The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Conservation of gene order, structure and sequence between three closely linked genes in Drosophila melanogaster and Drosophila virilis

In Drosophila melanogaster, the apparently unrelated genes anon-66Da, RpL14, and anon-66Db (from telomere to centromere) are located on a 5547 bp genomic fragment on chromosome arm 3L at cytological position 66D8. The three genes are tightly linked, and flanked by two relatively large genes with unknown function. We have taken a comparative genomic approach to investigate the evolutionary history of the three genes. To this end we isolated a Drosophila virilis 7.3 kb genomic fragment which is homologous to a 5.5 kb genomic region of D. melanogaster. Both fragments map to Muller's element D, namely to section 66D in D. melanogaster and to section 32E in D. virilis, and harbor the genes anon-66Da, RpL14, and anon-66Db. We demonstrate that the three genes exhibit a high conservation of gene topography in general and in detail. While most introns and intergenic regions reveal sequence divergences, there are, however, a number of interspersed conserved sequence motifs. In particular, two introns of the RpL14 gene contain a short, highly conserved 60 nt long sequence located at corresponding positions. This sequence represents a novel Drosophila small nucleolar RNA, which is homologous to human U49. Whereas DNA flanking the three genes shows no significant interspecies homologies, the 3'-flanking region in D. virilis contains sequences from the transposable element Penelope. The Penelope family of transposable elements has been shown to promote chromosomal rearrangements in the D. virilis species group. The presence of Penelope sequences in the D. virilis 7.3 kb genomic fragment may be indicative for a transposon-induced event of transposition which did not yet scramble the order of the three genes but led to the breakdown of sequence identity of the flanking DNA.     

Resolving the phylogenetic relationships and evolutionary history of the Drosophila virilis group using multilocus data

The Drosophila virilis group is one of the major lineages of Drosophila previously recognised and it has been used as a model for different types of studies. It comprises 13 species whose phylogenetic relationships are not well resolved. In the present study, six nuclear genes (Adh, fused, Gpdh, NonA, CG9631 and CG7219) and the mitochondrial ribosomal RNA genes (12S-16S) have been used to estimate the evolutionary tree of the group using different methods of phylogenetic reconstruction. Different competing evolutionary hypotheses have also been compared using the Approximately Unbiased test to further evaluate the robustness of the inferred trees. Results are, in general, consistent with previous studies in recovering the four major lineages of the group (D. virilis phylad, Drosophila montana subphylad, Drosophila kanekoi subphylad and Drosophila littoralis subphylad), although D. kanekoi, D. littoralis and Drosophila ezoana are here inferred to be more closely related to the D. virilis phylad than to the D. montana subphylad. The age of the crown group, estimated with a Bayesian method that assumes a relaxed molecular clock, is placed in the late Miocene (～ 10 Mya). The oldest lineages also appeared during this period (～ 7.5 to ～ 8.9 Mya), while the ages of the basal nodes of the montana subphylad and the virilis phylad are located in the early Pliocene (～ 4.9 and ～ 4.1 Mya). Major cladogenesis events correlate to geological and palaeoclimatic occurrences that most likely affected the freshwater and deciduous forests where these species are found. The inferred biogeographical history of the group, based on the statistical dispersal-vicariance analysis, indicates that the last common ancestor of the group had a Holarctic distribution from which the North American and the Eurasian lineages evolved as a result of a vicariant event.     

The tradeoff between reproductive and food resources in the crayfish Orconectes virilis

The conflict between mating and foraging is one of the many tradeoffs associated with reproduction. The relative attraction to sex pheromone and food stimuli by male virile crayfish (Orconectes virilis) were tested using two experiments. In the first experiment, the responses by males to pheromone and food stimuli alone were assessed. In the second experiment, males were exposed to both pheromone and food stimuli in a choice maze. To determine the effects of feeding motivation, the males in the choice experiment were fed either one day or seven days prior to observation. Male crayfish exhibited similar responses to the pheromone and food stimuli individually. Males exhibited a preference for the pheromone stimulus over the food stimulus in the choice trials, and that preference that was not influenced by starvation.     

Strategic differences in thermal adaptation between two Drosophila species,D. virilis and D. immigrans

Summary Preadult viability and developmental time at four different temperatures, heat and cold resistances of adult flies, effects of acclimatization on heat resistance, and preferred temperature of adult flies were compared between two species of Drosophila, D. virilis and D. immigrans. Four Japanese local populations were surveyed for each species. As compared with immigrans, virilis was higher in its ability to tolerate both heat and cold stresses and was viable over a broader temperature range. On the other hand, immigrans revealed a superior ability to acclimatize and a rigid preference for gradually changing thermal environment. Differences between geographical populations are remarkable for heat tolerance in virilis and cold tolerance in immigrans. In conclusion, thermal adaptation of virilis seems to be based on the high tolerance to extreme temperatures and that of immigrans mainly on the behavioural preference for viable temperatures.     

