ALBERT

Notes: Abstract Drosophila melanogaster (Meigen) was used to test the power of isofemale lines in preserving genetic variability. We performed experiments in two ways. One series consisted of measuring the genetic variability for three enzymatic loci in 32 isofemale lines, in the first and 23rd generations of culture. In the second series, we tested the capacity of the larvae to eliminate a parasitoid by encapsulation after eight years of laboratory breeding. In general, individual isofemale lines appeared to change during the 23 generations of the study, but the global frequency of these alleles among the 32 isofemale lines stayed relatively unchanged. The only rare allele observed was also conserved. Changes in allozyme frequencies at any one locus were independent of those at other loci. Genetic variation of a biological trait, the capacity of the larvae to encapsulate a parasitoid, also changed, but it could be restored to a level close to that of the starting population by mass hybridizing together individuals of each line.

Notes: Abstract Foraging parasitoids are thought to need more specific information than generalists on the presence, identity, availability, and suitability of their insect host species. In the present paper, we compare responses to host kairomones by two phylogenetically related parasitoid species that attack Drosophilidae and that differ in the width of their host range. As predicted, the behavioral response of the parasitoids to host kairomones reflected their difference in host range. The response of the specialist parasitoid Leptopilina boulardiwas restricted to contact kairomones from its natural hosts and one closely related species. In contrast, the generalist parasitoid Leptopilina heterotomaresponded to contact kairomones of a variety of Drosophilidae species.

Notes: Abstract The host-foraging behavior of female entomophagous parasitoids is commonly modified by positive associative learning. Typically, a rewarding experience (e.g., successful oviposition in a host) increases a female's foraging effort in a host microhabitat of the type associated with that experience. Less well understood are the effects of unrewarding experiences (i.e., unsuccessful foraging). The influence of unrewarding experience on microhabitat choice and residence time within a microhabitat was examined for the eucoilid parasitoid,Leptopilina heterotoma, in laboratory and greenhouse assays. As determined previously, females which oviposited successfully in either of two microhabitat types (fermenting apple or decaying mushroom) strongly preferred to forage subsequently on that microhabitat type. However, failure to find hosts in the formerly rewarding microhabitat caused females to reverse their preference in favor of a novel microhabitat type. The effect, though striking, was transient: within 1–2 h, the original learned preference was nearly fully restored. Similar effects of unrewarding experiences were observed with respect to the length of time spent foraging in a microhabitat. As determined previously, oviposition experience in a particular microhabitat type increased the time spent foraging in a patch of that microhabitat type. However, failure to find hosts in the patch caused the time a wasp spent in the next unoccupied patch of that type to decrease to almost nothing. In addition, there was a tendency for an unrewarding experience on a formerly rewarding microhabitat type to extend the time spent in a patch of a novel type. The function of the observed effects of unrewarding experiences is discussed.

Notes: Abstract Based on both previously published literature and results reported here, it appears thatDrosophila melanogaster meet the explicit assumptions of the Trivers and Willard offspring sex allocation model. However, contrary to the model's predictions, offspring sex ratio was not significantly affected when we manipulated factors that influence offspring quality. We suggest that contrary to implicit predictions of offspring sex ratio models,Drosophila may lack the genetic plasticity to readily alter sex ratio.

Notes: Abstract The influence of egg-laying experience on the response of females of the eucoilid parasitoid,Leptopilina heterotoma, to parasitized and unparasitizedDrosophila melanogaster host larvae was examined under more controlled conditions than those used in past studies. In laboratory assays, we precisely manipulated both the number of eggs laid by females and the kind of larvae (parasitized versus unparasitized) in which the eggs were laid. We found that the tendency to avoid laying eggs in parasitized hosts depended markedly on whether or not eggs had been laid previously, but depended little on whether those eggs had been laid in parasitized or unparasitized hosts. The observed effect of general egg-laying experience on avoidance of parasitized hosts may reflect responses to either changes in the wasp's internal state (perhaps, changes in egg load) or changes in the wasp's neural representation of the external environment (such as those presumed to occur during learning). In light of these results, we offer a tentative reinterpretation of several earlier studies.

Notes: Abstract Using wild-reared flies, we examined sexual selection on five phenotypic traits (thorax length, wing length, wing width, head width, and face width) inDrosophila buzzatii, by scoring copulatory status in nine mass mating cages. Only male face width was identified as a direct target of sexual selection in an analysis of selection gradient, while indirect selection was present on all other studied traits, as expected from their correlations with face width. In contrast to males, there was no indication of selection in females. Nor was there evidence of assortative mating. The suggested direct selection on face width seems to take place during licking behavior of the courtship and might be related to courtship feeding. This study suggests that courtship success gives rise to indirect selection on body size.

Notes: Abstract The effects of rearing and acclimation on the response of adultDrosophila to temperature were investigated in a gradient.D. melanogaster flies preferred a higher mean temperature and were distributed over a wider range of temperatures thanD. simulans flies. Acclimating adults at different temperatures for a week did not influence the response of either species. Adults reared at 28°C as immatures had a lower mean preference than those reared at cooler temperatures, suggesting that flies compensated for the effects of rearing conditions. Adults from tropical and temperate populations ofD. melanogaster andD. simulans did not differ in the mean temperature they preferred in a gradient, suggesting little genetic divergence for this trait within species. The species differences and environmental responses may be related to changes in optimal physiological conditions for the flies.

Notes: Abstract In this paper we examine theDrosophila melanogaster larval response to light. We survey the morphology of the larval visual and motor systems in relation to larval locomotory behavior and phototaxis. In addition, this paper proposes a model of sensorimotor transformation and examines the reversal in taxis occurring at theD. melanogaster larval wnadering stage.

Notes: Summary In order to test the validity of the prediction of the mating pattern of females from the sperm length distribution in males, three species ofDrosophila were analysed. Males in the three species are equally polygynous but females differ in the level of polyandry. A ‘low recurrence polyandry’ is observed in the sperm dimorphic speciesD. affinis while a ‘high recurrence polyandry’ is observed in the sperm monomorphic speciesD. latifasciaeformis andD. littoralis. These results are consistent with the hypothesis proposed previously that sperm dimorphism in males can only be maintained by a selective alternative in females (i.e. facultative female polygamy), whereas a stricter mating system (e.g., ‘obligatory’ polyandry) should only result in sperm monomorphism irrespective of the absolute value of sperm length.

Notes: Abstract Genetic variation has been found in males of aD. simulans population for their eagerness to hybridize withD. melanogaster females. In a search for traits involved in this hybridization, males ofD. simulans were tested for mating speed and sexual vigour. Between-male differences were detected in both sexual traits, but no relationship was noticed between them, nor with the frequency of hybridization. Thus male mating propensities appear to be unrelated to the breakdown of sexual isolation between these sibling species.

Notes: Summary The nucleotide sequence of a segment of the mitochondrial DNA from threeDrosophila species (D. erecta, D. eugracilis, andD. takahashii), belonging to different subgroups of themelanogaster group has been determined. The segment encompasses three complete tRNA genes (tRNAtrp, tRNAcys, and tRNAtyr) and portions of two protein-coding genes: the subunit 2 of the NADH dehydrogenase (ND2) and the subunit 1 of the cytochrome oxidase (COI). Comparisons also involve homologous sequences already known for four otherDrosophila species of themelanogaster group. Length differences were confined in the intergenic region where a long stretch of AT repeats was observed in one of the species analyzed. The three tRNA genes exhibit very different evolutionary rates, the most slowly evolving one, tRNAtyr, is adjacent to the 5′ end of COI; tRNAs in similar positions have been previously shown to evolve slowly because they are probably involved in transcript processing. Although the rate of synonymous substitutions was very similar between ND2 and COI genes there were strong discrepancies between them in terms of the number of nonsynonymous substitutions. Differences have also been found in G+C content of the genes, which are likely to be linked to different selective pressures. There is a reduction in G+C content in the region where selective constraints are reduced. This suggests the existence of different levels of constraints along the sequenced segment. An overall analysis of the types of substitutions showed a decrease in A+T content during the course of evolution of the species.

Notes: Summary The Drosophila Gart locus consists of two genes. One gene encodes three enzymes in the de novo purine nucleotide biosynthesis pathway [glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART)]. The second gene lies within an intron of the purine gene and encodes a cuticle protein. To investigate the evolution of the Gart locus, the Chironomus tentans homolog was cloned by screening a genomic DNA library with a polymerase chain reaction product. This study shows that the interesting structural features of this locus conserved in two distant Drosophila species are not found in the Chironomus homolog. These features include the cuticle protein gene nested within an intron and the existence of an alternative transcript to yield a monofunctional enzyme. In addition, the extremely rapid divergence of coding sequence seen for members of the tandemly duplicated AIRS domain in Drosophila is found to be much less rapid in Chironomus.

