ALBERT

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 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 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 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 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 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 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 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 influence of actinomycin on the neurosecretory Q and P cells of the brain of Enchytraeus was studied. Cytophotometrical measurements of Q cells in light mirocscopic preparations showed a significant decrease of PAF-positive material in the first hours after actinomycin application. At the ultrastructural level primary changes were established one to four hours after injection of actinomycin: In the nucleolus granular and fibrillar material became separated; there was a substantial increase of the fibrillar component. Concerning structural changes of the cytoplasm, Q and P cells differed especially with respect to the Golgi apparatus and the ribosomes. In the Q cells the Golgi apparatus had become greatly reduced shortly after actinomycin treatment. However, it persisted in P cells during all stages examined. Ribosomes became detached from membranes only in Q cells between 4 and 8 hours after injection. These data indicate that protein synthesis in P cells shows greater stability than in Q cells. The restitution of normal ultrastruoture during subsequent stages indicates that effects begin to subside 24 hours after a single injection.

Notes: Summary In the hypothalamus of albino rats submitted to positive air ions for 20 to 40 days, supra-optic and paraventricular nuclei exhibit various signs of hypo-activity: a slight decrease of nuclear and nucleolar volumes, an increase of the amount of neurosecretory granules in these neurones, while the migration of the neurosecretory material in the axons is diminished, almost no staining substance being detected along the axons. Simultaneously, the chrome-haematoxylin positive material is greatly reduced in the neurohypophysis, mainly after 40 days. Considering these data and other results, either unpublished or previously reported by various authors, it appears that the hypo-activity observed in the hypothalamo-neurohypophysial system may be correlated with a hyposecretion of antidiuretic hormone (ADH). There is no increase of diuresis; instead, an antidiuresis was noted which tends to disappear after about two weeks of treatment. This finding suggests that the hypothalamo-hypophysial response observed may be due to a disturbance of water metabolism induced by another antidiuretic factor. Besides, it was established that a treatment with positive air ions induces a significant increase of the serotonin level of the blood (Krueger). As an antidiuretic effect is directly exerted by serotonin on the kidneys, it may be responsible for the antidiuresis observed in this experiment. The inhibition of hypothalamic nuclei according to histological criteria would represent a homeostatic response trying to compensate the antidiuresis initially produced by serotonin, the neurohypophysial storage of ADH delaying the efficiency of this hypothalamic inhibition. All the available data on the modifications of water and electrolyte metabolism detected after a positive aero-ionization may be explained satisfactorily by an increase of the serotonin level of the blood, progressively counteracted by a decrease of ADH.

Notes: Summary The effects of adenohypophysectomy, and of several experimental interventions on the hypothalamo-pituitary-adrenal cortical axis have been studied in relation to the fine structure of the median eminence in the pigeon. 1. In control animals, the following morphological features of the caudal median eminence (C.M.E.) distinguish it from the rostral median eminence (R.M.E.): a) the absence in both external layers of the C.M.E. of large (1,600–1,900 Å) electron-dense granules, b) the presence in the C.M.E. of a small number of medium-size (1,200–1,400 Å) granules, and c) the predominance in the C.M.E. of small (600–800 Å) dense-core vesicles. 2. Adenohypophysectomy leads to: a) almost complete disappearance of electron-dense granules in both external layers of the R.M.E., and b) “emptying” of numerous axons and a relative increase in the number of large (1,600–1,900 Å) granules in the C.M.E. 3. Insulin shock produces modifications similar to those of adenohypophysectomy. The depletion of electron-dense granules from the axons is, however, restricted to the most anterior part of the R.M.E., and, in the C.M.E., the relative increase in the number of larger granules affects the 1,200–1,400 Å and the 1,600–1,900 Å size granules. 4. Metopirone enhances the number of medium-size (1,200–1,400 Å) granules in the external layers of both the R.M.E. and the C.M.E. and causes a significant increase in the number of synaptic-like vesicles in the C.M.E. 5. Prednisolone treatment leads to a marked enrichment of the external layers of the R.M.E. with 1,200–1,400 Å granules, and of the external layers of the C.M.E. with 1,000 Å granules. These results have been discussed with special reference to the hypothalamic control of the adrenocorticotropic function, especially reviewing the role of the 1,200–1,400 Å granules.

