinacoderm is formed, the sponge primordium is released through the open micropyle. The primordium develops into a new sponge, into which archaeocytes, histoblasts and scleroblasts migrate.

Notes: Summary Gynandromorphs with female XX-and male XO-areas result from the loss of an unstable ring-X-chromosome in the early cleavage mitoses of ring/rod-X-chromosome heterozygotes. The phenotypes of the recessive alleles on the rod-X-chromosome are expressed in the XO-areas. 377 larval gynandromorphs of the genotypeR(1)2, In(1)w vC /y w sn3Iz50e mal were examined and scored for the phenotypes of 13 paired and 10 unpaired structures (Table 2, Fig. 2). This was possible mainly by the cell-autonomous expression of aldehyde oxidase activity in soft tissues and by the comparison of the distribution of enzyme activity in wildtype and gynander larvae. The distances between pairs of structures were calculated in sturt-units (Tables 3 and 4). A morphogenetic fate map with the presumptive areas of larval structures was constructed (Fig. 3). The relative positions of the structures agree well with Poulson's fate map (Fig. 4). In addition, the distribution of phenotypes was scored in 380 adult gynandromorphs Table (5). The fate map (Fig. 5) which was constructed from these data is very similar to the fate map of larval structures. This similarity becomes even more pronounced if fate maps are constructed which contain only structures analogous in larva and imago (Table 6, Fig. 6). Therefore an attempt was made to set up an integrated morphogenetic fate map containing the presumptive areas of both larval and imaginal structures (Fig. 7). The possibilities of further blastoderm mapping are discussed.

Notes: Summary Cell division during embryonic development of the brine shrimp,Artemia salina has been studied by counting nuclei and mitotic figures. No cell division was observed during development of the encysted gastrula until about an hour before emergence of the embryo (a pre-nauplius) from the cyst, and even then only a few mitotic figures were observed. Following emergence, and during further development up to the stage II nauplius larva an increase of about 25% in the number of cells occurs. However, when the newly hatched larva is exposed to FUdR (10 μg/ml) cell division is largely inhibited, but observable development nevertheless proceeds normally. Evidently all processes involved with the development of the gastrula into a stage II nauplius larva can occur with far fewer cells than normally are present.

Notes: Summary Females homozygous for a newly isolated mutation induced by ethyl methane sulphonate,fs(1)K10, lay abnormally shaped eggs in which the dorsal appendages of the chorion are enlarged and fused ventrally. The eggs are usually not fertilized and development is never normal beyond the blastoderm stage. The mutant was mapped to the tip of the X-chromosome with a meiotic position of 1–0.5 and a cytological location between 2B17 and 3A3. Using germ line mosaics constructed by transplantation of pole cells, it was shown that the abnormal morphology and the sterility are obtained only when the germ line is homozygous for the mutant.

Notes: Summary TheDrosophila chorion is produced normally in isolated follicles in Robb's chemically defined culture medium. The complex architecture of the shell developed in vitro from follicles as young as early stage 10 is completely normal morphologically. In addition, the time required for in vitro development closely approximates that observed for in vivo development. Comparisons of insect culture media developed by Robb, Grace, Schneider, and Echalier show large variations in their ability to supportDrosophila chorion development.

Notes: Summary The bristle pattern of the second-leg basitarsus inDrosophila melanogaster was studied as a function of the number and size of the cells on this segment in well-fed and starved wild-type flies, in triploid flies, and in two mutants (dachs andfour-jointed) that have abnormally short basitarsi. The second-leg basitarsi of well-fed, wild-type flies from 22 otherDrosophila species were studied in a similar manner. There are typically 8 longitudinal rows of evenly-spaced bristles on the second-leg basitarsus, and in each row the number of bristles was consistently found to vary in proportion to the estimated number of cells along the segment, and the interval between bristles was found to vary in proportion to the average cell diameter on the segment. These correlations are interpreted to mean that the spacing of the bristles within each row is controlled developmentally, whereas the number of bristles is not. The interval between bristles is evidently measured either as a fixed number of cells or as a distance which indirectly depends upon cell diameter.

Notes: Summary Salivary gland cells of members of theDrosophila melanogaster group (from four different subgroups) were examined electron microscopically and histochemically during the late larval period of development. The secretory product, which is supposed to be utilized as ‘glue’ at the time of puparium formation, appears, by analogy to Palade and Jamieson's results, to be synthesized partially in the rough endoplasmic reticulum (RER) and partially in the Golgi complex. The latter is also the usual site of the packaging of the product into secretory granules, except in the case of one of the secretory granule components ofD. lucipennis. The phylogenetic relationships among the subgroups, implied by the morphological appearance of the secretory granules, fit well with the existing phylogenetic relationships within the group. The secretory granules of each species have their own morphological features; granules of species of the same subgroup share some of these features. Secretion occurs from the cells via exocytosis during which the morphology of the secretory granules changes. Light microscope examination of PAS (Periodic Acid-Schiff reaction) stained glands shows a strong positive reaction in most species, with the exception of the species of thesuzukii subgroup which show a weak, or a negative reaction (D. rajasekari). Electron histochemical localization of polysaccharides in the secretory granules was possible inD. melanogaster and the species of theananassae subgroup.

Notes: Summary The generalogical relationships of photoreceptor cells within the compound eye ofDrosophila have been studied using cell labelling, with either3H-thymidine or recessive mutations, during the third larval stage. It has been found that photoreceptor and secondary pigment cells arise from different precursor cells. Under the present experimental conditions, precursors of receptor cells give rise to about 8 elements which differentiate as R cells of two different groups. One of the cells differentiates as R7 and the remaining as any one of the R1 to R6. The last cells behave initially as equivalent, and can differentiate within the same or within different, but neighbouring, ommatidia. The class of R1 to R6 cell in which each one of these elements differentiates, seems to depend on the time of its origin. The implications of these findings for the formation of the ommatidial pattern are discussed.

Notes: Summary We report on the size distribution of clones marked by mitotic recombination induced by several different doses of X-rays applied to 72 h oldDrosophila larvae. The results indicate that the radiation significantly reduces the number of cells which undergo normal proliferation in the imaginal wing disc. We estimate that 1000 r reduces by 40–60% the number of cells capable of making a normal contribution to the development of the adult wing. Part of this reduction is due to severe curtailment in the proliferative ability of cells which nevertheless remain capable of adult differentiation; this effect is possibly due to radiation-induced aneuploidy. Cytological evidence suggests that immediate cell death also occurs as a result of radiation doses as low as 100 r. The surviving cells are stimulated to undergo additional proliferation in response to the X-ray damage so that the result is the differentiation of a normal wing.