Notes: Abstract Runs of identical amino acids encoded by triplet repeats (homopolymers) are components of numerous proteins, yet their role is poorly understood. Large numbers of homopolymers are present in the Drosophila melanogaster mastermind (mam) protein surrounding several unique charged amino acid clusters. Comparison of mam sequences from D. virilis and D. melanogaster reveals a high level of amino acid conservation in the charged clusters. In contrast, significant divergence is found in repetitive regions resulting from numerous amino acid replacements and large insertions and deletions. It appears that repetitive regions are under less selective pressure than unique regions, consistent with the idea that homopolymers act as flexible spacers separating functional domains in proteins. Notwithstanding extensive length variation in intervening homopolymers, there is extreme conservation of the amino acid spacing of specific charge clusters. The results support a model where homopolymer length variability is constrained by natural selection.

Notes: Summary The Threonine-Glycine (Thr-Gly) region of the period gene (per) in Drosophila was compared in the eight species of the D. melanogaster subgroup. This region can be divided into a diverged variable-length segment which is flanked by more conserved sequences. The number of amino acids encoded in the variable-length region ranges from 40 in D. teissieri to 69 in D. mauritiana. This is similar to the range found within natural populations of D. melanogaster. It was possible to derive a Thr-Gly “allele” of one species from that of another by invoking hypothetical Thr-Gly intermediates. A phylogeny based on the more conserved flanking sequences was produced. The results highlighted some of the problems which are encountered when highly polymorphic genes are used to infer phylogenies of closely related species.

Notes: Summary Increasing data onDrosophila alcohol dehydrogenase (ADH) sequences have made it possible to calculate the rate of amino acid replacement per year, which is 1.7×10−9. This value makes this protein suitable for reconstructing phylogenetic relationships within the genus for those species for which no molecular data are available such asScaptodrosophila. The amino acid sequence ofDrosophila lebanonensis is compared to all of the already knownDrosophila ADHs, stressing the unique characteristic features of this protein such as the conservation of an initiating methionine at the N-terminus, the unique replacement of a glycine by an alanine at a very conserved position in the NAD domain of all dehydrogenases, the lack of a slowmigrating peptide, and the total conservation of the maximally hydrophilic peptide. The functional significance of these features is discussed. Although the percent amino acid identity of the ADH molecule inDrosophila decreases as the number of sequences compared increases, the conservation of residue type in terms of size and hydrophobocity for the ADH molecule is shown to be very high throughout the genusDrosophila. The distance matrix and parsimony methods used to establish the phylogenetic relationships ofD. lebanonensis show that the three subgenera,Scaptodrosophila, Drosophila, andSophophora separated at approximately the same time.

Notes: Summary We have analyzed ∼18 kb of DNA in and upstream of thedefective chorion-1 (dec-1) locus of the eight known species of themelanogaster species subgroup ofDrosophila. The restriction maps ofD. simulans, D. mauritiana, D. sechellia, D. erecta, andD. orena are shown to have basically the restriction map ofD. melanogaster, whereas the maps ofD. teissieri andD. yakuba were more difficult to align. However, the basic amount of DNA and sequence arrangement appear to have been conserved in these species. A small deletion of varying length (65–200 bp) is found in a repeated sequence of the central transcribed region ofD. melanogaster, D. simulans, andD. erecta. Restriction site mapping indicated that thedec-1 gene is highly conserved in themelanogaster species subgroup. However, sequence comparison revealed that the amount of nucleotide and amino acid substitution in the repeated region is much larger than in the 5′ translated region. The 5′ flanking region showed noticeable restriction site polymorphisms between species. Based on calculations from the restriction maps a dendrogram was derived that supports earlier published phylogenetic relationships within themelanogaster species subgroup except that theerecta-orena pair is placed closer to themelanogaster complex than toD. teissieri andD. yakuba.

Notes: Summary The abundance of the transposable elementmariner differs dramatically in the genomes of the closely related speciesDrosophila simulans, D. mauritiana, D. sechellia, andD. melanogaster. Natural populations ofD. simulans andD. mauritiana have 1–10 and 20–30 copies per diploid genome, respectively, and the insertion sites are polymorphic. The element has not been found inD. melanogaster. In this paper we show thatD. sechellia, a species endemic to the Seychelles Islands, contains only twomariner elements that are at fixed sites in the genome. One element, inserted in chromosome 2R at 51A1–2, contains three deletions and is missing much of the 3′ end. The other element, inserted in chromosome 3L at 64A10–11, is the full length of 1286 bp. Although the sequence of the full-length element is polymorphic in populations ofD. sechellia, at least some of the sequences are closely related to elements fromD. simulans andD. mauritiana that are known to be active. However, judging from the progeny of crosses betweenD. sechellia andD. simulans, the biological activity of the full-lengthD. sechellia element appears to be low, either because of the nucleotide sequence of the element or because of its position in the genome, or both.

Notes: Abstract Nucleotide sequences of 72 species of Drosophilidae were determined for divergent D1 and D2 domains (representing 200 and 341 nucleotides respectively in D. melanogaster) of large ribosomal RNA, using the rRNA direct sequencing method. Molecular phylogenetic trees were reconstructed using both distance and parsimony methods and the robustness of the nodes was evaluated by the bootstrap procedure. The trees obtained by these methods revealed four main lineages or clades which do not correspond to the taxonomical hierarchy. In our results, the genus Chymomyza is associated with the subgenus Scaptodrosophila of the genus Drosophila and their cluster constitutes the most ancient clade. The two other clades are constituted of groups belonging to the subgenus Sophophora of the genus Drosophila: the so-called Neotropical clade including the willistoni and saltans groups and the obscura-melanogaster clade itself split into three lineages: (1) obscura group + ananassae subgroup, (2) montium subgroup, and (3) melanogaster + Oriental subgroups. The fourth clade, the Drosophila one, contains three lineages. D. polychaeta, D. iri, and D. fraburu are branched together and constitute the most ancient lineage; the second lineage includes the annulimana, bromeliae, dreyfusi, melanica, mesophragmatica, repleta, robusta, and virilis groups. The third lineage is composed of the immigrans and the cardini, funebris, guaramunu, guarani, histrio, pallidipennis, quinaria, and tripunctata groups. The genera Samoaia, Scaptomyza, and Zaprionus are branched within the Drosophila clade. Although these four clades appear regularly in almost all tree calculations, additional sequencing will be necessary to determine their precise relationships.

Notes: Summary We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.

Notes: Summary Thedec-1 eggshell gene inDrosophila melanogaster encodes follicle cell proteins required for proper eggshell assembly. As shown by Southern and Northern analyses thedec-1 gene occurs in four alleles (Fcl-4) among wild-type strains. Its second exon has a distinct feature in the form of 12 repeats with 78–91 nucleotides; the first five show nearly 100% homology. DNA sequence comparison of the repeated region of the alleles revealed that the length polymorphisms are caused by changes in the numbers of the first five repeats. The results suggest that the alleles have been generated by unequal intragenic crossing-over and/or slippage during DNA replication and that the allelic length variants have arisen independently. The possiblilty that the most common allele,FC1, has a selective advantage over the other alleles is discussed.

Notes: Abstract Approximately 2 kb corresponding to different regions of the mtDNA of 14 different species of the obscura group of Drosophila have been sequenced. In spite of the uncertainties arising in the phylogenetic reconstruction due to a restrictive selection toward a high mtDNA A+T content, all the phylogenetic analysis carried out clearly indicate that the obscura group is formed by, at least, four well-defined lineages that would have appeared as the consequence of a rapid phyletic radiation. Two of the lineages correspond to monophyletic subgroups (i.e., afftnis and pseudoobscura), whereas the obscura subgroup remains heterogeneous assemblage that could be reasonably subdivided into at least two complexes (i.e., subobscura and obscura).