Notes: Summary The Herring bodies in the posterior lobe of the bovine hypophysis are very large (2–600 μ) and can be classified into three types. The type I Herring body contains an accumulation of neurosecretory granules. These Herring bodies are very scarce and should not be confused with the numerous, but small, axonal swellings which also contain neurosecretory granules. The type II Herring body is characterized by the presence of a varying number of normal, moderately electron dense and “empty” vesicles, autophagic vacuoles, multilamellate bodies and occasional mitochondria. These Herring bodies are frequently observed. The type III Herring body is typified by the presence of dense vesicles connected to tubular formations which contain material of variable electron density, of filaments, and of long slender and very numerous mitochondria. The presence of multilamellate bodies and autophagic vacuoles suggests that the type II Herring body is in a degenerating phase. This concept is further substantiated by the similarity between this type of Herring body and transected neurosecretory axons in which degeneration is occurring. A similar comparison suggests that the type III Herring body is undergoing a regenerative process. Our current concept of the structure and function of Herring bodies is revised in the discussion.

Notes: Summary The corpora cardiaca of adult Locusta migratoria consist of two well separated areas, a fact which permits the differentiation between intrinsic and extrinsic neurosecretory material. In the neural area three types of electron dense “classical” neurosecretory granules, and a fourth more lucent type can be distinguished according to size. In the non-neural “glandular” area typical endocrine cells mingle with only one type of neurosecretory axons.

Notes: Summary The ultrastructural features of the paraventricular neurones of the non-treated rat are presented comparing them with those of the supraoptic neurones. No striking differences are seen between the general electron microscopic characteristics of the paraventricular and supraoptic neurones. The importance of adequate fixation to obtain good preservation of the neurones is emphasized, since inadequate fixation can cause e.g. artefactual appearance of “dark neurones”. The previously presented classification of the neurosecretory neurones into two categories (e.g. “light and dark neurones”) on the basis of the number of ribosomes is not considered justifiable, since their number can vary to a very great extent even within a single cell. The synthesis of neurosecretory products in the paraventricular neurones obviously follows the general mode of the synthesis of secretory proteins: ribosomes—RER—Golgi complex—secretory vesicles. On the basis of the localization of heavy metal deposits after osmium impregnation and demonstration of acid phosphatase the Golgi complex of the paraventricular neurones is found to be polarized. The direction of the polarity is discussed. The substructures of the dense cores of the neurosecretory granules and of the contents of the lysosomal dense bodies are nearly identical. Therefore it is considered impossible to determine positively the nature of the dark condensed material within the Golgi complex. The characteristics of the immature neurosecretory granules and the possibility of releasing neurosecretory products into the cytoplasm already within the perikarya are speculated.

Notes: Summary 1. Electron microscopical investigations of the neurohypophysis in rat and trout reveal that exocytosis of neurosecretory elementary granules from the nerve endings occurs only rarely. The authors are of the opinion that hormone release in the neural lobe follows mainly the “membrane-release” pattern. 2. Exocytosis is not performed by tangential fusion of the elementary granule membrane and the plasmalemma of the nerve ending (axolemma). Administering the goniometer technique one can observe the appearance of a stalk-like structure connecting the two membranes. The basis of the stalk in the axolemma corresponds to the site of the stoma through which the core of the vesicle leaves the nerve ending. 3. The mechanism of the origin of small clear vesicles (diameter 500 Å approx.) near the axolemma of the neurosecretory terminal has not been elucidated. The authors did not observe equivalents of a compensatory endocytosis in the vicinity of granules released by exocytosis.

Notes: Summary The identification and distribution of endocrine cells in embryonic adenohypophysis of Seyllium canicula, and chronology of cellular differentiation and histogenesis of this gland have been investigated in this work. More clearly than in the adult, the presence of four populations of endocrine cells, each one located in a different lobe, is demonstrated. Secretion granules of rostral lobe cells are glycoprotidic, their modal diameter is 170 nm; median lobe ones, of same dimensions, are protidic or polypeptidic. The nemo-intermediate lobe big granules (modal diameter: 260 nm) are glycoprotidic, the ventral lobe small granules (modal diameter: 120 nm), protidic or polypeptidic. At stage 10 mm, Rathke's pouch cells are recognized from stomodeal epithelium by enigmatic inclusions, the “odd bodies” of Schechter. At stage 35 mm, preoptic and tuberal nucleus cells include neurosecretory granules of same diameter varying about 100 nm. At this same stage, granules appear simultaneously in the four hypophyseal lobes, including the neuro-intermediate lobe where no neurosecretory fiber is present. Direct innervation of endocrine cells of this lobe is accomplished at stage 45 mm and is accompanied by changes of granules dimensions. The end of embryonic development is characterized by increasing granular load of endocrine cells.