Notes: Summary Immature ovaries ofDrosophila mercatorum were injected into young larvae and into adult males ofD. mercatorum, D. melanogaster, D. hydei, D. virilis, andZaprionius vittiger. These homo- and heteroplastic transplantations allow normal vitellogenesis to occur in the donor ovary. By SDS gel electrophoresis, we identified the major species-specific yolk proteins of mature eggs (stage 14) which were exclusively of donor-specific origin. Other experiments withD. hydei andZ. vittiger showed that, when females were used as hosts, the host-specific yolk proteins became incorporated into the donor eggs. When two immature ovaries, one ofD. mercatorum and one ofD. hydei, were co-cultured in males, again only the donor-specific yolk proteins were found in the mature eggs implying that these yolk proteins were not released into the host hemolymph. A parthenogenetic strain ofD. mercatorum was used to demonstrate the ability of transplanted immature ovaries to produce viable eggs which can give rise to fertile adults. The role of the species-specific yolk proteins is discussed with respect to the dual origin of these proteins during normal vitellogenesis, i.e., an autonomous synthesis within the ovary itself in addition to the well-known production by the fat body. Further experiments with pupae as hosts indicate that even in the absence of juvenile hormone and in the presence of high doses of ecdysone, vitellogenesis can proceed within the donor ovary. Based on these experiments, a new hyopthesis on the hormonal control of vitellogenesis inDrosophila is presented. We propose that yolk proteins derived from the fat body are controlled by juvenile hormone, whereas the independent and autonomous vitellogenesis within the ovary itself is controlled by endogenously synthesized ecdysone.

Notes: Summary We estimate the number of blastoderm cells which generate the thoracic imaginal discs ofDrosophila. At hatching the wing disc is twice the size of the haltere disc, but the results suggest that both discs develop from a similar number of blastoderm cells. Two homeotic mutations, which transform the haltere into wing, affect embryonic growth but not the primordial number. All the segmental primordia may be of similar size and each may be similarly subdivided into a larger anterior, and a smaller posterior polyclone.

Notes: Summary The embryonic organization of the sexually dimorphic genital disc was studied in genetic mosaics resulting (a) from early loss of a chromosome or (b) from mitotic recombination. (a) Early Loss of a Chromosome. Three types of mosaics were produced — purely female mosaics, purely male mosaics, and gynandromorphs. They show that the genital disc arises from a group of cells in the ventral region of the embryo somewhat larger than that giving rise to a single foreleg (Table 2). Within this group of cells three regions can be distinguished that are present in both sexes: an anterior, a medial, and a posterior one, with distances of only 3–4 sturts between adjacent regions. The anterior region gives rise to the female genitalia, the medial region to the male genitalia, and the posterior region forms the analia of both sexes and the parovaria of the female (Figs. 2 and 3). The relative positions of the three regions were deduced from sturt distances (Tables 1 and 5), and from frequencies of mosaicism (Table 2). (b) Mitotic recombination was induced at the blastoderm stage in order to produce twin spots in the external genitalia and analia of purely male and female flies. Clone sizes indicate that these structures arise from a small number of precursor cells (Table 4). Clones overlapped right and left sides, but no clones were found extending over analia and genitalia. However, within either the analia or the genitalia of each sex, no clonal restrictions could be observed, and the clones comprised structures that were up to 12 sturts apart. A comparison of clone sizes and sturt distances in the foreleg and in the genital disc indicates that equal gynandromorph distances involve equal numbers of cells in different regions on the ellipsoid egg (Fig. 5). The results obtained from all mosaics provide a consistent picture of the embryonic organization of the genital disc. This becomes apparent in the summarized fate maps (Fig. 4), where the map derived from normal gynandromorphs can be produced by a simple superposition of the male and the female maps. The data are also discussed with respect to mechanisms of sexual differentiation in the genital disc.

Notes: Summary Mutations of the bithorax complex result in segmental transformations in the thorax and abdomen ofDrosophila. The haltere discs from larvae homozygous forbx 3 orpbx are transformed so that the discs contain cells that will produce wing cuticle as well as cells that produce haltere cuticle. The pattern regulation behavior of these discs has been examined. The fate maps of the two discs were established, and then the regulative behavior of a number of fragments from both types of mutant discs was established by culturing the fragments in vivo prior to metamorphosis. The most important conclusion from this work is that the cells producing, haltere cuticle and wing cuticle within the same disc share the same positional information and that they communicate during pattern regulation.

Notes: Summary Pairs of eye-antennal discs, attached to the cephalic ganglia, were cultured in vitro with a concentration of β-ecdysone optimal for imaginal differentiation. The eye-antennal discs fused to form a vesicle inside which the antennae were partially everted, and on the inner surface of which imaginal structures differentiated. The epithelium of the discs was continuous, and an integrated pattern of bristles and hairs differentiated in vitro. In particular, the median ocellus, a unified structure derived partially from each disc, differentiated normally.

Notes: Summary l(1)su(f)mad-ts (mad) is a new temperature-sensitive (ts) lethal mutant ofDrosophila melanogaster which produces duplicated legs after temperature pulse treatment during larval development. The ts-lethality was studied in temperature experiments and genetic mosaics. Temperature pulses given during two distinct TSPs of larval development result in two different types of leg pattern duplication. “Total” differ from “partial” duplications with respect to the affected leg compartments and the orientation of the planes of symmetry which are perpendicular to the dorso-ventral and the proximo-distal leg axes in total and partial duplications, respectively. Genetic mosaic studies indicate (i) disc autonomy of leg pattern duplication, (ii) clonal separation of the anlagen of the two pattern copies, and (iii) clonal restriction along the antero-posterior compartment border in the two pattern copies of totally duplicated legs. The results suggest thatmad leg pattern duplication is caused by a change in positional information rather than by cell death and subsequent regeneration. Our data are compatible with the assumption that during normal development the leg disc cells acquire information about their position within the disc with respect to the different leg axes independently and at different times.

Notes: Summary Two possible mechanisms are considered for the occurrence of experimentally or genetically induced duplications of bristles: extra cell division of a bristle mother cell versus determination of more than one mother cell. From a clonal analysis it appears that duplications induced by actinomycin-D arise by the latter mechanism, whereas those found in the mutantspl seem to arise by the former mechanism.

Notes: Abstract Naphthylamidase activity was studied in rat molar and incisor teeth at different stages of development. L-leucyl-4-methoxy-2-naphthylamide, L-alanyl-4-methoxy-2-naphthylamide, L-leucyl-2-naphthylamide and DL-alanyl-2-naphthylamide were used as substrates and Fast blue B and Fast Garnet GBC as diazonium salts. Naphthylamidase was not demonstrable in the teeth during enamel matrix formation. After the termination of this stage, naphthylamidase was present in the ameloblasts in their distal ends close to the enamel surface. The enzyme activity retained this localization until the dental epithelium fused with the oral epithelium at the time of tooth eruption into the oral cavity. Naphthylamidase was not found in other dental tissues, but marked activity was found in osteoclasts at the resorbing surfaces of alveolar bone surrounding the developing and erupting teeth and in certain areas of the connective tissue.

Notes: Abstract The effect of tetracycline HCl on synthesis of calcium sulphate statoliths inAurelia is reported. Tetracycline inhibits synthesis of statoliths and nematocysts when administered at an early stage of strobilation. The tetracycline, however, is not incorporated into the developing statoliths or nematocysts. As the tetracycline does not combine with the calcium of the calcium sulfate dihydrate statoliths ofAurelia, an explanation for its inhibitory effects on statoliths and nematocyst differentiation apparently does not rest with an incorporation-related factor. In vitro studies of four inorganic calcium systems and tetracycline revealed that calcium sulfate dihydrate (gypsum) did not incorporate tetracycline nor did its isostructural equivalent, calcium hydrogen phosphate dihydrate (brushite). Calcium carbonate and calcium phosphate (apatite) did incorporate tetracycline. The explanation for these different behaviors of calcium can be found in the crystal structure of the respective compounds, namely, whether or not the Ca ion is readily available to react with tetracycline.