Notes: Abstract Polymerase chain reaction (PCR)-amplified, sequenced, and digitally typed intergenic spacers (IGSs) of the ribosomal (r)DNA in D. melanogaster reveal unexpected features of the mechanisms of turnover involved with the concerted evolution of the gene family. Characterization of the structure of three isolated IGS length variants reveals breakage “hot spots” within the 330-base-pair (bp) subrepeat array found in the spacers. Internal mapping of variant repeats within the 240-bp subrepeat array using a novel digital DNA typing procedure (minisatellite variant repeat [MVR]-PCR) shows an unexpected pattern of clustering of variant repeats. Each 240-bp subrepeat array consists of essentially two halves with the repeats in each half identified by specific mutations. This bipartite structure, observed in a cloned IGS unit, in the majority of genomic DNA of laboratory and wild flies and in PCR-amplified products, has been widely homogenized yet is not predicted by a model of unequal crossing over with randomly placed recombination breakpoints. Furthermore, wild populations contain large numbers of length variants in contrast to uniformly shared length variants in laboratory stocks. High numbers of length variants coupled to the observation of a homogenized bipartite structure of the 240-bp subrepeat array suggest that the unit of turnover and homogenization is smaller than the IGS and might involve gene conversion. The use of PCR for the structural analysis of members of the rDNA gene family coupled to digital DNA typing provides powerful new inroads into the mechanisms of DNA turnover affecting the course of molecular evolution in this family.

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 Genomic clones ofDrosophila andTetrahymena histone H2A variants were isolated using the corresponding cDNA clones, (van Daal et al. 1988; White et al. 1988). The site corresponding to the initiation of transcription was defined by primer extension for bothDrosophila andTetrahymena genomic sequences. The sequences of the genomic clones revealed the presence of introns in each of the genes. TheDrosophila gene has three introns: one immediately following the initiation codon, one between amino acids 26 and 27 (gln and phe), and one between amino acids 64 and 65 (glu and val). TheTetrahymena gene has two introns, the positions of which are identical to the first two introns of theDrosophila gene. The chicken H2A.F variant gene has been recently sequenced and it contains four introns (Dalton et al. 1989). The first three of these are in the same positions as the introns in theDrosophila gene. The fourth intron interrupts amino acid 108 (gly). In all cases the sizes and the sequences of the introns are divergent. However, the fact that they are in conserved positions suggests that at least two of the introns were present in the ancestral gene. A phylogenetic tree constructed from the sequences of the variant and major cell cycle-regulated histone H2A proteins from several species indicates that the H2A variant proteins are evolutionarily separate and distinct from the major cell cycle-regulated histone H2A proteins. The ancestral H2A gene must have duplicated and diverged before fungi and ciliates diverged from the rest of the eukaryote lineage. In addition, it appears that the variant histone H2A proteins analyzed here are more conserved than the major histone H2A proteins.

Notes: Summary The study of individual genes is essential to a comprehensive understanding of genome evolution. The wealth of information on alcohol dehydrogenase (Adh) inDrosophila makes this gene particularly suitable for such analysis. We have characterized more than 4 kb of the genomicAdh region inDrosophila ambigua and compared this region toDrosophila mauritiana andDrosophila pseudoobscura. The presence of two genes,Adh and 3′ORF (open reading frame), has been confirmed and some of their essential features have been inferred from primary structural analysis. Inter- and intraspecific comparisons have led us to support that both genes may have diverged from an ancient precursor. They appear to be evolving independently, and show a species-specific pattern. TheAdh in theobscura group species lacks amino acids three and four when compared to the species of themelanogaster group and has accumulated most of its amino acid replacements in the third exon. Neither characteristic is observed when any other group species are compared, which suggests that these may be particular features of the evolution of theobscura group. The 3′ORF is highly conserved among the three species analyzed, although variability in the length of the third exon and the nucleotide substitution rate, which is much higher than inAdh, are worth noting. According to our data, both mutation/fixation rates and the distribution of mutations vary over time, which makes it difficult to predict the evolutionary dynamics of specific genome regions.

Notes: Summary The transposable element mariner occurs widely in themelanogaster species group ofDrosophila. However, in drosophilids outside of themelanogaster species group, sequences showing strong DNA hybridization with mariner are found only in the genusZaprionus. the mariner sequence obtained fromZaprionus tuberculatus is 97% identical with that fromDrosophila mauritiana, a member of themelanogaster species subgroup, whereas a mariner sequence isolated fromDrosophila tsacasi is only 92% identical with that fromD. mauritiana. BecauseD. tsacasi is much more closely related toD. mauritiana than isZaprionus, the presence of mariner inZaprionus may result from horizontal transfer. In order to confirm lack of a close phylogenetic relationship between the genusZaprionus and themelanogaster species group, we compared the alcohol dehydrogenase (Adh) sequences among these species. The results show that the coding region of Adh is only 82% identical betweenZ. tuberculatus andD. mauritiana, as compared with 90% identical betweenD. tsacasi andD. mauritiana. Furthermore, the mariner gene phylogeny obtained by maximum likelihood and maximum parsimony analyses is discordant with the species phylogeny estimated by using the Adh genes. The only inconsistency in the mariner gene phylogeny is in the placement of theZaprionus mariner sequence, which clusters with mariner fromDrosophila teissieri andDrosophila yakuba in themelanogaster species subgroup. These results strongly suggest horizontal transfer.

Notes: Summary We have studied the evolutionary changes occurring in the noncoding regions around the developmentally important fushi tarazu (ftz) gene in a total of 11 species in the genus Drosophila. Previous molecular developmental studies have identified DNA elements both 3′ and 5′ to the coding region which are important in proper regulation of expression of the Drosophila melanogaster ftz gene. We show here that these same elements are the most evolutionarily conserved regions in the vicinity of the gene homologs. Parts of some control elements are more conserved than exonic sequences. Not only is there sequence conservation, but the relative position, orientation, and distances among the control elements remain conserved. One quite significant difference does exist between the two major subgenera studied, Sophophora and Drosophila: namely, an inversion of the ftz unit with respect to other genes in the Antennapedia complex, ANT-C. As a comparison, we applied similar analysis to a “housekeeping” gene-rosy (ry), or Xdh. In contrast, DNA sequences 5′ to the ry coding region revealed little evolutionary conservation. These studies bear out the proposition that functionally important DNA sequences remain more conserved through evolutionary time than do less functionally important sequences. This proposition could be tested in the present case because we could predict a priori from the developmental studies which DNA regions should be most conserved.

Notes: Abstract During the evolution of the Y chromosome of Drosophila hydei, retrotransposons became incorporated into the lampbrush loop pairs formed by several of the male fertility genes on this chromosome. Although insertions of retrotransposons are involved in many spontaneous mutations, they do not affect the functions of these genes. We have sequenced gypsy elements that are expressed as constituents of male fertility gene Q in the lampbrush loop pair Nooses. We find that these gypsy elements are all truncated and specifically lost those sequences that may interfere with the continuity of lampbrush loop transcription. Only defective coding regions are found within the loop. Gypsy is not transcribed in loops of many other Drosophila species harboring the family. These results suggest that any contribution of gypsy to the function of male fertility gene Q does not depend on a conserved DNA sequence.

Notes: Abstract The present study shows that the wing beat frequency of Drosophila is visually controlled and modulated in response to different optomotor stimuli. Whereas rotational large field stimuli do not appear to modulate wing beat frequency, single rotating vertical stripes increase or decrease wing beat frequency when moving back-to-front or front-to-back, respectively. Maximal modulations occur at lateral stripe positions. Expansion stimuli eliciting the landing response cause a marked increase in wing beat frequency. Parameters of this frequency response depend in a graded fashion on certain stimulus properties, and the frequency response co-habituates with the landing response. Several results indicate that the frequency response is an integral component of the landing response, although it can also occur when the characteristic front leg extension is not observed. The complex spatial input integration underlying the frequency response and other motor components of the landing response cannot easily be explained by a system of large field integration units, but might indicate the existence of local expansion detectors.

Notes: Abstract 1. Wildtype Oregon-R Drosophila melanogaster were trained in the ambient magnetic field to a horizontal gradient of 365 nm light emanating from one of the 4 cardinal compass directions and were subsequently tested in a visually-symmetrical, radial 8-arm maze in which the magnetic field alignment could be varied. When tested under 365 nm light, flies exhibited consistent magnetic compass orientation in the direction from which light had emanated in training. 2. When the data were analyzed by sex, males exhibited a strong and consistent magnetic compass response while females were randomly oriented with respect to the magnetic field. 3. When tested under 500 nm light of the same quantal flux, females were again randomly oriented with respect to the magnetic field, while males exhibited a 90° clockwise shift in magnetic compass orientation relative to the trained direction. 4. This wavelength-dependent shift in the direction of magnetic compass orientation suggests that Drosophila may utilize a light-dependent magnetic compass similar to that demonstrated previously in an amphibian. However, the data do not exclude the alternative hypothesis that a change in the wavelength of light has a non-specific effect on the flies' behavior, i.e., causing the flies to exhibit a different form of magnetic orientation behavior.