Notes: Summary The cerebral ganglion and the ventral nerve cord of Lumbricus terrestris have been studied with the electron microscope. The results are as follows: In the neuropile small granular vesicles (300 to 500 Å) occur in some varicose nerve fibres after fixation with potassium permanganate. This indicates the presence of noradrenaline. Sometimes only a few of the vesicles produce a positive reaction. After incubation with α-methyl-noradrenaline the numbers of nerve terminals with small granular vesicles greatly increase, indicating the presence of dopamine and/or 5-hydroxytryptamine. In this case the reaction is now complete. The number of small granular vesicles is largest in the terminal swellings. These findings are consistent with histofluorescence, chemical, and microspectrofluorometric analyses, which have demonstrated noradrenaline, dopamine, and 5-hydroxytryptamine in neurones in the central nervous system. Large granular vesicles (600 to 900 Å) are to be found in some perikarya, not identical with neurosecretory cell bodies. In this case the granular vesicles in the axon are smaller and fewer. This indicates a simultaneous proximo-distal transport and gradual decrease in size of the granular vesicles. The intraneuronal distribution of the vesicles is in agreement with the distribution of the fluorophores in the fluorescent neurones. Neurosecretory neurones are found most likely not to contain monoamines.

Notes: Summary 1. The effects of TSH and PTU treatment with thyroid stimulating hormone and propylthiouracil on the structure of median eminence of the pigeon have been studied by light- and electron microscopy. 2. By light microscopy it has been found that the aldehydefuchsin positive material of the external layers of the anterior median eminence differs functionally from that of the neural lobe. 3. The light- and electron microscopic investigations revealed that the anterior and posterior median eminence differ both structurally and functionally. Specifically after the experimental interventions the considerable reaction can be observed only in the anterior median eminence. 4. Electron microscopically it appears possible that the aldehyde-fuchsin positive material of the anterior median eminence may be associated with the 1200–1500 Å—size granules. 5. The quantitative changes observed in the granular composition after the treatments show a close correlation with thyroid function, namely after treatment with thyroid stimulating hormone the relative quantity of the 1200–1500 Å—size granules increases markedly, while administration of propylthiouracil has the opposite effect and increases the relative quantity of the 1000 Å—size dense-core vesicles. They presumably may play a role in the control of TSH-secretion.

Notes: Summary The purpose of this study is to investigate the ultrastructural features of perivascular cells as found in the neural lobe of the hypophysis. In particular, an inquiry was made into the nature of the relationship of such cells to neurosecretory fibers and endings. The latter, in fact, are often invaginated within the perivascular cells and enveloped by their processes; furthermore, they often reveal a certain number of empty granules as well as characteristics of degenerative nature. In the course of this study the localization of the perivascular cells has been investigated as well as that of their processes within the extensive interlobular network typical of the hypophysial neural lobe of rodents. Based on the data gathered, the hypothesis is put forward that the perivascular cells play an important role in the turnover of neurosecretory endings, both under physiological and experimental conditions, contributing thereby to the release of post-hypophysial hormones.

Notes: Summary The neurohypophysis of sexually mature male and female Acipenser güldenstädti Brandt and Acipenser stellatus Pallas was studied light and electron microscopically. The recessus hypophysei lined with ependymal cells of two main types, “narrow” and “wide”, are in the center of the neurohypophysial “roots”. Processes from both cell types run radially to the basement membrane of the connective tissue layers abutting on the hypophysial intermediate lobe. Protrusions penetrating deep into the recessus hypophyseus are found in the apical parts of the “wide” cells. Pituicytes are rare in the neurohypophysis. The ultrastructure of both ependymal cell types and of the pituicytes is described. Nonmyelinated “Gomori-positive” (peptidergic) neurosecretory A1 and A2 type fibres and their terminals containing elementary neurosecretory granules (1400–1800 Å and 1000–1400 Å respectively) are the main structural elements of the neurohypophysis. Some “dark” and single myelinated neurosecretory fibres have been found. The adrenergic fibres (type B) were described earlier (Polenov et al., 1972a). The structural peculiarities of the neurohypophysis are discussed in functional and comparative-morphological terms.