Notes: Abstract A serial longitudinal study was undertaken to determine the activities of the alkaline and acid phosphatases in the long bones and mandibles. The optimum pH of the two enzymes was recorded at 10.2 and 5.4 for alkaline and acid phosphatase, respectively. Synchronized and randomized litters of rats were killed, 1 litter daily, starting at day 1 to day 25 post partum. Samples were analyzed for protein concentration and activity of the phosphatases. A pattern of low and high activity was observed in both bony tissues, as well as a pattern of low alkaline phosphatase activity during acid phosphatase activity peaks, and vice versa. The observed peaks suggest a correspondence between phosphatase activity and the other biochemical changes occurring in the growing bone, i.e., organic matrix and mineral formation. A separate study, considering the possibility of rhythmic features of the enzyme activity suggests that there may be a small diurnal rhythm at an early age.

Notes: Abstract In homogenates of resting rapeseeds no lipase activity (glycerolester hydrolase, EC 3.1.1.3) could be detected using a titrimetric assay procedure. Following a 30-h lag-phase after imbibition, lipase activity increased sharply, reaching its maximum at day 4 after sowing. Simultaneously triglyceride content of the cotyledons decreased sharply. At any time during the 11-day period of seedling growth examined, only an alkaline lipase activity with a pH optimum around 9 was present. White light had essentially no effect on the development of lipase activity. However, the disappearance of lipase activity from the cotyledons after fat utilization was found to depend on nitrogen nutrition of the seedlings. The activities of the glyoxysomal enzymes catalase and malate synthetase showed the usual rise and fall patterns with peak activities at day 4 after sowing, independently of the mineral nutrition of the seedlings. About 90% of the lipase activity was associated with a microsomal membrane fraction. Resolution of this fraction by sucrose density gradient centrifugation (62,000 g for 14 h) yielded three distinct membrane fractions. Maximum activities of membrane marker enzymes were recovered from the gradients at following densities: The major portion of microsomal protein and lipase activity at 1.085 kg/l; microsomal malate synthetase and phosphorylcholineglyceride transferase at 1.116 kg/l; NADH-cytochrome c reductase and phosphorylcholinecytidyl transferase at 1.133 kg/l. Evidently in rapeseed cotyledons lipase activity is associated only with a discrete microsomal membrane fraction which sediments differently from membrane fractions of the endoplasmic reticulum.

Notes: Abstract Some of the morphological and physiological parameters of stipe growth or elongation inCoprinus radiatus were investigated. During the development of the fruit body the number of cells in a row in the growing portion of the stipe doubled during the development of the button, and again during the phase of rapid stipe elongation. Also during the stage of rapid elongation the cells in the upper 2/3 of the stipe increased 6–8 fold in length. The existence of a growth regulator synthesized in the cap and exerting control over the stipe was demonstrated through decapitation experiments. The cap appears to be required for normal stipe development until the stipe reaches about 1/4 of its final length. Through decapitation and cap-stipe exchanges it was found that the cap produced growth regulator up to the time of autodigestion; however, the stipe responded to the regulator only during a brief period at the onset of elongation.

Notes: Abstract Observations of sporophore development in fresh and glutaraldehydeosmium sequentially-fixed material ofProtophysarum phloiogenum show the following sequence. Small plasmodia cease streaming and round up. Food vacuoles collect in the lower center of the cytoplasmic mass. As the cytoplasm rises the food vacuolar contents are excluded from the plasmalemma and become the stalk core. A continuous, fibrous peridium and stalk tube enclose cytoplasm and stalk core respectively. Capillitial formation just precedes spore cleavage. Sporophore development is marked by autophagic activity and calcium deposition. Stalks of dried herbarium specimens of seven additional species have been examined. A mature stalk morphology very similar toProtophysarum with recognizable remnants of microorganismal food material is seen in all of them. It is thought that this marker is indicative of non-stemonitaceous stalk development.

Notes: Abstract In cultures ofacanthamoeba castellanii the stationary growth phase in which trophozoites develop to cysts, was induced in three ways: by transferring cells from a logarithmic growing culture into a nutrient-free medium, by growth in nutrient medium to high cell density and by inhibition of the mitochondrial DNA synthesis with ethidium bromide. In all cases, the intracellular concentration of adenosine-3′,5′-monophosphate (cAMP) rises by a factor of two to three from the end of the logarithmic phase to the beginning of the stationary phase. The results show that this rise may be more a consequence of an increased adenylate cyclase activity than of a diminished cAMP phosphodiesterase activity. No extracellular cAMP could be measured in cultures of the logarithmic and stationary growth phase, perhaps because of the extracellular cAMP phosphodiesterase. Because theophylline also induces the stationary phase and encystation, cAMP seems to be of importance for the development ofAcanthamoeba castellanii, especially for the degradation of glycogen.

Notes: Abstract The organism shows long-conical, colourless cells of bacterial size, affixed with the broader basis and cutting off buds from the free apex in numbers as high as 30, which form a chain and later separate. Multiplication is entirely by budding. Some resemblance to Cyanophycean cells, in particular to the exosporangia of Chamaesiphon, seems to be cytologically not verified. The organism grows exclusively on living cells of the diatom Cymbella cesati, and is fixed to them principally on the edges of the valvae. In spite of the presence of many other similar diatom species, the organism represents a remarkable case of extremely specialized epiphytism. There are no signs of damage of the “host” cell, in cell division the epiphyte is taken over by the epithecae of the daughter cells. Systematically the species because of practical reasons can be included into the Caulobacterales sensu Henrici and Johnson (1935). A second species, obviously belonging in here, is represented by Chamaesiphon hyalinus Scherffel (1907).

Notes: Abstract The present paper describes intracellular changes in ribonuclease specific activity during Ca2+-induced sporangium formation in the water mold Achlya bisexualis. The enzymes undergo a decrease in activity prior to crosswall formation followed by an increase in activity during spore cleavage. As spore discharge occurs the RNase activity again decreases. A large percentage of the nuclease activity is associated with a lysosomal-like fraction of the cell, but there is also considerably activity associated with nuclear and microsomal fractions. Addition of cycloheximide or actinomycin D at various times during development prevents further decrease or increase in the enzyme activity. Mixing of cell extracts from different developmental stages provides evidence that inhibitors or activators of the enzyme activity are not responsible for the activity levels evident at the different stages. There is a change in the total levels of presumptive mRNA during Ca2+-induced sporangial formation which appears to be associated with the patterns of RNase activity. Utilizing total cellular RNA and Poly(A)+ RNA with the crude ribonuclease preparations, no substrate specificity could be ascertained.

Notes: Abstract Mutagenesis with nitrosoguanidine yielded two classes of ring trap mutants in the predacious HyphomyceteDactylella brochopaga: strains which could make no traps and those with a proportion of giant, functional traps. A third strain, derived from a trapless strain made abnormally small functional traps. The giant traps are described, together with developmental abnormalities they sometimes display. The characteristics of the chief mutant strains are discussed.

Notes: Abstract The concentration of adenosine tri-, adenosine di-, and adenosine monophosphate in cells of Acanthamoeba castellanii was measured during the logarithmic growth phase and the stationary growth phase in which trophozoites were transformed into cysts. This developmental process was induced in three ways: by growth in nutrient medium to high cell density, by transferring cells in the logarithmic phase into a nutrient-free medium, and by mixing logarithmically growing cells with ethidium bromide. In all cases, encystment is accompanied with a reduction of total adenosine phosphate content to about 85%, mainly because of a depletion of cellular ATP. The value of the adenosine phosphate energy charge in logarithmically growing amoebae is 0.83. During development the energy charge becomes stabilized at different values (between 0.58 and 0.81), characteristic to the mode of encystation. A possible functional relationship between changes of the adenosine phosphate concentration and developmental processes of the amoeba is discussed.