Notes: Abstract Neuromuscular preparations from third instar larvae of Drosophila are not well-maintained in commonly used physiological solutions: vacuoles form in the muscle fibers, and membrane potential declines. These problems may result from the Na∶K ratio and total divalent cation content of these physiological solutions being quite different from those of haemolymph. Accordingly haemolymph-like solutions, based upon ion measurements of major cations, were developed and tested. Haemolymph-like solutions maintained the membrane potential at a relatively constant level, and prolonged the physiological life of the preparations. Synaptic transmission was well-maintained in haemolymph-like solutions, but the excitatory synaptic potentials had a slower time course and summated more effectively with repetitive stimulation, than in standard Drosophila solutions. Voltage-clamp experiments suggest that these effects are linked to more pronounced activation of muscle fiber membrane conductances in standard solutions, rather than to differences in passive muscle membrane properties or changes in postsynaptic receptor channel kinetics. Calcium dependence of transmitter release was steep in both standard and haemolymph-like solutions, but higher external calcium concentrations were required for a given level of release in haemolymph-like solutions. Thus, haemolymph-like solutions allow for prolonged, stable recording of synaptic transmission.

Notes: Abstract Bang-sensitive mutants of Drosophila melano gaster (bas 1, bssMW1, eas2, tko25t) display seizure followed by paralysis when subjected to mechanical shock. However, no physiological or biochemical defect has been found to be common to all of these mutants. In order to observe the effects of bang-sensitive mutations upon an identified neuron, and to study the nature of mechanically induced paralysis, we examined the response of a mechanosensory neuron in these mutants. In each single mutant and the double mutant bas 1 bssMW1, the frequency of action potentials in response to a bristle displacement was reduced. This is the first demonstration of a physiological defect common to several of the bang-sensitive mutations. Adaptation of spike frequency, cumulative adaptation to repeated stimulation (fatigue) and the time course of recovery from adaptation were also examined. Recovery from adaptation to a conditioning stimulus was examined in two mutants (bas 1 and bss MW1), and initial recovery from adaptation was greater in both mutants. Quantification of receptor potentials was complicated by variability inherent in extracellular recording conditions, but examination of the waveform and range of amplitudes did not indicate clear mutant defects. Therefore the differences observed in the spike response may be due to an alteration of the transfer from receptor potentials to action potential production. DNA sequence analysis of tko and eas has indicated that they encode apparently unrelated biochemical products. Our results suggest that these biochemical lesions lead to a common physiological defect in mechanoreceptors. Although this defect does not provide a straightforward explanation for bang sensitivity, the altered cellular process may lead to bang sensitivity through its action in different parts of the nervous system.

Notes: Summary Single-fly polymerase chain reaction amplification and direct DNA sequencing revealed high levels of length polymorphism in the threonine-glycine encoding repeat region of theperiod (per) gene in natural populations ofDrosophila melanogaster. DNA comparison of two alleles of identical lengths gave a high number of synonymous substitutions suggesting an ancient time of separation. However detailed examination of the sequences of different Thr-Gly length variants indicated that this divergence could be understood in terms of four deletion/insertion events. InDrosophila pseudoobscura a length polymorphism is observed in a five-amino acid degenerate repeat, which corresponds tomelanogaster's Thr-Gly domain. In spite of the differences betweenD. melanogaster andD. pseudoobscura in the amino acid sequence of the repeats, the predicted secondary structures suggest evolutionary and mechanistic constraints on theper protein of these two species.

Notes: Summary Characterization of sequences homologous to theDrosophila melanogaster gypsy transposable element was carried out inDrosophila subobscura (gypsyDS). They were found to be widely distributed among natural populations of this species. From Southern blot and in situ analyses, these sequences appear to be mobile in this species.GypsyDS sequences are located in both euchromatic and heterochromatic regions. A completegypsyDS sequence was isolated from aD. subobscura genomic library, and a 1.3-kb fragment which aligns with the ORF2 of theD. melanogaster gypsy element was sequenced. Comparisons of this sequence in three species (D. subobscura, D. melanogaster, and D. virilis) indicate that there is greater similarity between theD. subobscura-D. virilis sequences than betweenD. subobscura andD. melanogaster. Molecular divergence ofgypsy sequences betweenD. virilis andD. subobscura is estimated at 16 MY, whereas the most likely divergence time of these two species is more than 60 MY. These data strongly suggest thatgypsy sequences have been horizontally transferred between these species.

Notes: Summary In the twelve Drosophila obscura group species studied, belonging to the affinis, obscura, and pseudoobscura subgroups, the mitochondrial DNA length ranges from 15.8 to 17.2 kb. This length polymorphism is mainly due to insertions/deletions in the variable region of the A+T-rich region. In addition, one species (D. tristis) possess a tandem duplication of a 470-bp fragment that contains the replication origin. The same duplication has occurred at least twice in the Drosophila evolutionary history due to the fact that the repetition is analogous to repetitions found in the four species of the D. melanogaster complex. By comparing the nucleotide sequence of the conserved region in D. ambigua, D. obscura, D. yakuba, D. teissieri, and D. virilis, we show the presence of a secondary structure, likely implied in the replication origin, which could favor the generation of this kind of duplications. Finally, we propose that the high A and T content in the variable region of the A + T-rich region favors the formation of less-stable secondary structures, which could explain the generation of minor insertion/deletions found in this region.

Notes: Summary DNA-DNA hybridization studies of insects, more specificallyDrosophila and cave crickets, have revealed interesting patterns of genome evolution that contrast markedly with what has been seen in other taxa, especially mammals and birds. Insect genomes are composed of sections of single-copy DNA with extreme variation in rates of evolutionary change. This variation is more extreme than between introns and exons; introns fall into the relatively conserved fraction of the genome. Attempts to calculate absolute rates of change inDrosophila DNA have all led to estimates some 5–10 times faster than those found in most vertebrates; this is true even for the more conservative part of the nuclear genome. Finally we point out that morphological similarity, chromosomal similarity, and/or ability to form interspecific hybrids is often associated with quite high levels of single-copy DNA divergence in insects as compared to mammals and birds.

Notes: Abstract From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the C↔T substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.

Notes: Abstract The muscle-myosin heavy-chain (mMHC) gene of Drosophila hydei has been sequenced completely (size 23.3 kb). The sequence comparison with the D. melanogaster mMHC gene revealed that the exonintron pattern is identical. The protein coding regions show a high degree of conservation (97%). The alternatively spliced exons (3a-b, 7a-d, 9a-c, 11a-e, and 15a-b) display more variations in the number of nonsynonymous and synonymous substitutions than the common exons (2, 4, 5, 6, 8, 10, 12, 13, 14, 16, 17, and 19). The base composition at synonymous sites of fourfold degenerate codons (third position) is not biased in the alternative exons. In the common exons there exists a bias for C and against A. These findings imply that the alternative exons of the Drosophila mMHC gene evolve at a different, in several cases higher, rate than the common ones. The 5′ splice junctions and 5′ and 3′ untranslated regions show a high level of similarity, indicating a functional constraint on these sequences. The intron regions vary considerably in length within one species, but the corresponding introns are very similar in length between the two species and all contain stretches of sequence similarity. A particular example is the first intron, which contains multiple regions of similarity. In the conserved regions of intron 12 (head-tail border) sequences were found which have the potential to direct another smaller mMHC transcript.

Notes: Summary Previous studies have demonstrated that the expression of the α-amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the α-amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.

Notes: Summary A 2.1-kb SStI fragment including the rp49 gene and the 3′ end of the δ-serendipity gene has been cloned and sequenced in Drosophila pseudoobscura. rp49 maps at region 62 on the tip of chromosome II of this species. Both the coding and flanking regions have been aligned and compared with those of D. subobscura. There is no evidence for heterogeneity in the rate of silent substitution between the rp49 coding region and the rate of substitutions in flanking regions, the overall silent divergence per site being 0.19. Noncoding regions also differ between both species by different insertions/deletions, some of which are related to repeated sequences. The rp49 region of D. pseudoobscura shows a strong codon bias similar to those of D. subobscura and D. melanogaster. Comparison of the rates of silent (K S ) and nonsilent (K a ) substitutions of the rp49 gene and other genes completely sequenced in D. pseudoobscura and D. melanogaster confirms previous results indicating that rp49 is evolving slowly both at silent and nonsilent sites. According to the data for the rp49 region, D. pseudoobscura and D. subobscura lineages would have diverged some 9 Myr ago, if one assumes a divergence time of 30 Myr for the melanogaster and obscura groups.