Notes: Summary During sexual development ofPlatynereis dumerilii, the number of neurones exhibiting secretory activity rises in parallel with the growth and maturation of the germ cells. The cytological differentiation of neuroglandular properties is accompanied with an increased uptake of tritiated cystine. The first neurones developing the characteristics of secretory cells and incorporating cystine appear just before the oocytes enter the second growth phase which corresponds with vitellogenesis. When plotted against the oocyte diameter the number of labelled neurosecretory cells yields an ascending line, which becomes a plateau once the oocytes approach a critical size. This critical size defines the developmental stage beyond which decerebration does not prevent normal maturation. The neuronal synthetic activity rises again when the oocytes differentiate the cortical structure of ripe eggs. The curve displays its maximum slope at the onset of metamorphosis. During the epitokous transformation the synthetic activity decreases rapidly and is almost totally depressed when swarming is imminent. In animals induced to regenerate their posterior segments after they have passed the critical stage, the number of labelled neurosecretory cells becomes reduced to the precritical level. According to this, regeneration in young precritical animals containing only few endocrine neurones does not stimulate the development of further secretory neurones. Thus, the time course during maturation and the changes caused by regeneration are reciprocal to the activity of the brain with respect to the release of juvenile hormone. The question is thus raised as to whether the declining titer of juvenile hormone may be accompanied or caused by an increased production of an antagonistic principle related to maturation.

Notes: Summary The sinus gland of Gammarus oceanicus, like that of other crustaceans, is composed of three elements: neurosecretory axons, glial cells and stromal sheath. Five neurosecretory axon types are identified on the basis of granule diameter, shape, and electron density, and axon matrix density. Exocytosis appears to be the major release mechanism of neurosecretory material. The preterminal regions of neurosecretory axons contain axoplasmic reticulum and neurotubules. Their arrangement in the axon and relationship with one another suggest a transport function. Multilamellar bodies are found in the terminal regions of neurosecretory axons. They arise from mitochondria and may be involved in granulolysis.

Notes: Summary Two new types of structure of the cytoplasm of an autonomic neuron were described: (1) Inclusions of the rough ergastoplasmic reticulum, which were named “ergastosomes”, contain finely granular material with moderately electron opaque staining with both glutaraldehyde-OsO4 and KMnO4 fixations. A short comment was made on the nature of the contents of the ergastosomes. (2) Tubular formations of endoplasmic reticulum were found in the axons and/or dendrites of the paracervical ganglion.

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 Neurohypophyses from sexually mature female sturgeons, Acipenser güldenstädti Brandt, killed before, soon after spawning, and during down-stream migration were studied light and electron microscopically. Ovaries were examined only under a light microscope. A large amount of neurosecretory material (3.5–4.5 arbitrary units) is found in the neurohypophysial “roots” during up-stream migration to spawning grounds. Neurosecretory fibres and their terminals are replete with elementary neurosecretory granules. Side by side with the latter some terminals contain single residual granules and few “synaptic” vesicles. Soon after spawning the amount of neurosecretory material decreases markedly in most individuals (content up to 1–3 units), and elementary granules are few in the fibres and their terminals. The number of residual granules increases, and “synaptic” vesicles are especially numerous. Granulated, disintegrating neurosecretory granules and granule-shadows occur in the preterminal parts of the fibres. The number of pituicytes increases because some “light” tanycytes seem to migrate from the ependymal into the subependymal layer. The restoration of neurosecretory material (reaching again up to 3.5–4.5 units) occurs within a month after spawning. The number of elementary granules increases in fibres and terminals while that of residual granules and “synaptic” vesicles decreases. These changes in the neurohypophysis of females after spawning are due to discharge of great amounts of peptide neurohormones into the general circulation after spawning. Spawning is regarded as a “physiological” stress for these females.

Notes: Summary The dynamics of the “A” cells of the parsintercerebralis of Oncopeltus fasciatus over the first eight days of adult life was studied by microspectrophotometry of sections stained either with aldehyde fuchsin or alcian blue 8 GX. The data show that the two stains differ in their selectivity as they record different events in the history of the cells. A hypothesis is proposed that the aldehyde fuchsin is more sensitive to the presence of a “carrier” protein in the cell, whereas alcian blue 8 GX is more sensitive to the presence of the “active principle” in the cell.