Notes: Abstract The development of peritrichous flagella and, consequently, swarming of Vibrio alginolyticus depend on a complex relationship between temperature, salt concentrations and pH. At temperatures above 28°C V. alginolyticus did not develop peritrichous flagella unless certain minimal concentrations of NaCl are present: the higher the temperature, the higher the NaCl concentrations required for peritrichous flagella synthesis. This requirement for NaCl at high temperatures is much more pronounced at pH 9 than at pH 6. High temperatures and low concentrations of NaCl also inhibited swarming of cells already armed with peritrichous flagella. Other cations, such as Li+, K+ and Mg2+, replaced NaCl only at temperatures below 28°C.

Notes: Abstract It could be shown that extracts of growing cultures of Acanthamoeba castellanii contained a cellulose degrading system. Reducing sugars are split off by one component of this system at an optimum of pH 4, another enzyme changes the viscosity at an optimum of pH 6, and a third component is a β-glucosidase with an optimum at pH 3.5. At pH 4 the cellulose degradation products are cellobiose and glucose; at pH 6 higher molecular weight oligosaccharides are produced. During the development from trophozoites to cysts in a nutrient-free medium, the activities of both cellulases decline: Prior to the start of cellulose synthesis only 30%, and in cysts only 10% of the original existing activities are detectable. The biological function of the cellulase enzyme system is discussed together with a consideration of the fact that excystment takes place without digestion of the cyst wall in which the cellulose is deposited.

Notes: Abstract The cultivation of Physarum polycephalum amoebae in two media with different protein contents revealed a regulation of aminopeptidases and proteases depending on the albumin content of the medium: in growing amoebae and plasmodia the aminopeptidases have similar isoenzyme patterns and relative activities against nitroanilides. One alanine and four leucine aminopeptidase isoenzymes were found within the slightly acid pH range. During growth amoebae secrete—different from plasmodia—leucine aminopeptidase into the medium with low protein content. In an albumin-rich medium additional alanine aminopeptidase activity was found. Out of nine plasmodial proteases four were found in amoebae too. Only one band (pI 3.6) was present in the protein-poor medium. No protease activity could be detected in the proteinrich medium.

Notes: Abstract Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3′-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.

Notes: Abstract Individuals of an alcohol dehydrogenase-negative strain of Drosophila melanogaster designated Adh n5 are resistant to ethanol poisoning at low but not at high temperatures. The basis for this behavior is that Adh n5 flies contain a mutant form of alcohol dehydrogenase which is less heat stable than that of wild-type flies. The mutation in Adh n5 maps at, or very close to, the presumptive structural locus and is not complemented by any of 11 other alcohol dehydrogenase-null mutants.

Notes: Abstract 3-Hydroxykynurenine is virtually absent from st larvae but accumulates during adult development in the puparium. Over the period of adult emergence, the accumulated 3-hydroxykynurenine is excreted so that st adults contain none. Larvae of st fed on tryptophan-C 14 medium produce labeled 3-hydroxykynurenine, at a reduced rate, perhaps, compared to wild type. Xanthurenic acid levels in st pupae are similar to those in wild type. Thus the failure of st larvae to accumulate 3-hydroxykynurenine does not seem to be due either to an inability to synthesize this compound or to an excessive rate of its conversion to xanthurenic acid. Rather, it appears that the mechanism of 3-hydroxykynurenine storage during larval life is defective, so that this compound is excreted at an abnormally high rate. The inability of the pigment cells of the eyes of st to synthesize xanthommatin may result from a similar defect in their ability to take up or store 3-hydroxykynurenine.

Notes: Abstract A marked decrease in the amount of the A2 component of phenol oxidase occurs in the speck locus of Drosophila melanogaster. The amount of A2 in speck is restored to a normal amount in the presence of the suppressor mutant, su(s) 2.

Notes: Abstract Particulate fractions from the heads of Drosophila melanogaster catalyze the conversion of o-aminophenols to phenoxazinones. This particulate enzyme is stimulated by Mn2+. It has a number of features which distinguish it clearly from the Mn2+-dependent activity found in the soluble fraction. The particulate enzyme has a characteristic developmental pattern, showing a marked increase in activity at about the time of onset of xanthommatin synthesis. In addition, it is much reduced in activity in a number of xanthommatin-deficient mutants (v, cn, st, cd, and w). We believe that the head particulate enzyme is involved in xanthommatin biosynthesis and that the developmental onset of synthesis of this pigment is brought about by the synthesis or activation of this enzyme.

Notes: Abstract Allozyme polymorphisms at seven loci have been studied in nine natural populations of Drosophila melanogaster from the Saône and Rhône valleys sampled in 1973 and 1974. A great deal of polymorphism was observed; an individual was on the average heterozygous at 20.2% of its loci. The populations were genetically very homogeneous throughout the region sampled. The number of ovariolae per female varied from one group of populations to another depending on their geographical separation. Yet the number of ovariolae remained constant from one year to the next. The results show that migration alone cannot explain the homogeneity of the allozyme frequencies. It seems reasonable to conclude that selection plays a major role in maintaining the homogeneity of populations living in proximal biotopes.

Notes: Abstract The activities of the enzymes aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) were determined in adult females from a wild-type strain and from eight different alleles of the X-linked mutation rudimentary (r) of Drosophila melanogaster. The alleles chosen span the genetic map of the r locus. The characteristics of the DHOase-catalyzed reaction which converts carbamyl aspartate to dihydroorotate are briefly described. Of all of the r strains tested, only one, r 9, has wild-type levels of aspartate transcarbamylase and dihydroorotase activities. The other seven show either intermediate or very low levels of activity for both enzymes. The lowered ATCase and DHOase activities observed in mutants which do not map in the region of the structural gene for these enzymes are interpreted in light of recent evidence that ATCase and DHOase are part of a three-enzyme complex.

Notes: Abstract Three of the major protein species present in the hemolymph of Drosophila melanogaster larvae just prior to pupation are absent from second instar larvae but accumulate rapidly during the third instar. This article describes the purification and characterization of one of these, larval serum protein (LSP) 2, using an immunological assay. It is a homohexamer of molecular weight about 450,000, with a polypeptide molecular weight of 78,000–83,000. Fast and slow electrophoretic variants of this protein map between the markers vin and gs, at 36–37 on chromosome 3.

Notes: Abstract Only one molecular form of trehalase (E.C. 3.2.1.28) was detectable in adult Drosophila melanogaster by polyacrylamide gel electrophoresis and isoelectric focusing. An examination of duplication- and deletion-bearing aneuploids exhibiting do sage sensitivity indicated that the enzyme is encoded by a gene, Treh +, located between 55B and 55E of the second chromosome. The tissue-specific soluble and particulate forms of trehalase appear to be manifestations of a single protein encoded by a single gene.