Notes: Abstract In an effort to understand the forces shaping evolution of regulatory genes and patterns, we have compared data on interspecific differences in enzyme expression patterns among the rapidly evolving Hawaiian picture-winged Drosophila to similar data on the more conservative virilis species group. Divergence of regulatory patterns is significantly more common in the former group, but cause and effect are difficult to discern. Random fixation of regulatory variants in small populations and/or during speciation may be somewhat more likely than divergence driven by selection. Within the picture-winged group, we also have compared enzymes that fulfill different metabolic roles. There are highly significant differences between individual enzymes, but no obvious correlations to functional categories.

Notes: Abstract The region of the genome of D. lebanonensis that contains the Adh gene and the downstream Adh-dup gene was sequenced. The structure of the two genes is the same as has been described for D. melanogaster. Adh has two promoters and Adh-dup has only one putative promoter. The levels of expression of the two genes in this species are dramatically different. Hybridizing the same Northern blots with a specific probe for Adh-dup, we did not find transcripts for this gene in D. lebanonensis. The level of Adh distal transcript in adults of D. lebanonensis is five times greater than that of D. melanogaster adults. The maximum levels of proximal transcript are attained at different larval stages in the two species, being three times higher in D. melanogaster late-second-instar larvae than in D. lebanonensis first-instar larvae. The level of Adh transcripts allowed us to determine distal and proximal initiation transcription sites, the position of the first intron, the use of two polyadenylation signals, and the heterogeneity of polyadenylation sites. Temporal and spatial expression profiles of the Adh gene of D. lebanonensis show qualitative differences compared with D. melanogaster. Adh and Adh-dup evolve differently as shown by the synonymous and nonsynonymous substitution rates for the coding region of both genes when compared across two species of the melanogaster group, two of the obscura group of the subgenus Sophophora and D. lebanonensis of the victoria group of the subgenus Scaptodrsophila. Synonymous rates for Adh are approximately half those for Adh-dup, while nonsynonymous rates for Adh are generally higher than those for Adh-dup. Adh shows 76.8% identities at the protein level and 70.2% identities at the nucleotide level while Adh-dup shows 83.7% identities at the protein level and 67.5% identities at the nucleotide level. Codon usage for Adh-dup is shown to be less biased than for Adh, which could explain the higher synonymous rates and the generally lower nonsynonymous substitution rates in Adh-dup compared with Adh. Phylogenetic trees reconstructed by distance matrix and parsimony methods show that Sophophora and Scaptodrosophila subgenera diverged shortly after the separation from the Drosophila subgenus.

Notes: Abstract Interspecific sequence comparison of the highly repetitive Drosophila gene mastermind (mam) reveals extensive length variation in homopolymer domains. The length variation in homopolymers is due to nucleotide misalignment in the underlying triplet repeats, which can lead to slippage mutations during DNA replication or repair. In mam, the length variation in repetitive regions appears to be balanced by natural selection acting to maintain the distance between two highly conserved charge clusters. Here we report a statistical test of the null hypothesis that the similarity in the amino acid distance between the charge clusters of each species arose by chance. The results suggest that at mam there is a juxtaposition of length variability due to molecular drive and length conservation maintained by natural selection. The analysis of mam allows the extension of current theories of drive-selection interaction to encompass homopolymers. Our model of drive-selection equilibrium suggests that the physical flexibility, length variability, and abundance of homopolymer domains provide an important source of genetic variation for natural populations.

Notes: Abstract We describe the kinetics of odorant response in the maxillary palp of Drosophila, and show that the rate of recovery from odorant stimulation is affected by mutation of the rdgB (retinal degeneration B) gene. We use immunocytochemistry to confirm that the rdgB gene product is expressed in the maxillary palp. rdgB has recently been shown to encode a protein with Ca2+-binding sites and sequence similarity to rat brain phosphatidylinositol transfer protein; it is located near the rhabdomeric membranes in photoreceptor cells, where it has been suggested to play a role in membrane transport. The delay in recovery kinetics that we observe in olfactory tissue may reflect a defect in membrane restoration at the conclusion of the olfactory transduction cascade. The use of common molecules in the physiology of two olfactory organs, and in both visual and olfactory physiology, is discussed.

Notes: Abstract Maxillary palps have been proposed as secondary olfactory organs, after the antennae, in Drosophila melanogaster. Our study tries to establish the quantitative importance of both organs as olfactory information mediators. Dose-response curves for three odorants: ethyl acetate, propionaldehyde and benzaldehyde were carried out for comparing olfaction in either complete animals or flies surgically deprived of antennae. Antennaless flies tested in our behavioral assay showed indifferent, attractant and repellent responses depending on concentration, similarly as normal flies do. However, they clearly displayed less sensitivity than normal flies. The range of concentrations they were able to perceive was correlated to antennal sensitivity approximately by a factor 1∶10 for ethyl acetate and benzaldehyde, and between 1∶10 and 1∶100 at high concentrations of propionaldehyde. A complementary experiment was performed to test changes in olfactory behavior produced by removing maxillary palps in the presence of antennae. At high concentrations of odorant, responses to ethyl acetate and propionaldehyde experienced small changes when both palps were removed. Results are compatible with a summation model of all olfactory information reaching the brain either through antennae or palps.

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 spl mutation of the N gene causes, among other phenotypic traits, the lack of a few ommatidia, roughness and a general reduction in the size of the compound eye; these defects are drastically enhanced by the dominant mutation E(spl) D. We have studied cellular and developmental aspects of the phenotypic interaction between spl and E(spl) D. We found that the initial clustering of photoreceptor cells is affected in eye imaginal discs of spl larvae causing the defects visible in the adult eye. The degree of disorganization of the spl/Y; E(spl) D/ + eye disc is much higher, only a few photoreceptor cells are able to group with representatives of the other cell types and differentiate normally. BrdU incorporation shows that the proliferation pattern of the spl/Y; E(spl) D/ + disc cells during the third instar is normal. Abundant cell death occurs posteriorly in the mutant discs, which accounts for their small size. Finally, we found that in the eye imaginal disc the transcription of m8, the E(spl) gene, responsible for the enhancement of the spl phenotype caused by the E(spl) D mutation, is restricted to the morphogenetic furrow, where the ommatidial cells start grouping with each other to take on their future developmental fates; the m8 transcription rate is highly increased in E(spl) D eye discs. All these observations indicate that the assembly of the ommatidial cells is affected in the spl/Y; E(spl) D/ + disc and that the other abnormalities are morphogenetic consequences of the defective cell grouping.

Notes: Summary Arginine kinase (AK) is present throughout the life cycle of Drosophila melanogaster, but there is a sharp, transient peak of AK activity during the prepupal period and a second period of elevated activity at the time of eclosion of the adult. Imaginal discs show the greatest increase in AK activity at the prepupal stage of those tissues assayed. The prepupal peak is not seen when the temperature-sensitive ecdysoneless mutant ecd-1 is shifted to 29° C at mid-third instar larval stage. The peak in activity reappears when ecd-1 is either shifted back to 20° C after 60 h at 29° C or is fed 20-hydroxyecdysone. At the restrictive temperature, imaginal discs from ecd-1 larvae progressively lose AK activity, whereas discs from 20-hydroxyecdysone-fed larvae have a marked increase in AK activity at stage P3 of the prepupal period. These data suggest that the prepupal peak is regulated by the hormone 20-hydroxyecdysone.

Notes: Summary We have devised a protocol for the cloning of our cell lines, and have demonstrated that a cloned line may contain cells of widely differing morphology — epithelial, fibroblast-like, and lamellocyte-like. These different morphologies must therefore represent diversity in the microenvironment of the culture rather than diversity in the cellular origin of the line.