Notes: Summary The neurosecretory Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation stimulating hormone. Previously it has been shown that neuronal and non-neuronal inputs are involved in the regulation of their activity. The degree of autonomy of these cells has been investigated by studying with morphometric methods the ultrastructure of CDC maintained in vitro. CDC of isolated cerebral ganglia which were cultured for 7 days show a considerable rate of synthesis, transport and release of neurohormone. Apparently these processes can proceed in the absence of neuronal and hormonal inputs from outside the cerebral ganglia. Completely isolated CDC, however, do not show neurosecretory activity in vitro; active Golgi zones, indicating the formation of neurosecretory elementary granules, are absent from such cells. Isolation does not seem to affect general cell functions such as protein synthesis and respiration. It is suggested that a neuronal input, originating within the cerebral ganglia, is necessary for the stimulation of CDC neurosecretory activity. Techniques are described for the isolation and culture of neurosecretory cells of L. stagnalis.

Notes: Summary Both primary and secondary (tongue) bars of the pharyngeal gill basket are covered by epithelial cells that are continuous with the cells that line the atrium. Anterior and posterior faces of the gill bars are covered with lateral ciliated cells, which possess a single cilium, ringed by microvilli, and an elaborate basal mitochondria-rootlet apparatus. Pharyngeal faces of the gill bars are covered with ciliated pharyngeal cells, atrial faces by mucus secreting atrial cells. The surface epithelium rests on a stromal septum, a flattened tube of basal lamina which dilates to form the visceral blood vessel (along the pharyngeal face) and skeletal blood vessel (along the atrial face). This basal lamina surrounds paired skeletal rods which run through the longitudinal axis of the gill bars near the atrial face. Between the skeletal rods and atrial cells of primary gill bars is a coelomic channel lined by epithelioid coelomic cells. Neuronal processes, some with neurosecretory granules, are located among the bases of the atrial cells. Some axons may contact lateral ciliated cells where the latter meet atrial cells, but synaptoid endings have not been found here or elsewhere in the gill bars. Nervous tissue has not been identified among lateral ciliated cells even though ciliary activity of these cells is supposedly regulated by atrial nervous tissue.

Notes: Summary The neurosecretory system of the freshwater snail Bulinus truncatus was investigated. With the Alcian blue-Alcian yellow (AB/AY) staining method at least 10 different types of neurosecretory cells (NSC) were distinguished in the ganglia of the central nervous system. The differences in staining properties of the NSC — with AB/AY the cells take on different shades of green and yellow — are borne out at the ultrastructural level: the NSC types contain different types of neurosecretory elementary granules. The neurosecretory system of B. truncatus is compared to that of Lymnaea stagnalis, the species which has received the most attention among the pulmonates. It appears from the comparison that the systems of both species show many similarities, although some differences are also apparent.

Notes: Summary Six principal types of neurons are recognized in the median neurosecretory-cell group of the pars intercerebralis in the house cricket, Acheta domesticus. Four of the six cell types are neurosecretory neurons. The majority of the 900 cells contain granules 1500–3000 Å in diameter and are designated as type I. These neurons, which are associated with the nervus corporis cardiaci I (NCCI), undergo asynchronous secretory cycles. Two cell types (II and IV), which contain 700–1600 Å granules, terminate in a neurohemal area on the ventral median surface of the brain, a site not previously recognized as neurohemal in insects. Neurosecretory axons that lie within and below the perineurial sheath in the cerebral neurohemal area contain typical synaptoid figures, but no evidence of exocytosis was found. A single pair of neurosecretory cells (type III) has been recognized only from light microscope sections. Two distinct types of vesicle-bearing axons occur in protocerebral neuropile. The majority contain electron-dense and dense-cored vesicles 600–1000 Å in diameter. Some axons contain granules of type I cells and may be collaterals or loops of NCCI axons.