Drosophila virilis and its cosmopolitan relatives in urban islands

Populations of brewery Drosophila have been studied for 2 years. Species distributions diner between breweries, and are very different from those found in woodland and fruitmarkets. It is suggested that breweries form 'urban islands'. One of the commonest species found in breweries appears to be Drosophila virilis , which has not previously been found in Britain. Tests on various characteristics, including courtship song, have shown conclusively that this species is a strain of D. virilis.     

The structure of the normal puparium and in segmentation disorders in Drosophila

In Drosophila puparium is a cuticular mould of external structures formed by the larval cells and can, therefore, preserve traces of developmental defects which appeared in embryogenesis. In this way ontogenetic relationship between embryonic defects and developmental defects in adult individuals can be established. The normal pattern of segmentation in puparia of Drosophila melanogaster was established by comparing segmentation defects in adult flies and corresponding puparia. Boundaries of segment and compartments have been determined, imaginal rudiments of abdominal segments (nests of histoblasts) and rudiments of trachea have been localized on puparia.     

Purification and enzyme stability of alcohol dehydrogenase from Drosophila simulans, Drosophila virilis and Drosophila melanogaster adhS.

Three alcohol dehydrogenases from Drosophila simulans, Drosophila virillis and Drosophila melanogaster adhS (which possesses an alloenzyme with slow electrophoretic mobility) were purified essentially to homogeneity. The purification procedure involves a new step of affinity chromatography, which efficiently lowers the amount of contaminants in the final preparation, producing a very stable enzyme. The purification procedure developed consists of a salmine sulphate precipitation, two CM-Sepharose CL-6B colume-chromatography steps, an affinity-chromatography step and a Sephacryl gel filtration. A minimum of 30-fold purification is obtained and the yield is not less than 34%. The isoelectric points and molar absorption coefficients were determined.     

Activity of phenol oxidases at the puparium formation stage in development of nineteen lozenge mutants of Drosophila melanogaster

Phenol oxidase activity of activated A₁ and A₂ (A₃) electrophoretic components from 19 lozenge mutant and three lozenge double mutant strains was compared to that of wild type flies. Melanin production by the activated A components with tyrosine as substrate was compared to activity in the same acrylamide gels with dopa as substrate. Melanin production decreased, first in the A₁ band and then in the A₂ (A₃) band, with increased morphological expression of the mutant genes. No melanin bands were obtained with either substrate in five of the more severely affected mutants. A possible correlation between phenol oxidase activity and quinone production necessary for normal development of eyes, female accessory sex organs, and claws is discussed.Supported by PHS grant AM-08331-05.     

Inheritance of cold shock tolerance in hybrids of Drosophila virilis and Drosophila lummei

The genetic basis of the difference in cold shock tolerance between the southern temperate Drosophila virilis and its boreal relative D. lummei is studied. After adult eclosion, the parental stocks, reciprocal F₁ and backcross hybrids were pretreated for eight days at 18°C or at 6°C. The cold shock used consisted of fast cooling to-10°C and exposure to this temperature for varying lengths of time. D. lummei tolerated such exposure for 40–50% longer than did D. virilis (100–135% after acclimation). Reciprocal F₁ females, differing only in their maternal cytoplasm deviated significantly from each other, and the reciprocal F₁ males even more so, the contribution of the X chromosome being three to four times that of the cytoplasm. The cold resistance scores of the hybrid males were more extreme than those of the parental stocks. Autosomally heterozygous males with the X chromosome and cytoplasm of virilis were the weakest flies studied. The reciprocal males (X chromosome and cytoplasm of lummei) survived better than the parental lummei stock. The reciprocal differences decreased after cold temperature acclimation. The roles of the four major autosomes were analyzed by backcrossing the reciprocal F₁ males with females of the virilis marker stock. The third chromosome of lummei as heterozygous contributed most to cold tolerance, while the other autosomes had a rather weak effect in the opposite direction (virilis homozygotes survived better), which disappeared after acclimation at 6°C. Some of the cold susceptibility of F₁ hybrids disappeared in chromosomally identical backcross flies, indicating complex cytoplasmchromosomal interactions.     

Ovarian defects in hybrids of the species pair Drosophila virilis and Drosophila texana

In interspecific matings between Drosophila virilis and Drosophila texana female sterility is observed in F₂ hybrid females. A previous study has shown that no vitellogenin synthesis occurs in the fat body of sterile hybrid females. The results presented in this paper show that hybrid ovaries of sterile females transplanted into the abdomens of females of the parental species are not able to develop upon maturity. With few exceptions, the hybrid ovaries remained alive in the host environment, but their oocytes failed to develop to vitellogenic stages. Thus, in hybrid females between Drosophila virilis and Drosophila texana sterility is the result of defects in both the two main developmental processes of egg maturation, the synthesis of vitellogenins in the fat body and the uptake of vitellogenins by the ovary. Dev Genet 20:47–52, 1997. © 1997 Wiley-Liss, Inc.     