Notes: Summary The development of a leg segment of the fruitflyDrosophila melanogaster was analyzed in order to determine whether the orderliness of the segment's bristle pattern originates via waves of cellular interactions, such as those that organize the retina. Fly development was perturbed at specific times by either teratogenic agents (gamma rays, heat shock, or the drug mitomycin C) or temperature-sensitive mutations (l(1)63, l(1) Notchts1, orl(1) shibire ts1 ), and the resulting abnormalities (e.g., missing or extra structures) were mapped within the pattern area. If bristles develop in a linear sequence across the pattern, then they should show sensitivity to perturbations in the same order, and wavefronts of cuticular defects should result. Contrary to this prediction, the maps reveal no evidence for any directional waves of sensitivity. Nevertheless, other clues were uncovered as to the nature and timing of patterning events. Chemosensory bristles show earlier sensitivities than mechanosensory bristles, and longer bristles precede shorter ones. The types and sequence of cuticular abnormalities imply the following stages of bristle pattern development: (1) scattered inception of bristle mother cells, each surrounded by an inhibitory field, (2) alignment of the mother cells into rows, (3) differential mitoses, (4) assignment of cuticular fates to the mitotic progeny, (5) polytenization of the bristle cells, (6) fine-tuning adjustments in bristle spacing, and (7) signalling from bristle cells to adjacent epidermal cells, inducing them to form “bracts”.

Notes: Summary The arrangement of bristles on a leg segment of the fruitflyDrosophila melanogaster was studied in various mutants that have abnormal numbers of bristles on this segment. Eighteen mutations at six different genetic loci were analyzed, plus five double or triple mutant combinations. Recessive mutations at theachaete-scute locus were found to affect distinct groups of bristles:achaete mutations remove mechanosensory bristles, whereasscute mutations remove mainly chemosensory bristles. Mechanosensory bristles remain uniformly spaced along the longitudinal axis unless their number decreases below a certain threshold, suggesting that spacing is controlled by cell interactions that cannot function when bristle cells are too far apart. Above a certain threshold, bristle spacing and alignment both become irregular, perhaps due to excessive force from these same interactions. Chemosensory bristles occupy definite positions that are virtually unaffected by removal of individual bristles from the array. Extra chemosensory bristles develop only near the six normal sites. At two of the six sites the multiple bristles tend to exhibit uniform longitudinal spacing — a property confined to mechanosensory bristles in wild-type flies. To explain the various mutant phenotypes the following scheme is proposed, with different mutations directly or indirectly affecting each step: (1) spots and stripes are demarcated within the pattern area, (2) one bristle cell normally arises within each spot, multiple bristle cells within each stripe, (3) incipient bristle cells inhibit neighboring cells from becoming bristle cells, and (4) the bristle cells within each stripe become aligned to form rows and then repel one another to generate uniform spacing.

Notes: Abstract Extracellular matrix and membrane proteins and their correct secretion probably are key elements in morphogenesis and differentiation in Drosophila. In this study, we have analysed the effects of monensin, a Na+-H+-ionophore which blocks normal secretion, applied during cellular blastoderm formation on further development. Normal cell morphology and intercellular contacts are lost and the extracellular matrix becomes disorganized. Gastrulation is blocked and abnormal foldings can be observed. Cuticle phenotypes showed different degrees of ventral, dorsal, head and posterior defects. The results are discussed in the context of what is known about membrane and extracellular matrix proteins in Drosophila.

Notes: Abstract Glial cells are of significant importance for central nervous system development and function. In insects, knowledge of the types and development of CNS glia is rather low. This is especially true for postembryonic glial development. Using bromodeoxyuridine incorporation and enhancer trap lines we identified a reproducible spatial and temporal pattern of DNA replicating cells in the abdominal larval CNS (A3-7 neuromeres) ofDrosophila melanogaster. These cells correspond to embryonically established glial cells in that region. Except for a specific subfraction, these cells apparently do not divide during larval life. Similar patterns were found in two otherDrosophila species,D. virilis andD. hydei.

Notes: Abstract The Drosophila proneural genes specify neuronal determination among cells within the ectoderm. Here we address the question of whether proneural genes also affect the specification of glia, the most abundant cell type in the nervous system. We provide evidence that the proneural gene daughterless is essential for the formation of two major classes of PNS glia. In contrast, the proneural genes in the achaete-scute complex have no detectable effect on the specification and differentiation of these PNS glia and certain CNS glia. We also show that, as with neuronal development, glial determination is restricted by the neurogenic genes neuralized, Delta, and the genes of the Enhancer of split complex. Finally, we demonstrate that prospero, a gene involved in neuronal differentiation, also affects glial development. These results demonstrate extensive overlap in the genetic control of glial and neuronal development.

Notes: Abstract Effects of the essential oils (EOs) extracted from eleven aromatic plants belonging to the Lamiaceae family (common in the Greek flora) were examined upon three different developmental stages ofDrosophila auraria. All of the EOs examined exhibited insecticidal effects, either by preventing egg hatching, or by causing the death of larvae and adult flies. In several cases, malformation and/or prohibition of puparium formation was also observed.

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 By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca2+-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca2+ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca2+. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca2+ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins.

Notes: Summary The genecrumbs (crb) ofDrosophila melanogaster provides an essential function for the embryonic development of ectodermally derived epithelia. Complete loss of function alleles of thecrb gene are recessive embryonic lethals and lead to a disorganization of the primordia of these epithelia, followed by cell death in some tissues. Incrb mutant embryos, different organs are affected to a different extent. Some tissues die almost completely (as the epidermis, the atrium and the pharynx) while others partially survive and conserve their basic epithelial structure (as the tracheal system, the oesophagus, the proventriculus, the salivary glands, the hindgut and the Malpighian tubules). Degeneration is first visible at stage 11 and continues successively throughout development. There is evidence that the loss of epithelial cell polarity may be the cause for the degeneration of these tissues, suggesting that thecrb gene product is involved in stabilizing the apico-basal polarity of epithelial cells. As previously shown, thecrb protein is specifically expressed on the apical side of embryonic epithelia in a reticular pattern outlining the borders of the cells. Here we demonstrate that thecrb protein shows the same subcellular localization in epithelial cells of imaginal discs and in follicle cells, indicating a similar function ofcrb during the development of embryonic, imaginal and follicle epithelia. Clonal analysis experiments indicate that the genecrb is not cell-autonomous in its expression, suggesting that the gene product may act as a diffusible factor and may serve as a signal in a cell-cell communication process. This signal is thought to be required for the formation and/or maintenance of the cell and tissue structure of the respective epithelia.

Notes: Summary In holometabolous insects, the steroid molting hormone 20-OH-ecdysone (ecdysterone) orchestrates the diverse developmental events of metamorphosis, in large part by regulating gene expression. In Drosophila, the Broad Complex (BR-C) is one of the first loci to be induced by ecdysterone at the end of larval life, and is essential for translating the hormonal signal into the behavioral and anatomical events which herald the onset of metamorphosis. BR-C products are believed to act by binding to and modifying the transcriptional activities of other hormone-sensitive genes. In addition to abnormalities of the epidermis, BR-C mutants dying during metamorphosis manifest a syndrome of multiple internal tissue defects which represent a failure of the larval-to-adult transition. We have reported features of central nervous system metamorphosis requiring BR-C function, notably morphogenetic movements and optic lobe organization. In this paper we describe defective development of salivary glands, flight muscles, and gut in BR-C mutants, including: persistence of larval salivary glands; failure of the adult salivary glands to extend into the thorax; abnormalities of midgut transition and of proventriculus structure and location; and absence of dorsal-ventral indirect flight muscles. Some of these abnormalities represent defects in programmed cell death. Distinct patterns of phenotypes were seen in mutants of each of the three lethal complementation groups comprising the BR-C. The patterns of phenotypes suggest overlapping but distinct functions encoded by this complex locus.

Notes: Summary InDrosophila imaginal discs, pattern formation requires the activity of three positional information systems, antero-posterior (A/P), dorso-ventral (D/V) and proximo-distal (P/D). Three genes,Decapentaplegic, Distal-less androtund (rn), involved in pattern formation along the P/D axis have been characterized. Thern gene is required in a sub-distal region, localized at a similar position along the P/D axis in all appendages; it encodes two major transcripts, m1.7 and m5.3, both expressed in the central region of all the major imaginal discs. The present study of these transcripts in severalrn mutant favours m5.3 as encodingrn morphogenetic function in the imaginal discs. The fine characterization of its distribution partitions all major imaginal discs in domains along the P/D axis. The ventral and dorsal discs appear to be similarly but not identically organized: two P/D domains are evident in the wing and haltere discs whilst the leg and antenna discs appear to be composed of at least three. We also show that m5.3 is sex-regulated in the genital disc and thatrn function is required for proper development of a sub-distal structure of the female genitalia. This suggests that the primordia of the female genitalia may be organized in a similar way to the other imaginal discs, and strongly supports the hypothesis thatrn function is specific to pattern formation along the P/D axis and that it may be involved in the establishment or maintenance of this pattern.