Notes: Summary Neurohypophyses of sexually mature Acipenser güldenstädti Brandt and Acipenser stellatus Pallas maintained in hypertonic solutions were studied by means of light and electron microscopy. The fish were caught in the Ural and transferred into basins containing either freshwater (controls) or 32‰, 22‰ and 17‰ NaCl solutions respectively. The neurohypophyses of control animals kept in freshwater for two days contained a large amount of “Gomori-positive” neurosecretory material (4 arbitrary units). An abundance of neurosecretory granules of different kinds, predominantly elementary ones, was observed electron microscopically in neurosecretory fibres and terminals of these fish. A decrease in the amount of neurosecretory material was found in all experimental animals: up to 1–2 units in A. güldenstädti after 3.5–10 h in 32‰ NaCl, and up to 3.5 units in A. stellatus after 4.5–48 h in 22‰ or 17‰ NaCl. The number of neurosecretory granules decreased considerably in the fibres and terminals in all experimental series, but that of granulated, disintegrating granules and “granule-shadows” increased markedly, especially in preterminal parts of axons. An increased number of hypertrophied mitochondria, lamellar bodies, residual granules and especially “synaptic” vesicles was seen. These changes speak for an active discharge of neurohormones into the general circulation, a phenomenon well known to be characteristic of stress responses. Pathological ultrastructural changes (breakdown of terminals, extracellular wide cisternae) were visible in A. güldenstädti neurohypophyses at the end of the experiments. Also tanycytes — “narrow” ependymal cells — and pituicytes underwent noticeable changes.

Notes: Summary In the protocerebrum of Limulus polyphemus, the horseshoe crab, neurosecretory cells are grouped within the curvature of the central body, an association area for inputs from lateral, median, and ventral eyes. Scattered neurohemal terminals face circulatory sinusoids within the brain. Efferent fibers, originating from unspecified, presumably neurosecretory cells in the central nervous system, course through the optic nerves and terminate on pigment and receptor cells of the eyes. Their terminals have a synaptoid structure and their granules have the characteristic density and crystalline substructure of neurosecretory granules. Transection of the lateral optic nerve leads to degranulation and gradual disappearance of terminals and to accumulation of granules at the proximal ends of cut fibers. The Limulus ocular innervation presents a striking example of directed delivery of neurosecretion to pigmentary effector cells, whose adaption is usually controlled by circulating neurohormones.

Notes: Summary The pericardial organ-anterior ramifications complexes of Uca pugilator and Callinectus sapidus were studied by transmission electron microscopy. Five morphologically distinguishable groups of granules and two groups of vesicles were identified. These granules and vesicles are present in approximately the same proportions in the pericardial organs and anterior ramifications of both species. Two of the granule groups are never mixed in the same axon terminals and are believed to represent different hormone-protein complexes. The remaining granule and vesicle groups are believed to be products of neurosecretory hormone release. Evidence that at least some of these granules and vesicles arise from intraaxonal release of neurosecretory material is presented.

Notes: Summary Using whole mount preparations, nerve cells at the cut surface (head region) and also the peduncular-basal disk region were studied during various stages of regeneration (zero hour — 96 h) in Hydra. Leucomethylene blue staining technique allows excellent stainability of nerve cells and thus a statistical count of them at the specified stages of regeneration was obtained. Within 1 h after transection a significant increase (P 〈 0.001) in release of neurosecretory droplets occurs. Between 4 and 15 h of regeneration the number of perikarya remains relatively constant, but the number of processes increases by 200%. This indicates that neurons in Hydra have the capacity to regenerate their processes. Their growth in length is dramatically illustrated at 18 hrs. of regeneration. This degree of anastomosing arrangement is not observed in any other stage of regeneration. After this time the majority of processes break down releasing the neurosecretory materials. A study of the number of perikarya, the number of neurites and the number of neurosecretory droplets in regenerating animals reveals a continuous increase in the number of nerve cells and neurosecretory droplets released for up to 24 h. With the accumulation of nerve cells at the cut surface (24 h of regeneration) there is a simultaneous appearance of tentacle outpushings. The tentacles increase in number and length during the subsequent periods of regeneration. As the regenerative process approaches completion (72–96 h) the number of neurosecretory droplets released decreases, approaching the pre-transection levels as seen in normal animals. It is suggested that the neurosecretory material may act as a “trophic” agent which stimulates differentiation of interstitial cells into nerve cells and thus influences the regenerative process.