Vitellogenetic defects in hybrids of the species pair Drosophila virilis and Drosophila texana

In interspecific matings between the species Drosophila virilis and Drosophila texana, female sterility can be observed in F₂ backcross females and in F₂ hybrid females. The results presented in this report show that the female sterility, whenever it exists, is due to prevention of vitellogenin synthesis in the fat body, but other abnormalities such as defects with the hybrid ovaries are not excluded. The observation that sterility appears among females from backcrosses suggests that incompatibilities between interspecific genes may cause female sterility even in the presence of a complete habloid genome from one or the other species. Yet, the parallel observation that female sterility appears only in hybrid females with recombinant chromosomes indicates that sterility results when conspecific combinations of genes on the same chromosome are broken by interspecific recombination. © 1996 Wiley-Liss, Inc.     

A telomeric satellite in Drosophila virilis and its sibling species

Telomere elongation by telomerase is the most widespread mechanism among eukaryotes. However, alternative mechanisms such as homologous recombination between terminal satellite DNAs are probably used in lower dipteran insects and in some plants. Drosophila melanogaster uses the very unusual telomere elongation pathway of transposition of telomere-specific retrotransposable elements. The uniqueness of this telomere elongation mechanism raises the question of its origin. We, therefore, analyzed sequences located at telomeres of fairly distantly related Drosophila species, and in this paper we describe the characterization of complex satellite DNA sequences located at the telomeres of D. virilis and other species in the virilis group. We suggest an involvement of these DNA satellites in telomere elongation by homologous recombination similar to that found in lower dipterans. Our findings raise the possibility that telomere elongation by specific retrotransposons as found in D. melanogaster and its sibling species is a recent event in the evolution of dipteran insects.     

Temporal relationships between biotransformation, detoxication, and chlordecone potentiation of chloroform-induced hepatotoxicity

Exposure to chlordecone (CD, Kepone) is known to increase the hepatotoxicity of chloroform (CHCl3) in rats. A time-course analysis was conducted relating several indices of biotransformation capacity with the ability of CD to potentiate CHCl3-induced hepatotoxicity. Male Sprague-Dawley rats were given a single administration of corn oil alone or CD (50 mg/kg, po) dissolved in corn oil. At 2, 4, 8, 16, 20, 24, or 32 days posttreatment, groups of rats were killed and their livers were analyzed for (i) cytochrome P-450, NADPH-dependent cytochrome c reductase, cytochrome b5 and glutathione content or (ii) in vitro irreversible binding of 14CHCl3-derived radiolabel to microsomal protein. Similarly treated rats were challenged (2-32 days posttreatment) with CHCl3 (0.5 mL/kg po); 24 h later, liver damage was assessed by plasma alanine aminotransferase (ALT), plasma ornithine carbamyl transferase (OCT), plasma bilirubin, and hepatic glucose-6-phosphatase. CD potentiation was maximal 2 days posttreatment; and enhanced susceptibility to CHCl3 persisted up to 20-24 days post-CD treatment. In a parallel study animals treated with chlordecone were killed 8, 16, 20, 24, or 32 days later. Blood, kidney, liver, and adipose tissue samples were taken and analyzed for chlordecone content. The results suggest that a general temporal correlation exists between biotransformation rate (microsomal 14C binding), chlordecone content, and the severity of liver injury; the other parameters monitored do not appear to relate directly to the potentiation.     

Mitochondrial DNA in Drosophila. An analysis of genome organization and transcription in Drosophila melanogaster and Drosophila virilis

Transfer RNAs of Escherichia coli were separated by two-dimensional polyacrylamide gel electrophoresis, and the relative abundance of each of the 26 known tRNAs thus separated was measured on the basis of molecular numbers in cells. Based on this relative abundance, the distributions of cognate codons in E. coli genes (lacI, rpA, asnA, recA, lpp and four ribosomal protein genes) and in coliphage (MS2, φX174 and λ) genes were examined. A strong positive correlation between the tRNA abundance and the choice of codons, among both synonymous codons and those corresponding to different amino acids, was found for all E. coli protein genes that had been sequenced completely. However, the correlation was less significant for the phage genes. The relationship between tRNA abundance and its usage (namely anticodon usage) was examined by regression analysis. The degree of the relationship found for individual E. coli genes differed from gene to gene: those of r-protein genes and recA were higher than those of trpA, lacI and asnA. The dependent relationship of tRNA usage on its content for the first two genes seems to be greater than that expected from the proportional relationship between the two variables; i.e. these genes selectively use codons corresponding to major tRNAs but nearly avoid using those of minor tRNAs.