Notes: Summary We have devised techniques to culture whole, dissected embryos of Drosophila melanogaster. We examine multiple aspects of the morphological and physiological development of the epidermis, musculature, nervous system, and internal organs in this cultured preparation, and show that in vitro development closely parallels normal embryogenesis. These techniques permit a wide range of experimental manipulations during embryogenesis and allow us to extend observations through late embryonic stages, after cuticle deposition. Applications of this technique are presented.

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 We have produced monoclonal and polyclonal antibodies against an antigen that is asymmetrically distributed in mature oocytes of Drosophila melanogaster. During late oogenesis and early embryogenesis the antigen undergoes dramatic changes in its cellular localization: until about 2.5 h before completion of oogenesis it is homogeneously distributed in the cytoplasm, then it becomes localized in granules that are more numerous in posterior than in anterior peripheral positions of the ooplasm. The germ plasm is void of the antigen. Shortly after egg deposition the antigen is released from the granules and forms a shallow temporary gradient in the egg. Later during embryogenesis the antigen is associated with the yolk-containing cytoplasm. At the syncytial blastoderm stage it is also detected in the peripheral nuclei. Preliminary evidence suggests that the antigen is an ecdysteroid-related molecule. Five different anti-ecdysone antisera were found to bind to the same antigen or to an antigen with the same localization as our monoclonal antibody. In pattern mutants affecting anteroposterior polarity, the described asymmetrical distribution of the antigen is abnormal. In the mutant BicD, for example, which leads to the formation of two abdomina of opposite polarity, the antigen-containing granules are distributed homogeneously in mature oocytes.

Notes: Summary Cytoplasm removal/transplant techniques applied to Drosophila cleavage-stage embryos induced changes in anteroposterior polarity. Removal of anterior cytoplasm or anterior transplantation of posterior cytoplasm caused the anterior formation of posterior (telson) structures, and the replacement of anterior cytoplasm with posterior cytoplasm induced double-abdomen embryos, as reported by Frohnhöfer et al. [J Embryol Exp Morphol 97 (suppl):169–179 (1986)]. Changing the conditions of anterior cytoplasm removal we showed that greater volumes, earlier stages, and removal from the periphery were efficient. In addition we found that double-cephalon embryos are induced by replacing posterior cytoplasm with anterior cytoplasm, while removal of posterior cytoplasm or the posterior transplantation of anterior cytoplasm was without effect. However, introduction of anterior cytoplasm into the posterior of nanos embryos, which are mutants not developing abdominal segments, caused the formation of double-cephalon embryos. Similarly, double-abdomen embryos are produced by introducing posterior cytoplasm into the anterior of bicoid embryos, which are mutants not forming cephalic and thoracic structures. These results are compatible with the initial involvement of separate anterior, posterior and terminal cytoplasmic factors deduced from mutant analysis (Nüsslein-Volhard and Roth 1989).

Notes: Summary We have searched for dominant modifiers, i.e., enhancers and suppressors, of the compound eye phenotype of split, a recessive viable allele of Notch. Among the spl modifiers found, we have detected mutations in loci whose functions were previously known to cooperate with Notch in embryonic neurogenesis, such as daughterless, master mind, Delta and Hairless. In addition, other spl modifier mutations have been found in loci that were not previously known to interact with Notch, such as scabrous, glass, roughened eye, and several other genes that have not yet been assigned to known loci. The phenotypes associated with mutations in some of these latter loci suggest the participation of the corresponding genes in embryonic neurogenesis. We show that in some cases the observed interactions are due to genetic haplo-insufficent expression of the genes, whereas allele-specific interactions with spl are observed in master mind and Delta alleles. From this observation, we propose a direct functional association between the proteins encoded by Notch, Delta and master mind.

Notes: Summary Diplo-X flies homozygous for the transform-er-2 ts (tra-2 ts) mutation develop into females at 16° C, while they develop into males at 29° C (Belote and Baker 1982). By means of this conditional mutation, we have carried out a detailed analysis of the development of the genital disc. Temperature shifts between 16 and 29° C, in both directions, and temperature pulses at 29° C, have been applied during the larval growth of tra-2 ts homozygous diplo-X flies, and the external derivatives of the genital disc have been analysed. Genital discs shifted from 16 to 29° C rapidly lose their capacity to differentiate female genital structures, while they become able to differentiate male genital structures whose inventory is more complete the earlier in larval development the temperature shift is carried out; moreover, duplicated male genital structures were observed. In the shift from 29 to 16° C, the genital disc loses its capacity to differentiate male genital structures, while it becomes able to differentiate female genital structures. The inventory of male structures is smaller, and the inventory of the female structures is more complete, the earlier in larval development the temperature is shifted. No duplicated female or male genital structures were observed in the downshift experiment. With respect to the analia, the shift from 16 to 29° C resulted in the quick formation of pure male anal plates, while in the opposite shift the formation of pure female anal plates occurred gradually. Moreover, the time course for the dorsal and ventral anal plates to show normal female phenotype was different: when the dorsal anal plates were completely normal, it was still possible to find incomplete ventral anal plates. In the pulse experiment at 29° C, the genital disc is able to differentiate both female and male genital structures, although the inventory of the latter ones was not complete. In addition, the capacity of the genital disc to differentiate male genital structures depended on the duration of the temperature pulse. The anal plates were always female, although they showed a reduction in their size, the ventral female anal plate being more affected than the dorsal one. No male anal plates were observed. The results have revealed that the genital disc follows a sequence in its capacity to differentiate female or male adult structures. We suggest that this sequence reflects the sequence of determination events occurring in the genital disc during its larval growth. In addition, results shown here provide evidence for the existence in the female genital primordium of a set of cells capable of giving rise either to female genital structures (ventral vaginal plates) or to male genital structures (hypandrium and penis apparatus). We also present evidence supporting the previous idea of two primordia for the anal plates.

Notes: Summary In order to study the regulation of spatial and temporal expression of the homeotic gene Antennapedia (Antp) in Drosophila melanogaster, we have constructed fusion genes which contain Antp sequences linked to the reporter gene lac Z of Escherichia coli. In one case of P-element transformation, a fusion gene construct integrated into the endogenous Antp gene close to one of the two promoters (P1). The spatial expression from the reporter gene in this transformant line, as analysed by the detection of β-galactosidase activity, was found to exactly mimic the normal expression from the P1 promoter of the Antp gene. We have used this unique transformant as a tool for studying the expression of the P1 promoter in embryonic, larval and adult development. Parallel lines transformed with the same fusion gene construct did not confer a correct P1 pattern of expression. The position in the genome was, therefore, crucial for the expression pattern of the reporter gene. Experiments aiming at the detection of autoregulatory control of Antp gene expression were designed. The results did not, however, support models of positive or negative autoregulation of P1 expression by Amp protein.

Notes: Summary Drosophila imaginal disc cell lines show a characteristic pattern of aggregation in culture, which appears to be due to cell-cell rather than cell-substrate interactions. We have examined the distribution of PS integrins in wing and leg cell lines, and find that these integrin homologues are expressed preferentially in aggregates. Cell sheets, small cell clumps and chains of cells express antigen at points of cell-cell contact only.

Notes: Summary Segment polarity genes define the cell states that are required for proper organization of each metameric unit of the Drosophila embryo. Among these, the gooseberry locus has been shown to be composed of two closely related genes which are expressed in an overlapping single-segment periodicity. We have used specific antibodies raised against the protein product of the gooseberry proximal (gsb-p) gene to determine the spatial distribution of this antigen in wild type embryos, and to monitor the effects of segment polarity mutants on the pattern of the gsb-p protein distribution. We find that the gsb-p protein accumulates beneath each posterior axonal commissure in the progeny of neuroblasts deriving from the epidermal compartments of wingless (wg) and engrailed (en) expression. The results of this analysis support the idea that gsb-p has a specific role in the control of cell fates during neurogenesis, and indicate that en and wg provide critical positional cues to define the domain in which gsbp will be activated. Furthermore, these data suggest that, in order to be expressed in the embryonic CNS, gsb-p may preliminarily require activity of the gooseberry-distal gene in the epidermis.