Notes: Summary Major ultrastructural changes in neurons were studied during sequential periods of hypostomal regeneration in Hydra. Some neurons remain unaffected except that at certain periods following amputation, they become more active in neurosecretory production. Other neurons in various stages of differentiation were also observed. Most emphasis was placed on degenerating neurons showing a loss of organelles and destruction of the perikarya. Certain large, membrane-bounded structures (up to 1.7 μ in diameter) suggested tentatively to be of a lysosomal-like nature, may be partly responsible for the degenerative process. The neurites of these cells first assume a beaded appearance and/or contain bulbous endings. The eventual isolated fragments of neurites contain typical membrane-bounded neurosecretory droplets (850/1700 Å in diameter) which disintegrate forming particulate materials (350 Å in diameter). Following complete disruption of the neurites, some of the granules accumulate in the extracellular spaces before they are disintegrated. From the data presented in this and the preceding paper, it is suggested that these particles, derived from neurosecretory droplets, may be responsible for the stimulation of: 1) interstitial cell differentiation into neurons, 2) the increased activity in neurosecretory production in normal cells, 3) the conspicuous increase in neurite length and consequently the exaggerated degree of anastomosis, and 4) the possible capacity of neurons to regenerate neurites.

Notes: Summary The lower pituitary stalk has been studied in rats given hypertonic saline for four days and in rats with congenital diabetes insipidus (GDI), that is, in two groups of animals in which there is evidence of an increase in the transport of polypeptides down the axons of the tractus hypophyseus. A quantitative ultrastructural analysis of this tract, the first yet made, has demonstrated that in normal animals it is composed of unusually small axons, over half of which are less than 300 nm in diameter. In this tract there is a significant increase in axonal diameter and microtubular content in animals given hypertonic saline for four days. In adult rats with CDI, these changes are even more marked, a feature which is correlated with the fact that in these animals the transport of polypeptides has probably been abnormal since birth. An alteration in the microtubular content of neurons has not previously been observed either in physiologically stressed animals or in diseased animals; such alterations suggest the participation of microtubules in polypeptide transport. The narrow diameter of axons in the tractus hypophyseus of the normal animal raises the possibility of the extravesicular transport of such polypeptides.

Notes: Summary The infracerebral complex consists of: (a) two types of ependymoid infracerebral cells located on the ventral surface of the brain, adjacent to a coelomic sinus and blood vessels; (b) a noncellular stroma, the neural lamella, which separates the infracerebral cells from cerebral nervous tissue; (c) two types of cerebral neurosecretory endings (one with many mitochondria) which abut on the neural lamella opposite from the infracerebral cells. The infracerebral a cells become columnar and develop abundant apical caveolae and Golgi elements in older worms. The infracerebral b cells, filled with electron opaque granules, resemble neurosecretory cells; their axon-like processes contain microtubules and make synaptoid contacts with the neural lamella. Both cell types are present in young worms of about 10 segments but the b cells, relatively abundant in younger worms, are outnumbered by a cells in mature individuals. However, during reproductive development neither a nor b cells display any structural changes which would indicate that they represent glandular tissue. Basal processes originating both from a and b cells form entanglements which become progressively more intricate during maturation. Neurosecretory endings are present in 10-segment worms and become very numerous in mature worms but at no time do they penetrate the neural lamella and contact the infracerebral cells. It is unlikely that the infracerebral cells per se constitute a gland but they are strategically located to influence the release of hormones from neurosecretory endings.

Notes: Summary A major function of the larval salivary glands of Drosophila melanogaster is known to be the production of a mucopolysaccharide that serves as an adhesive during puparium formation. In order to localize the mucosubstances during development substrate histochemical methods were used, and the site of acid phosphatase was demonstrated by the ultrahistochemical lead-salt method. It could be shown that the “glue”-granules in the corpus cells of larval salivary glands as well as the large secretion vacuoles in the prepupal corpus cells give a positive β-amylase-resistent PAS-reaction, which indicates neutral mucosubstances. Granular PAS-positive deposits in the larval and prepupal collum cells were reduced after preincubation with β-amylase and may represent glycogen, which has also been seen in electron micrographs of these cells. The Hale-reaction gave a weak indication that acid mucosubstances are present in the larval “glue” granules and in the large prepupal secretory vacuoles. After digestion of sialic acid with α-neuraminidase the weak indication was absent showing that the acid mucosubstances had been sialomucines. Ultrahistochemical demonstration of acid phosphatase indicated the presence of this enzyme in Golgi fields and lysosomal structures. Acid phosphatase seems to be missing in the large secretion vacuoles of the prepupal salivary gland. It is concluded, that the large vacuoles in the corpus cells of prepupal salivary glands represent a secretion product, obviously a mucosubstance. The lysosomal structures, containing acid phosphatase, may be accumulated in preparation for the autolysis of the gland which begins about two hours after the pupal moult, i.e. 15 hours after puparium formation.