Notes: Abstract Sepiapterin synthase, the enzyme system responsible for the synthesis of sepiapterin from dihydroneopterin triphosphate, has been partially purified from extracts of the heads of young adult fruit flies (Drosophila melanogaster). The sepiapterin synthase system consists of two components, termed “enzyme A” (MW 82,000) and “enzyme B” (MW 36,000). Some of the properties of the enzyme system are as follows: NADPH and a divalent cation, supplied most effectively as MgCl2, are required for activity; optimal activity occurs at pH 7.4 and 30 C; the K m for dihydroneopterin triphosphate is 10 µm; and a number of unconjugated pterins, including biopterin and sepiapterin, are inhibitory. Dihydroneopterin cannot be used as substrate in place of dihydroneopterin triphosphate. Evidence is presented in support of a proposed reaction mechanism for the enzymatic conversion of dihydroneopterin triphosphate to sepiapterin in which enzyme A catalyzes the production of a labile intermediate by nonhydrolytic elimination of the phosphates of dihydroneopterin triphosphate, and enzyme B catalyzes the conversion of this intermediate, in the presence of NADPH, to sepiapterin. An analysis of the activity of sepiapterin synthase during development in Drosophila revealed the presence of a small amount of activity in eggs and young larvae and a much larger amount in late pupae and young adults. Sepiapterin synthase activity during development corresponds with the appearance of sepiapterin in the flies. Of a variety of eye color mutants of Drosophila melanogaster tested for sepiapterin synthase activity, only purple (pr) flies contained activity that was significantly lower than that found in the wild-type flies (22% of the wild-type activity). Further studies indicated that the amount of enzyme A activity is low in purple flies, whereas the amount of enzyme B activity is equal to that present in wild-type flies.

Notes: Abstract Four glycolytic enzymes in Drosophila melanogaster have been genetically and/or cytogenetically mapped. The structural gene for aldolase (Ald) has been genetically mapped to 3-91.5 and cytogenetically localized to 97A-B. Tpi, the structural gene for triosephosphate isomerase, has been genetically mapped to 3-101.3 and cytogenetically localized to 99B-E. Utilizing closer-flanking markers than the previous mapping, Pgk, the structural gene for 3-phosphoglycerate kinase, has been mapped to 2-5.9; cytogenetically it was found to lie in the interval between 22D and 23E3. The cytogenetic location of Pgm, the structural gene for phosphoglucomutase which has been located genetically at 3-43.4, was determined to be in 72D1-5.

Notes: Abstract A biochemical comparison was made between α-glycerophosphate dehydrogenase allozymes from Drosophila melanogaster. Enzymes extracted from the three major genotypes were indistinguishable in terms of their pH optima and thermal stabilities. Distinctive differences were observed for three parameters; temperature dependence of specific activity, temperature dependence of Km, and reaction rate constancy over a physiological temperature range. These results are discussed in terms of a model of balancing selection and the existence of spatial and temporal allele frequency clines in natural populations.

Notes: Abstract Experiments utilizing standard techniques of cell fractionation and disc electrophoresis have revealed the presence of three distinctly different enzymes which catalyze the oxidation of d-sorbitol in crude extracts of Drosophila melanogaster adults. These include (1) a soluble NAD-dependent sorbitol dehydrogenase (NAD-SoDHs), (2) a mitochondrial NAD-dependent sorbitol dehydrogenase (NAD-SoDHm), and (3) a soluble NADP-dependent sorbitol dehydrogenase (NADP-SoDH). The structural gene for NAD-SoDHs has been mapped to a locus between 65.3 and 65.6 on the third chromosome by means of an electrophoretic variant and a low-activity allele. Through the use of segmental aneuploidy, this gene has been localized to the region limited by salivary bands 91B–93F. Because mutants which alter either the activity or electrophoretic mobility of the soluble NAD-dependent enzyme have no significant measurable effect on the mitochondrial or NADP-dependent forms, it is suggested that the enzymes in this system are coded for autonomously by different genes.

Notes: Abstract The activity levels of alcohol dehydrogenase and α-glycerophosphate dehydrogenase were compared among nine species of Drosophila representing three phylogenetic groups. For any given life stage, interspecific variability in activity level was much greater for ADH than for α-GPDH. Patterns of ontogenetic expression of enzyme activity were also much more variable among species for ADH than for α-GPDH. These results are consistent with the interpretation that α-GPDH is involved with a relatively uniform adaptive function among species, whereas ADH levels may reflect variable adaptive capabilities. There is a significant correlation between ADH activities and survivorship on alcohol-treated media for these nine species.

Notes: Abstract Our earlier evidence concerning the complexity of the activation process for Drosophila phenol oxidase has been confirmed by separation and purification of six proteins involved. This is a minimal number required in a reaction series or cascade to yield active enzyme, and at least two more proteins are known to be involved. A simpler system involving only the last step with one precursor and one activation as has been reported in the literature is consistent with the cascade picture, but the whole complex system must be considered when dealing with genetic and developmental regulation of pigmentation and sclerotization. Details are given for partial or complete purification of six of the proteins involved and evidence for their modes of interaction is presented.

Notes: Abstract An improved thin-layer chromatography technique is described for the separation of fluorescent compounds found in extracts of heads of Drosophila melanogaster. Eighteen to twenty fluorescent spots are resolved, two of which are xanthurenic acid and 3-hydroxykynurenine, and the remaining spots are presumably pteridines. Of these, nine have been identified and quantitated directly on the chromatograms with a fluorometer. One of the spots present on the chromatogram apparently has not been described previous to this work. Characteristics of this substance, termed “quench spot,” are presented, several of which indicate that it may be a pteridine or pteridine derivative.

Notes: Abstract Glutamine-dependent CPSase, ATCase, and DHOase from Drosophila, the first three enzymes in pyrimidine biosynthesis, show coordinate variation in activity throughout development. The three activities were highest in first instar larvae and decreased as development proceeded. The three activities cosediment in sucrose gradients as a single peak with a relative sedimentation coefficient of approximately 30S. CPSase, ATCase, and DHOase copurify during (NH4) 2SO4 fractionation and during DEAE-cellulose and hydroxylapatite chromatography.

Notes: Abstract It has been shown that crude extracts of Drosophila melanogaster adults contain three distinctly different enzymes which catalyze the oxidation of d-sorbitol into d-fructose. These include (1) a soluble NAD-dependent sorbitol dehydrogenase (NAD-SoDHs), (2) a mitochondrial NAD-dependent sorbitol dehydrogenase (NAD-SoDHm), and (3) a soluble NADP-dependent sorbitol dehydrogenase (NADP-SoDH). Developmental studies have shown that the activities of all three of these enzymes are lowest during the larval stages while highest levels are seen during or shortly prior to the adult period. With respect to NAD-SoDHs, studies of tissue distribution in adults have shown that highest activity is associated with thoracic musculature in both sexes and with organs of the male reproductive system. The developmental profile of this enzyme reveals a significant increase in activity at between 40 and 60 hr after hatching. This time interval corresponds closely to that during which the paternally derived NAD-SoDHs gene is expressed. An additional increase in activity is seen in male pupae at 160 hr and in female adults at 210 hr. The rapid increase in males takes place immediately following the developmental period during which the testes attach to their respective duct systems. NADP-SoDH activity is concentrated among organs of the thorax and abdomen in both sexes. Males show significantly higher levels of this enzyme during the late pupal and early adult periods. In contrast to the patterns of distribution seen for NAD-SoDHs and NADP-SoDH, 91–92% of the total NAD-SoDHm activity in adults is localized to the thoracic musculature. The developmental profile of this enzyme reveals a significant increase in activity during the late pupal and early adult periods, when flight muscle mitochondria are known to be proliferating and undergoing structural maturation.