Notes: Summary A blastoderm fate map has been prepared for Drosophila, using mosaics of a temperature-sensitive mutation, shibire (shi). The mutation can cause abnormal flight muscle morphology, inducible only by a short heat pulse in early metamorphosis. Thus muscle lineage and development are unperturbed until the heat pulse in the early pupa. The developmental focus of the shi muscle phenotype maps to the ventral thorax at the expected site of thoracic mesoderm, and probably indicates the blastoderm progenitors of the adult flight muscle. The fate map provides greater detail than previously available for the dorsolongitudinal fibers (DLM) of flight muscle, showing wide separation of the fibers of flight muscle. DLM fibers a and b map close together, and far anterior to fibers e and f, which also map together. On a fate map, common developmental focus indicates a common blastoderm origin. Thus, the observed pattern for DLM fibers suggests that the blastoderm progenitors for each of these syncytial fiber pairs (a, b; e, f) include only one or two cells. It follows that there is usually a single genotype within each fiber pair (a, b; e, f), and that this genotype is directly reflected in the fiber phenotype. In a large number of cases, DLM fibers a and b differ in phenotype from other DLM fibers, in parallel with their other differences (e.g., timing of development in pupa, innervation, motor activity). The separation of fate map locations of the developmental focus for DLM fibers within mesoderm suggests that specific fibers of flight muscle may, in normal development, originate in all three thoracic mesodermal parasegments.

Notes: Summary We have investigated the action of the moulting hormone 20-hydroxy ecdysone (20-HOE) on our leg and wing imaginal disc cell lines. At the morphological level, cells stop dividing and there is some cell death. The remaining cells elongate and aggregate, often producing long processes which form connections between different aggregates. 20-HOE acts within the first one or two days of a passage, at an optimum concentration of 10 ng/ml, this being about 1/100 of the optimum for ecdysone. One cloned wing cell line, C9, has been found to be relatively insensitive to the action of 20-HOE. We have been able to select for resistance to 20-HOE by growing cells in gradually increasing concentrations of hormone followed by passages in hormone-free medium. This has enabled us to isolate a wing cell line C1.8R from its parent cloned line C1.8+. This shows no response to 20-HOE, and cell growth continues even at hormone concentrations as high as 150 ng/ml. We have measured chitin synthesis by the incorporation of radioactive glucosamine into a cell fraction resistant to extensive alkali hydrolysis. The residue was incubated with chitinase, which resulted in a 50% reduction in labelled product. Treatment with 10 ng/ml of 20-HOE dramatically increased chitin synthesis in line C1.8+, but had no effect in the line C1.8R, selected for resistance to hormone.

Notes: Summary We have screened for dominant enhancers and suppressors of the wing phenotype associated with two Delta alleles: Dl 9P39, an amorphic allele, and Dl FE32, an antimorphic allele. The interactions of some of the modifiers with Delta are due to haplo-insufficient expression of the corresponding genes. Although not explicitly shown for the remaining cases, we assume that haploin-sufficiency is also the basis for the relationships of these genes to Delta, since no allele specific interactions were observed. The modifiers found define 22 genes with pleiotropic expression, which can be classified into two groups: genes required for wing vein pattern formation and for neurogenesis, and genes which are not required for neurogenesis. Among the genes of the first group, Hairless and Star were previously known to participate in neural development. One further modifier was found which may correspond to a new neurogenic gene. The second group of genes is larger and includes already known loci, e.g., Plexate, blistered, plexus, etc, as well as other previously unidentified genes, which function during wing morphogenesis.

Notes: Summary Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the β-galactosidase expression pattern in transformed lines carrying different lengths of 5′ flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5′ flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5′ flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5′ flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5′ flanking DNA is not necessary for embryonic survival and development to adult flies.

Notes: Summary In Drosophila, mutations in a class of genes, the neurogenic genes, produce an excess of neurons. This neural hyperplasia has been attributed to the formation of more than the normal number of neuronal precursor cells at the expense of epidermal cells. In order to find out whether the neurogenic genes only act at this intial step of neurogenesis, we studied the replication pattern of the sensory organ precursor cells by monitoring BrdU incorporation in embryos mutant for Notch (N), Delta (Dl), mastermind (mam), almondex (amx), neuralized (neu), big brain (bib) and the Enhancer of split-Complex (E(spl)-C). Using temperature sensitive alleles of two of the neurogenic genes, DI and N, we also induced an acute increase of replicating sensory precursors by shifting briefly to the restricted temperature. We have found that the loss of function of all the seven neurogenic loci that were tested causes an increase in replicating sensory precursor cells, consistent with the model that these neurogenic genes normally participate in the process of restricting the number of neuronal precursors. Whereas the temporal pattern of replication appeared normal in mutants of five of the seven neurogenic loci, in N and mam embryos replicating PNS cells are present beyond the time when they normally undergo replication. Experiments with colchicine suggest that many of these late replicating cells may be newly emerging precursors and probably not additional cell divisions of already recruited precursors. Thus, different neurogenic genes may be required over different periods of time for the specification of sensory precursor cells.

Notes: Abstract We have followed the normal development of the different cell types associated with the Drosophila dorsal vessel, i.e. cardioblasts, pericardial cells, alary muscles, lymph gland and ring gland, by using several tissue-specific markers and transmission electron microscopy. Precursors of pericardial cells and cardioblasts split as two longitudinal rows of cells from the lateral mesoderm of segments T2-A7 (“cardiogenic region”) during stage 12. The lymph gland and dorsal part of the ring gland (corpus allatum) originate from clusters of lateral mesodermal cells located in T3 and T1/dorsal ridge, respectively. Cardioblast precursors are strictly segmentally organized; each of T2-A6 gives rise to six cardioblasts. While moving dorsally during the stages leading up to dorsal closure, cardioblast precursors become flattened, polarized cells aligned in a regular longitudinal row. At dorsal closure, the leading edges of the cardioblast precursors meet their contralateral counterparts. The lumen of the dorsal vessel is formed when the trailing edges of the cardioblast precursors of either side bend around and contact each other. The amnioserosa invaginates during dorsal closure and is transiently attached to the cardioblasts; however, it does not contribute to the cells associated with the dorsal vessel and degenerates during late embryogenesis. We describe ultrastructural characteristics of cardioblast differentiation and discuss similarities between cardioblast development and capillary differentiation in vertebrates.

Notes: Abstract We analysed spatial patterns of expression of a lacZ reporter gene in the gut of Drosophila larvae that had been transformed with a P-element-lacZ vector to identify regional differences in gene expression. lacZ-positive epithelial cells formed distinct domains with discrete transverse and longitudinal boundaries along the gut tube. Boundaries were often found at sites at which morphological boundaries were not obvious. The gut epithelium was subdivided into 36 compartments by the boundaries. We refer to these novel compartments as “tissue compartments”. The lacZ-positive domain of each strain appeared as a single tissue compartment or as a combination of several tissue compartments. The tissue compartment is considered to be a unit of regional differentiation. The spatial organization of the tissue compartments may represent the “floor plan”, determined by genes that control the regional differentiation of this nonsegmental organ.

Notes: Abstract The sensory precursors for labellar taste bristles develop from the labial disc in three distinct temporal waves occurring at 0 h, 8 h and 14 h of pupal development. In each temporal wave, transcripts for the achaete (ac) and scute (sc) genes are expressed in overlapping patterns in cells of the disc epithelium prior to the appearance of sensory mother cells (SMCs). No bristles form in mutant flies in which the ac and sc genes are absent. When the sc gene alone is deleted, a set of seven bristles fail to form. Pulses of ubiquitous sc + expression during pupal development, in a strain mutant for both ac and sc, can result in flies with all the labellar bristles at their correct positions. sc + pulses at times corresponding to the initiation of each of the waves of SMC specification in the disc was sufficient to restore bristle pattern. Bristles were not induced at ectopic positions and times as a result of the ubiquitous expression of sc +. These results suggest that the proneural genes ac and sc do not themselves set the pattern of the labellar bristles. Instead, they are required for the elaboration of the pattern set by other gene products. We also show that the formation and positioning of the later waves of bristles can take place even in the absence of bristles normally specified earlier.

Notes: Abstract Using intracellular horseradish peroxidase injection we traced the developmental fate of early gastrula cells of the procephalic region in the stage 16/17 embryo. Morphogenetic movements in the developing brain are described in three dimensions. The results are related to head segmentation, and an early gastrula fate map of pregnathal head segments is proposed.

Notes: Abstract Glial cells are of significant importance for central nervous system development and function. In insects, knowledge of the types and development of CNS glia is rather low. This is especially true for postembryonic glial development. Using bromodeoxyuridine incorporation and enhancer trap lines we identified a reproducible spatial and temporal pattern of DNA replicating cells in the abdominal larval CNS (A3-7 neuromeres) of Drosophila melanogaster. These cells correspond to embryonically established glial cells in that region. Except for a specific subfraction, these cells apparently do not divide during larval life. Similar patterns were found in two other Drosophila species, D. virilis and D. hydei.