Notes: Summary Electron-microscope autoradiographs have been prepared from the neural lobes of the pituitary glands of rats which had received intracisternal injections of [35S] cysteine at various times before gland removal. The rate of appearance and disappearance of autoradiographically demonstrable radioactivity in the neural lobe closely paralleled that previously determined, biochemically, for radioactive hormones and neurophysins. Radioactivity was appreciably associated with the undilated parts of neurosecretory axons only during the first few hours after injection of the label. The axonal dilations were subdivided into those in which small vesicles could be seen (“endings”) and those in which no small vesicles could be seen (“swellings”). Radioactivity appeared first in “endings” and then in progressively larger and larger profiles of “swellings”. It appeared that newly arrived granules were found close to the limiting membrane of the nerve swelling and that as time progressed they moved deeper and deeper into the swelling. On the basis of the results, suggestions were made for an anatomical explanation of the readily-releasable pool of hormone which has been demonstrated pharmacologically.

Notes: Summary The effects of depleting brain catecholamines with a combined treatment of reserpine and α-methyl-p-tyrosine on serum corticosterone levels and release of immunoreactive neurophysin from the median eminence, in osmotically stressed and unstressed mallard ducks, were studied. Corticoid levels in salt loaded birds were more than three times that of unstressed birds. The combined treatment of reserpine and α-methyl-p-tyrosine significantly decreased the concentration of brain monoamines in all experimental groups and raised serum corticoid levels in non-stressed birds to the same level found in the osmotically stressed animals. Immunoreactive neurophysin in the zona externa of the median eminence was depleted in all birds subjected to either osmotic stress and/or reserpine treatment but not in unstressed control birds. These preliminary data indicate that catecholamines may exert an inhibitory influence on both ACTH release from the anterior pituitary and neurophysin from the median eminence and that these two events may in some way be interrelated in the duck.

Notes: Summary The ultrastructure of the sinus gland of the fiddler crab, Uca pugnax, was investigated and found to be similar to that in other crustaceans. Five types of neurosecretory axon terminals were tentatively identified on the basis of the size, shape, and electron density of granules within the axons. Release of neuro-secretory material appears to be by exocytosis.

Notes: Summary During stages 11 and 12, follicle cells surrounding the nurse cells produce lysosomes which presumably aid in the breakdown of the nurse cells. Accompanying a DNA reduction in nurse cell nuclei are several characteristic morphological changes including the appearance of intranuclear rod-like structures and nuclear granules about 300 Å in diameter. Similarities between structures seen in Drosophila nurse cell nuclei and those seen in other organisms are discussed.

Notes: Summary In continuation of previous investigations on the reaction of neurosecretory Herring bodies to osmotic stress, the alterations of these structures with increasing age were studied. The Herring bodies located in the vicinity of the supraoptic and paraventricular nucleus of young (3 months) and aged (15–20 months) rats were compared by means of light and electron microscopy. In old rats, the light microscope reveals a strong increase in the number of Herring bodies as visualized by neurosecretory stain whereas the electron microscope exhibits an accumulation of dense bodies and myelinated bodies inside these structures. These are interpreted as lysosomal residual bodies which are transported from the neurosecretory pericarya into the cell processes, thus accumulating with increasing age. Morphological and topographical considerations lead to the assumption that the storage sites are dilatations of dendrites.

Notes: Summary Neurosecretory nerve-endings are found in association with the ventral longitudinal muscles of the body wall in locust, stick insect and blowfly larva, and with toe muscles in the frog. In the stick insect the endings are less intimately associated with the muscles than in the locust or in the blowfly larva. In the locust and the stick insect and in the frog there are separate “neurosecretory” and “ordinary” motor endings but in the blowfly larva the characteristic features of motor and neurosecretory endings are combined in the same axon. The apparent sites of release of neurosecretory material show a characteristically rippled plasma membrane and are covered by only a flimsy layer of connective tissue material (stroma). In all three insects studied most of the neurosecretory material appears to be liberated into the haemolymph. Neurosecretory material appears to be released through the membrane at any point however, including, in the locust and in the blowfly larva, that part applied to the muscle. In the stick insect no endings have been seen as closely associated with the muscle as in the other two species. In the frog, as in the stick insect, the neurosecretory material is not liberated directly on to the muscle but into the connective tissue that lies between muscle fibres.