Notes: Abstract Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55–65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.

Notes: Abstract NADP-malic enzyme (NADP-ME) (E.C. 1.1.1.40) is situated in the cytosol of Drosophila melanogaster. Both the tissue activity and CRM level of NADP-ME parallel changes in the dosage of a gene, Men +, located in region 87C2-3 to 87D1-2 of the third chromosome. The tissue activity of NADP-ME is very high in early third instar larvae, providing about 33% of the NADPH at this life stage. The tissue activity declines during pupal development but increases as the adult ages. The concentration of NADP-ME CRM and tissue activity are coordinately increased in third instar larvae by dietary carbohydrate and decreased by dietary lipid.

Notes: Abstract Isoelectrofocusing of abdominal extracts of Drosophila melanogaster revealed the existence of two forms of sucrase (E.C. 3.2.1.26). One form exhibited an isoelectric point of 4.63±0.02 while the other form exhibited an isoelectric point of 4.83±0.02. The localization of the structural gene for sucrase is proposed on the basis of enzyme determinations in a series of duplication- and deletion-bearing aneuploids. We suggest that the sucrase structural gene lies between 31CD and 31EF on the left arm of chromosome 2 and that the two forms of abdominal sucrase derive from a common protein coded for by a single sucrase gene designated Sucr +.

Notes: Abstract Genetic and cytogenetic locations of the structural genes for the NAD-dependent malate dehydrogenases have been studied. The mitochondrial form (mMDH) is coded for by a gene (Mdh) found at 62.6 on the third chromosome and included in Df(3R)P14, which includes 90C2–91A3 in the salivary gland chromosomes. Based on its inclusion within several J (Jammed; 2–41.0) deficiencies, the structural gene (cMdh) for the cytoplasmic form (cMDH) was determined to lie in region 31B-E, confirming the earlier finding of Grell. Flies lacking any cMDH activity (cMdhn-γ10069/ Df(2L)J-der-27) were both viable and fertile.

Notes: Abstract When adult Drosophila are placed on medium containing 0.5% acetone, their level of alcohol dehydrogenase activity drops rapidly. At the same time, the proportion of activity in the various electrophoretic forms of the enzyme shifts; most of the activity becomes localized in what is ordinarily a minor form of the enzyme. Moreover, the loss of enzyme activity occurs in vivo as well, as shown by sensitivity to ethanol poisoning, insensitivity to pentenol treatment, and inability to utilize ethanol as an energy source. These observations are discussed in light of a model advanced for the origin of the multiple forms of alcohol dehydrogenase in Drosophila.

Notes: Abstract Several points of biochemical similarity between white and scarlet mutants suggest that both are defective in the transport of xanthommatin precursors. In both, accumulation of 3-hydroxykynurenine is negligible during larval life and occurs at only a slow rate during adult development. Larvae of both mutants also excrete 3H-3-hydroxykynurenine and 3H-kynurenine rapidly, which probably accounts for the normal levels of kynurenine during larval life. 3-Hydroxykynurenine levels are abnormal in all white mutants which were studied, although in two alleles which are strongly pigmented (w sat and w col) accumulation is enhanced rather than diminished. In w a, larval accumulation is normal but accumulation during adult development is greatly diminished, suggesting that this mutation has a tissue-specific effect. Similar levels were found in zeste females. Of the 11 other eye color mutants tested, abnormal levels of 3-hydroxykynurenine were found in eight. In four of these (claret, light, lightoid, and pink), larval accumulation is negligible, suggesting that these have defects in the kynurenine transport system like scarlet and white. In three others, however (brown, karmoisin, and rosy), accumulation during larval life is enhanced. In cardinal accumulation is normal during larval life but is excessive during adult development. This evidence supports the suggestion that the cd mutation blocks the final step of xanthommatin synthesis.

Notes: Abstract The suppressible eye color mutant purple (pr) of Drosophila melanogaster is known to be unable to synthesize a wild-type complement of pteridine eye pigments. This study measures the reduced levels of drosopterins, sepiapterin, and an unidentified presumed pteridine in pr and pr bw. Pteridine analyses in double mutants combining pr with one of three other eye color mutants sepia, Henna-recessive3, and prune2, suggest that the metabolic block in pr occurs prior to sepiapterin biosynthesis. Measurements of GTP and GTP cyclohydrolase in pr showed wild-type levels and indicate the metabolic block in pr to be at one of the steps converting dihydroneopterin triphosphate to sepiapterin. Quantitation of pteridines in suppressed purple [su(s) 2; pr and pr; su(pr) e3] shows restoration of pteridines to wild-type or nearly wild-type levels.

Notes: Abstract It is shown that the gene controlling the synthesis of the organ-specific S-esterase of Drosophila virilis ejaculatory bulbs is located on the second chromosome (at approximate position 192.1±map units). The cells of the genital imaginal disks are determined for the synthesis of S-esterase 10–12 hr after the second molt. The organ-specific esterase can be detected after adult emergence only. It is preceded by an increase in RNA content and by enhancement of RNA synthesis in the cells of the ejaculatory bulbs. Interstock differences were found in the level of the activity of S-esterase, which is under the control of the X chromosome, as well as in the time of expression of enzyme activity, which is controlled by the fifth chromosome. It is suggested that the specific phenotypic expression of this enzyme depends on the system of genes with regulatory expression at both the transcriptional and posttranscriptional levels. The genetic control of the synthesis of the S-esterase described is a convenient model for studying mechanisms of gene activity regulation in eukaryotes.

Notes: Abstract Starch and polyacrylamide gel electrophoresis were used to ascertain the substrate specificities of alcohol-oxidizing enzymes in 13 Drosophila species. The substrates used were a variety of long- and short-chain aliphatic alcohols, one aromatic alcohol, and benzaldehyde. Only one enzyme (product of a single-gene locus) showed significant NAD+-dependent alcohol dehydrogenase activity with short-chain aliphatic alcohols. The 13 species, belonging to four different Drosophila groups, all showed a similar complement of alcohol-oxidizing enzymes, although differences in electrophoretic mobility and in levels of activity existed from species to species. These findings are relevant to the adaptation of Drosophila to alcohol environments.

Notes: Abstract Several in vitro properties of partially purified form II RNA polymerase from Drosophila melanogaster embryo nuclei are described. The enzyme preparation is free from contaminating RNase, protein kinase, and polyphosphate kinase activities and can be used to study the incorporation of γ-32P-labeled nucleoside triphosphates. The enzyme exhibits a biphasic heat inactivation pattern which is probably related to differential lability of its two subforms. However, a considerable protection against heat inactivation is provided by the nucleoside triphosphates present in the in vitro reaction system such that the enzyme catalyzes RNA synthesis in a nearly linear mode for over 2 hr at 30 C. Two initiation inhibitors, rifamycin AF/013 and polyriboinosinic acid (poly[I]), were tested against this enzyme. Rifamycin AF/013 was found unsuitable for critical studies because of the high concentrations necessary for total inhibition (200 µg/ml) and particularly because of the obligate use of solvents which secondarily have a destabilizing effect on native DNA. Poly[I] was found to effectively block initiation at very low concentrations (1 µg/ml). The enzyme rapidly forms poly[I]-resistant preinitiation complexes on both double- and single-stranded DNA. These complexes decay with a half-life of 2.5–3 min. RNA synthesis from poly[I]-resistant complexes amounts to 10% of the total potential synthesis on both double- and single-stranded DNA. Enzyme-DNA saturation experiments indicate that the form II enzyme discriminates two types of sites on Drosophila DNA, tight binding and weak binding, from which RNA synthesis proceeds slowly and rapidly, respectively. The tight-binding sites appear to be analogous to those sites with which the enzyme is able to form poly[I]-resistant complexes.

