The ribosomal DNA of Calliphora erythrocephala: The cistron classes of total genomic DNA

The composition of the genome set of ribosomal DNA cistrons in Calliphora erythrocephala (a Dipteran fly) has been analyzed. In contrast to previously cloned fragments of the rDNA (see Beckingham & White, 1980), the great majority of the rDNA cistrons do not contain introns in the 28 S β coding region. In the strain of flies studied, however, most cistrons fall into two discrete length classes that are present in approximately equal amounts in the genome. These results from distinct size variants of the non-transcribed spacer in the cistron population.The major genome class of intron-containing (intron⁺) rDNA cistrons was found to constitute approximately 5% of all cistrons and to contain introns of 6·1 × 10³ base-pairs. Interestingly, the intron⁺ cistrons were shown to be clustered within the rDNA and to contain a different population of non-transcribed spacer/external transcribed spacer (NTS + ETS) regions to that seen amongst the intron^? cistrons. The implications of these findings in relation to the mechanisms that maintain homogeneity within tandemly repeated gene sets are discussed.Some evidence for the existence of intron sequence DNA outside the rDNA is presented.     

The metabolism of 3H-moulting hormone in Calliphora erythrocephala during larval development

The activity in whole insects for converting ³H-α-ecdysone to ³H-β-ecdysone after injection is low (half-maximal) in young last instar larvae, maximal in mature larvae, and minimal (fourth-maximal) at the white puparial stage. Because moulting hormone titre is low throughout the last larval instar and increases at the formation of the puparium it appears that hydroxylation at C-20 is not a key step in regulating β-ecdysone biosynthesis during larval development.The activity for catabolizing ³H-β-ecdysone is maximal in second instar larvae, about thirdmaximal throughout most of the third instar, and minimal at pupariation (thirtieth-maximal). Thus inactivation may play a rôle in regulating moulting hormone titre during larval development.     

In vitro synthesis of peritrophic membranes of the blowfly, Calliphora erythrocephala.

Tyrode solution containing added glutamine and Leloup's medium 1 has been used as a basic medium for the in vitro culture of the so-called proventriculus of adult Calliphora erythrocephala to elucidate some of the factors controlling the synthesis of peritrophic membranes (PM) in vitro. The formation rate was chosen as a quantitative criterion for the evaluation of the modifications of the incubation media.After systematic variation of osmolarity, pH, and temperature optimal formation rates were obtained in media with an osmolarity of 320 to 360 mOsmol, a pH of 6·8, and an incubation temperature of 27°C. Under these conditions the average rate of formation was in the modified Tyrode solution 3·0±1·1 mm PM/hr, and in Leloup's medium 3·6±0·8 mm PM/hr. In the modified Tyrode solution the formation of PM was complete after 5 to 7 hr, whereas in Leloup's medium it continued up to 24 hr. The addition of β-ecdysone caused an increase of the formation rate of PM to 4·5 to 5·5 mm PM/hr.The results obtained led to the hypothesis that an osmotically regulated enzyme system could be the limiting factor of the formation rate of peritrophic membranes, i.e. a system which could regulate the internal osmolarity of the formative cells by the interconversion of a bulk polymer and its monomer which are needed for the synthesis of PM.     

Juvenile hormone and juvenile hormone mimics inhibit proliferation in a lepidopteran imaginal disc cell line

The action of juvenile hormone (JH) and JH mimics have been examined in vitro by utilizing the imaginal disc-derived cell line, IAL-PID2. We have discovered that the cell line was responsive to JH and a variety of JH mimics. The most consistent response obtained in our studies was inhibition of cell proliferation, in the absence of 20-hydroxyecdysone (20E), which characteristically reduces cell proliferation in its own right in this cell line. JH-I, JH-III, methoprene, fenoxycarb, and farnesol significantly inhibited cell proliferation after 3 days of exposure of the cells in vitro to each of the compounds. Linoleic acid controls had no effect on proliferation in the cultures. The cell proliferation assay demonstrates the JH responsiveness of this cell line, but the concentrations of JH required were high compared to the concentrations of 20E needed for inhibition of proliferation in these cells.     

Effects of ecdysone and juvenile hormone on DNA metabolism of imaginal disks of Drosophila melanogaster

Imaginal disks of Drosophila melanogaster isolated en masse and incubated in Robb's tissue culture medium incorporate ³H-thymidine into nuclear DNA. Both α- and β-ecdysone stimulate the rate of ³H-thymidine incorporation into disk DNA. Concentrations of ecdysone that induce complete evagination of disks in vitro cause the initiation of DNA synthesis in some disk cells. Juvenile hormone has no effect on DNA synthesis in control disks. However, juvenile hormone blocks the ecdysone stimulation of DNA synthesis. It is proposed that juvenile hormone and ecdysone act in a balanced fashion to regulate DNA synthesis in imaginal disks.     

Effects of polyoxin D on in vitro synthesis of peritrophic membranes in Calliphora erythrocephala

The synthesis of peritrophic membranes in vitro in adult Calliphora erythrocephala is affected strongly by polyoxin D, an inhibitor of chitin synthesis. Depending on the polyoxin concentrations used, a reduction in membrane dry weight, an inhibition of chitin synthesis, and prominent changes in the fine structure of the membranes were found.     