Notes: Abstract Allozyme frequency data from natural populations of Drosophila buzzatii were analyzed for genotype-environment relationships. Allele frequency and heterozygosity at six loci polymorphic throughout eastern Australia and a number of environmental factors (both means and variabilities) were examined by a variety of multivariate techniques. Significant genotype-environment associations were found for five of the six loci, and after correcting for geographic location significant associations remained for Est-2 and Adh-1 gene frequencies and heterozygosities and for Pgm gene frequencies. The results are discussed in relation to selection and gene flow and provide the basis for laboratory studies to disentangle confounded effects of (1) environmental means and environmental variabilities and (2) allele frequency and heterozygosity, and thus to further test for and determine the nature of any natural selection at particular allozyme loci.

Notes: Abstract A locus has been found, an allele of which causes a modification of some allozymes of the enzyme esterase 6 in Drosophila melanogaster. There are two alleles of this locus, one of which is dominant to the other and results in increased electrophoretic mobility of affected allozymes. The locus responsible has been mapped to 3-56.7 on the standard genetic map (Est-6 is at 3-36.8). Of 13 other enzyme systems analyzed, only leucine aminopeptidase is affected by the modifier locus. Neuraminidase incubations of homogenates altered the electrophoretic mobility of esterase 6 allozymes, but the mobility differences found are not large enough to conclude that esterase 6 is sialylated.

Notes: Summary The mutagenic efficiency of ionizing radiations has been tested on different lines of Drosophila melanogaster. It has been shown that differential lethal effects are obtained when irradiated females from different lines are mated to flies carrying heterozygous lethal genes. The results seem not to be attributable to differential expression of the lethality in the various crosses performed with the irradiated flies. This might suggest that gene activity is involved in the expression of the mutagenic effects of radiations.

Notes: Summary Sex-dependent differences in the state of the nuclear chromatin of somatic cells were found in Daphnia pulex. It is suggested that the genome of Daphnia pulex has two developmental programmes based on identical chromosome sets. The female programme consistently functions under a wide range of ecological conditions, whereas the male programme is turned on by specific ecological stimuli. The genes controlling the activation and function of the male programme may be phenotypically latent for many parthenogenetic generations.

Notes: Summary As a test of the hypothesis that adult temperature stress is an important component of natural selection maintaining the α-gpdh polymorphism, we have looked for differential survival among genotypes subjected to (i) heat shock and (ii) cold shock. Factorial ANOVAR, taking account of genotype, sex and temperature-stress indicated that genotype did not contribute to the variance of survival proportion per vial. We have not therefore found evidence to support our hypothesis. Incidental to the above was a significant sex-temperature interaction. Thus, adult females showed higher survival than males under heat stress, while under cold stress, there was no indication of a survival difference between the sexes.

Notes: Summary The epicuticle of adult Tenebrio consists of two distinct layers named outer and inner epicuticle. The outer epicuticle is the first cuticular layer to be deposited in form of small patches on top of the microvilli. These initial patches are composed of four dense laminae (A, B, C1 and C2) separated by three light spaces. The outer epicuticle grows by densification of diffuse material at the edges of the patches until the entire area is covered. The thickness of outer epicuticle remains constant (175 Å) during the development of the pharate adult, lamina B however rapidly disappears. Thus, the adult outer epicuticle is fivelayered (three dense laminae: A, C1 and C2). After being deposited, the inner epicuticle shows a complex laminar structure interpreted to represent a highly organized molecular system. The laminae are masked during the formation of the first procuticle lamellae. During the deposition of the epicuticle, lamina A is covered by a component of the moulting fluid, forming an irregular dense layer which disappears after the resorption of this fluid. Perhaps this layer protects the new epicuticle from lytic enzymes of the moulting fluid. In adult animals, there is an additional superficial layer, the signification of which is not clear. The possibility of remains of cement or wax is discussed. The development of the surface patterns of the sternal and pleural cuticle is determined before the epicuticle formation by the shape of the epidermal surface. The rate of outer epicuticle deposition appears to depend on the size of the microvilli: epicuticle deposition seems to proceed faster over high microvilli.

Notes: Summary The development of the seminal vesicle from the Wolffian duct and of the prostate from the urogenital sinus has been studied in rat fetuses from day 14 of gestation to birth with the use of the electron microscope. Prior to the onset of androgen secretion, the cells of the urogenital sinus and the caudal part of the Wolffian duct have a simple undifferentiated appearance. After the onset of androgen secretion by the fetal testes at day 15, “intracytoplasmic confronting cisternae” of the granular reticulum appear in both urogenital sinus and Wolffian duct. Portions of the granular endoplasmic reticulum of the urogenital sinus become distended with a finely granular, moderately dense material. In the urogenital sinus, many hemidesmosomes are formed at the basal surface of the epithelium. Specializations of the extracellular materials are present opposite the hemidesmosomes. The formation of the seminal vesicles and the prostate begins at day 18–19 of gestation. The cells of the seminal vesicle are taller than the Wolffian duct cells from which they arise, the granular endoplasmic reticulum increases moderately in amount, and a patent lumen is formed. The cells of the fetal prostate do not differ greatly from those of the urogenital sinus from which they arise except that the prostatic cells initially lack hemidesmosomes. The fine structural changes are discussed in relation to the onset of fetal androgen secretion, the formation of the organs, and the functions of the cells in adult life.

Notes: Summary The differentiation of the podocytes was studied by scanning electron microscopy on kidneys of 10 days old rats. The results were compared with transmission electron microscopic pictures from the same kidneys. There are three ways of forming processes by the podocytes: 1. Slits within the cytoplasm give rise to cytoplasmic bridges which further divide themselves and finally build up a meshwork of processes within a cell. 2. Thick and sometimes very long processes originate from the cell border. Their smaller branches may interdigitate with those of distant podocytes or with other processes out of the same cell. 3. Finger-like processes of neighbouring cells interdigitate as soon as they develop. In spite of numerous desmosomal structures between neighbouring podocytes the cell processes and their interdigitations develop mostly independently from each other and only seldom after the expected rules of epithelial vicinity (type 3). These findings are interpreted as indication that flooded and growing capillaries induce the differentiation of podocytes and that they influence the orientation of their processes.

Notes: Summary The basic structure of the terminal sensilla of Locusta migratoria resembles that of Schistocerca gregaria. There are commonly six or ten neurons whose dendrites extend almost to the opening of the peg. Proximally the dendrites are clothed by a neurilemma cell which also encloses a basal cavity through which their ciliary region passes. The tormogen cell encloses the receptor-lymph cavity and actively secretes material into it. The receptor-lymph cavity and the basal cavity are quite separate. The development of new pegs at a moult is described. After apolysis the scolopale extends across the subcuticular space and protects the dendrites, which remain in a functional condition until shortly before ecdysis. As the trichogen cell grows out to form a new peg the tip is surrounded by a mass of electron-dense material, probably derived from the receptorlymph cavity. The function of this material is unknown. Regeneration of the dendrites is considered. The possible mechanism by which the tip of the peg opens and closes is considered and the general structure of the organule is discussed in relation to functioning.

Notes: Summary The fat body sheath which surrounds the ventral nerve cord of the adult stick insect, Carausius morosus, is absent in the hatchling. Since the haemolymph sodium ion compositions of the two stages are similar, it is suggested that the fat body sheath is not a site of a blood-brain barrier dealing with extra-axonal regulation of sodium. Similarities of ultrastructure in connectives of adults and hatchlings are demonstrated, and tight junctions are shown to occur in perineural cells of both stages. The possible significance of this to the blood-brain barrier of this insect is discussed.

Notes: Summary In the ovary of Chironomus during phase 1 of the fourth larval instar, polygonally flattened “inner cells” are surrounded by smaller “outer cells” which contain bacteroids and phagosomes. Irregular cell remnants (“germ line accompanying substances”) lie among the inner cells. At the beginning of ovariole formation in phase 3, two layers of outer cells are separated by the formation of fissures. The inner layer of these cells (follicle- and egg-passage epithelium) forms regular invaginations. Cell pairs, identified as oocytes and nurse cells by “synaptic complexes” or multiple chromatin structures, wander from inside into the invaginations. Frequently between the two cells are fusomes, which later close in a characteristic manner. During phase 5, an egg passage is formed as a fissure among the egg-passage cells. During phase 7, the egg passage cells are conspicuously full of glycogen. Shortly before vitellogenesis membrane systems and annulated lamellae appear in the region of the oocyte. Accessory nuclei are formed by a “tieing-off” of projections of the the oocyte nucleus. During phase 9, microvilli and pinocytotic vesicles can be seen at the periphery of the oocyte. The distal cells of the ovariole are of oocyte or nurse cell nature, but in Ch. melanotus they are not surrounded by follicle cells and are reduced during further ovariole growth. In spite of the extremely small number of nurse cells in the follicle, the Chironomus ovary apparently does not differ functionally from other polytrophic meroistic insect ovaries.

Notes: Summary Ultrastructural diversification of muscle fibers, with regard particularly to myofibrillar changes, has been investigated in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles of the rat during fetal and postnatal development in the presence and in the absence of motor innervation. The band pattern and the shape of the myofibrils were uniform in fetal and neonatal muscle fibers and underwent differential changes during the first weeks after birth, concomitantly with fiber type specialization. The most evident variations in myofibrillar structure arising in this period concern the thickness of the Z band and the arrangement of the myofibrils. Myofibril formation was at first not impaired by denervation of rat muscles performed in utero and, although focal disintegration of myofibrils and detachment and loss of orientation of filaments became apparent by one week, atrophic muscle fibers with well-organized myofibrils could be seen as late as 2 months after birth. However, denervated muscle fibers of EDL and soleus did not display any significant and consistent difference in myofibrillar band pattern and shape. No variation in mitochondrial content and sarcoplasmic reticulum development was likewise seen in muscle fibers of EDL and soleus after fetal denervation. The findings emphasize the importance of neuromuscular interactions in muscle differentiation.

Notes: Summary The tracheal organ of the mesothoracic tibia of Teleogryllus is located in a corresponding position to the tympanal organ of the prothoracic tibia. The mesothoracic organ contains an average of only 12 scolopidia, the location of which corresponds to that of the proximal group A and proximal main group in the prothoracid tympanal organ. There are no scolopidia corresponding to the distal group of the tympanal organ. The variability in number of scolopidia is much greater in the mesothoracic organ than in the prothoracic organ. The adult tracheal system of the mesothoracic leg resembles the early nymphal tracheal system in both pro- and meso-thoracic legs. The development of the tracheal organ is usually complete by the sixth instar. The mesothoracic tracheal organ of the adult is broadly equivalent to the prothoracic tympanal organ of a fifth instar animal.

Notes: Summary The incorporation of 3H-uridine into nuclear and mitochondrial RNA has been followed by electron microscope autoradiography during spermiogenesis in Drosophila. Nuclei and mitochondria are simultaneously labeled up to the beginning of the chromatin condensation. The nebenkern, characteristic of the first stages of spermiogenesis, is the most radioactive cellular component. During chromatin condensation, nuclear RNA synthesis ceases, but mitochondrial derivatives continue to be significantly labeled up to their complete paracrystalline transformation. These data show an autonomous RNA synthesis by mitochondria at the end of spermiogenesis.

Notes: Summary The ultrastructure and protein content of the five subfractions of the crude mitochondrial fraction from the brain stem of the 1-day old and adult rat was examined. The morphological composition of the subfractions after fixation in glutaraldehyde and osmiumtetroxide in the adult rat brain stem resembled that previously reported for the whole brain; synaptosomes sedimented in a sucrose gradient in subfractions C and D. In the 1-day old rat, mature synaptosomes were found in subfractions A, B, C and D; E contained mainly free mitochondria. 80–95% of the processes in the adult and 10–30% in the 1-day old rat contained synaptic vesicles which were of four types: (1) small agranular vesicles (2) large dense core vesicles (3) large agranular vesicles (4) coated vesicles. Pre- and postsynaptic membrane thickenings were demonstrated in many nerve-ending particles. In the subfractions of the 1-day old rat the protein content was one half and the distribution resembled that in the adult. Evidently nerve endings develop faster in the brain stem than in cortical areas; a serotoninor adrenergic origin of the early synaptosomes is suggested.

Notes: Summary Muscle spindles of limb muscles were deefferented in neonatal rats by sectioning ventral roots or by removal of the lumbosacral spinal cord. Ten to 56 days after the operation, muscle spindles were examined in the medial gastrocnemius, extensor digitorum longus and soleus muscles. The differentiation of muscle spindles was not affected by deefferentation. The number of spindles in the investigated muscles was not reduced. Intrafusal fibres increased in number from two at birth to four per spindle on the average, as in normal muscles. The characteristic ultrastructural distinctions of nuclear bag and nuclear chain fibres developed as under normal conditions. However, intrafusal fibres atrophied slowly after fusimotor denervation, their polar zones becoming reduced in diameter by about 25% in comparison with control fibre diameters. Spindle capsules, on the other hand, increased in size and attained diameters comparable with normal spindles, appearing even somewhat distended. As intrafusal fibres degenerate after complete denervation at birth (Zelená, 1957), but differentiate in the absence of fusimotor innervation, it can be concluded that sensory nerve terminals induce and support their development. It is assumed that the morphogenetic influence of sensory terminals is mediated by release and uptake of a trophic substance at the synaptic junction. The occurrence of light and dense core vesicles in the sensory terminals and of coated invaginations and vesicles at both the axonal and plasma membrane speak in favour of such a possibility.

Notes: Summary The development of the submandibular gland of the mouse was studied by means of electron microscopy, from the 14th day of gestation up to birth. In the first two days the acini are solid and their cells contain polyribosomes and a few lamellae of endoplasmic reticulum. Beginning with the 16th day secretory granules appear and rapidly fill an increasing number of cells. The different electron density of the granules makes it possible to distinguish 1. two types of granules, dense and pale, and sometimes intermediate ones, 2. “polymorphic” granules. The latter consists of electron dense and electron pale parts combined in different configurations. The possible significance of the various types of granules is discussed.