Ocellar interneurones in the blowfly Calliphora erythrocephala: Morphology and central projections

Summary The morphology and central projections of first-order ocellar interneurones were analysed in the blowfly, Calliphora erythrocephala after cobalt and horseradish-peroxidase labelling. Three classes of interneurones can be distinguished on the basis of axon diameters: large, medium and small neurones. In total there are 12 large, 10 medium and an unknown number of small interneurones. These interneurones connect the fused first-order ocellar neuropil (underlying the three ocelli) with various areas of the central nervous system. The large neurones terminate in three subregions of the posterior slope (ocellar foci); the medium neurones arborise in several regions of the lateral protocerebrum, in the posterior slope, the lobula, the ventral medulla, and in the pro- and mesothoracic ganglia. The thin fibers arborise in all the above regions (except in the thoracic ganglia), and in addition in the neuropil dorsal to the oesophagus and antero-ventral to posterior slope (tritocerebrum). The anatomy of the ocellar pathway in C. erythrocephala is compared with those in other studied insects. Possible interactions between ocellar interneurones and other pathways are discussed.     

Length variation in the non-transcribed spacer of Calliphora erythrocephala ribosomal DNA is due to a 350 base-pair repeat

The non-transcribed spacer regions in the ribosomal DNA cistrons of Calliphora erythrocephala vary in length. This length variation is shown to be due to variable numbers of small repeated units found in certain areas of the NTS² regions, as has been found in several other systems. In contrast to the other organisms, however, the length variation in C. erythrocephala leads to the formation of only two major size classes with a length difference of about 1 kb. We have investigated the nature of this length difference by means of electron microscope heteroduplex and restriction enzyme digestion analyses. We demonstrate that the length variation in C. erythrocephala is due to a variation in the number of 350 bp repeating units defined by the presence of sites for three restriction enzymes, HhaI, Sau3A and AluI. Furthermore, the existence of an XbaI site within one 350 bp unit and the lack of a HhaI restriction site within another 350 bp unit reveals that at least some sequence differences exist among the repeating units. These restriction site differences can be viewed as genetic markers and lead to the hypothesis that the longer NTS class originated from the shorter NTS class by an unequal crossover event that served to duplicate the region containing repetitive units. An interesting feature of our observation is that not all NTS length classes are equally represented, suggesting special rules for the unequal recombination events often hypothesized as the basis for such variation.     

Juvenile hormone acid methyltransferase activity in imaginal discs of Manduca sexta prepupae

The occurrence of a peak of juvenile hormone (JH) during the prepupal period has been noted in several lepidopterans. In Manduca sexta and Hyalophora cecropia this peak is known to prevent the precocious onset of adult differentiation in imaginal tissues. However, it has previously been observed in our laboratory that corpora allata (CA) of this age are incapable of making JH owing to a lack of the terminal synthetic enzyme, juvenile hormone acid methyltransferase (JHAMT). Since the CA are required for normal pupation, it is likely that JH acid is the product released by the prepupal CA. Therefore, we analyzed whether JH acid treatment would prevent precocious adultoid differentiation in allatectomized M. sexta larvae. JH acid injections were found to be as effective as JH in normalizing pupation, and acted in a time- and dose-dependent manner. This finding led to a question of whether injected or endogenous JH acid could be methylated to JH. Homogenates of several tissues from prepupae were assayed for the presence of JHAMT. Of the tissues assayed, only imaginal discs possessed significant levels of the enzyme. These results support our previously proposed mechanism for production of the prepupal JH peak in M. sexta.     

Protein synthesis in imaginal disks ofPlodia interpunctella during development in vivo and in vitro

Summary Wing imaginal disks were dissected from larvae ofPlodia interpunctella (Hübner) at various stages during the larval-pupal transformation. The wing-disk proteins separated by electrophoresis and scanned with a densitometer changed quantitatively but not qualitatively during development in vivo. Treatment of wing disks in vitro with β-ecdysone resulted in a 2-fold increase in synthesis of proteins after only 2 hr incubation. The maximum rate of protein synthesis was reached 16 hr after treatment with hormone. The pattern of proteins separated by electrophoresis of wing disks that were incubated in vitro with β-ecdysone did not change qualitatively. The major features of protein synthesis during wing-disk development in vivo were similar to those observed during β-ecdysone-induced development in vitro. Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA.     

Changes in acid mucopolysaccharides during the development of the blowfly, Calliphora erythrocephala.

Acid mucopolysaccharides have been isolated from different developmental stages of Calliphora. Hyaluronic acid and a ‘larval AMPS’ were identified during all developmental stages. During the later part of the development chondroitin and a poorly-sulphated keratin sulphate-like compound were also present. Chondroitin sulphate and heparan sulphate could be detected at all development stages and during the latter part possibly keratin sulphate. The variation of acid mucopolysaccharides during development is discussed.     

Ecdysteroid Titres and Cuticle Depositions in Embryos of the Dipteran Calliphora erythrocephala

Summary

Before the shortening of the germ band, embryos of Calliphora erythrocephala secrete an electron dense layer which resembles a thin atypical cuticle. After completion of the dorsal closure, a second, typical, cuticle is deposited. It is characterized by a thick procuticle and by the presence of hooked setae. This cuticle develops into the larval cuticle of the first instar.

We did not find in newly-laid eggs of Calliphora detectable amounts of either free or hydrolysable conjugated ecdysteroids. A marked rise in ecdysteroid concentrations, essentially attributable to free ecdysone and 20- hydroxyecdysone, occurs shortly before the deposition of the typical cuticle. After a transient decrease, the ecdysteroid titre rises again before hatching.

When eggs are mid-ligatured at blastula or early gastrula stages, the posterior embryonic half is able to build up a typical cuticle with hooked setae. This cuticulogenesis is therefore not dependent on the presence of the embryonic ring glands.