Differential regulation of duplicate alcohol dehydrogenase genes in Drosophila mojavensis

These studies report the existence of multiple forms of alcohol dehydrogenase in extracts of Drosophila mojavensis. The existence of these forms can be best explained by the hypothesis of a duplication for the Adh locus in D. mojavensis. Electrophoretic variants at each locus have been identified and crosses between individuals carrying alternative alleles at each locus result in F1 progeny with six bands of ADH. This pattern is consistent with these individuals being heterozygous at two loci. The loci have been named Adh-1 and Adh-2. Examination of the isozyme content during development shows that the two Adh genes are not coordinately controlled but have separate developmental programs. In embryos and first and second instar larvae only Adh-1 is expressed. At about the time of the second molt Adh-2 expression commences in some of the same cells that previously expressed and continue to express Adh-1. This is evidenced by the existence of an interlocus heterodimer in third instar larvae. Both genes are expressed throughout pupation. Shortly after emergence Adh-1 expression declines. In mature males only ADH-2 is present. In mature females both Adh-1 and Adh-2 are expressed but not in the same cells since the interlocus heterodimer is absent. Examination of specific tissues reveals that most of the larval ADH is found in fat body cells and as in most tissues of third instar larvae both Adh-1 and Adh-2 are expressed. The single exception appears to be larval gut which contains ADH-1 but little if any ADH-2. In mature males and female flies all ADH containing tissues have only ADH-2. However, mature ovaries contain substantial quantities of ADH-1 which is apparently deposited into eggs. Given the extensive amount of available information on the Adh gene-enzyme system of D. melanogaster and the tools that can be applied to the analysis of homologous systems, the ADH duplication of D. mojavensis, and its regulation may be a useful one for studying differential gene regulation in specific cell types.     

Ecological genetics of the Adh-1 locus of Drosophila buzzatii

The Adh-1 polymorphism of Drosophila buzzatii was studied in terms of the effects of the larval substrate (laboratory food and Opuntia stricta tissue inoculated with live yeast species) on larval survival, development time and subsequent adult size, using the three common Adh-1 genotypes. Studies with laboratory food yielded mixed results, but in general the Adh-1^b/Adh-1^b genotype was superior to Adh-1^c/Adh-1^c with the heterozygote intermediate. Studies with the natural substrate (Opuntia stricta tissue) using mono- and bicultures of four associated yeast species also showed that the Adh-1^b/Adh-1^b genotype develops faster and survives better than Adh-1^c/Adh-1^c. There were no genotype-by-yeast interactions which might explain the maintenance of the polymorphism. Drosophila buzzatii larvae develop faster and attain larger adult sizes when raised on bicultures of yeasts as compared with the corresponding monocultures.     

Sequence variation of alcohol dehydrogenase (Adh) paralogs in cactophilic Drosophila

This study focuses on the population genetics of alcohol dehydrogenase (Adh) in cactophilic Drosophila. Drosophila mojavensis and D. arizonae utilize cactus hosts, and each host contains a characteristic mixture of alcohol compounds. In these Drosophila species there are two functional Adh loci, an adult form (Adh-2) and a larval and ovarian form (Adh-1). Overall, the greater level of variation segregating in D. arizonae than in D. mojavensis suggests a larger population size for D. arizonae. There are markedly different patterns of variation between the paralogs across both species. A 16-bp intron haplotype segregates in both species at Adh-2, apparently the product of an ancient gene conversion event between the paralogs, which suggests that there is selection for the maintenance of the intron structure possibly for the maintenance of pre-mRNA structure. We observe a pattern of variation consistent with adaptive protein evolution in the D. mojavensis lineage at Adh-1, suggesting that the cactus host shift that occurred in the divergence of D. mojavensis from D. arizonae had an effect on the evolution of the larval expressed paralog. Contrary to previous work we estimate a recent time for both the divergence of D. mojavensis and D. arizonae (2.4 +/- 0.7 MY) and the age of the gene duplication (3.95 +/- 0.45 MY).     

Regulatory elements involved in Drosophila Adh gene expression are conserved in divergent species and separate elements mediate expression in different tissues.

Alcohol dehydrogenase (ADH) is expressed in a complex temporal and spatial pattern from tandem promoters (proximal and distal) in Drosophila melanogaster, and from two closely linked genes (Adh-1 and Adh-2) in D. mulleri. The expression patterns of Adh-1 and the proximal promoter, and Adh-2 and the distal promoter are similar, but not identical. We show that the mulleri Adh genes are appropriately expressed when introduced into the melanogaster genome, indicating that the cis- and trans-acting elements which regulate the corresponding promoters are functionally equivalent in the two species. By analyzing the expression of in vitro generated mutants of the mulleri Adh locus, we define at least three regulatory regions of the mulleri Adh genes and show that different control elements mediate the expression of Adh in different tissues.     

Gene expression and lysosomal content of silkworm peritracheal athrocytes

To examine the function of silkworm Bombyx mori L. athrocytes (nephrocytes), we constructed cDNAs of larval peritracheal athrocytes that were anatomically isolated from surrounding tissues. Larval expression levels of genes encoding hemolymph proteins, such as arylphorin, the 30K proteins, and lysozyme, were lower in peritracheal athrocytes than in the fat body, whereas genes involved in protein degradation were highly expressed in athrocytes. Real time RT-PCR revealed that a member of the Hsp40/Dnaj protein family, DjA2 (also known as Rdj2, Dj3, Dnj3, Cpr3, and Hirip4), an endocytic gene, was highly expressed in the peritracheal athrocytes compared to the fat body. Homologs of the Drosophila ATG1, ATG5, ATG6, and ATG8 genes had high expression levels in the peritracheal athrocytes. Observations using laser confocal microscopy with lysosomal fluorescent probes showed that silkworm athrocytes, including pericardial cells, suboesophageal body, and peritracheal athrocytes, were rich in lysosomes, in contrast to other tissues. Peritracheal athrocytes had lysotracker-positive spots at all times from the fourth larval molt to the pupa. Of these, molting larval and pupal peritracheal athrocytes had larger spots. Starvation for 24h induced greater lysotracker staining, but the number of spots decreased. Silkworm peritracheal athrocytes are lysosome-rich tissues and may function in the degradation of proteins.     

Hid can induce, but is not required for autophagy in polyploid larval Drosophila tissues

The major cell death pathways are apoptosis and autophagy-type cell death in Drosophila. Overexpression of proapoptotic genes in developing imaginal tissues leads to the activation of caspases and apoptosis, but most of them show no effect on the polytenic cells of the fat body during the last larval stage. Surprisingly, overexpression of Hid induces caspase-independent autophagy in the fat body, as well as in most other larval tissues tested. Hid mutation results in inhibition of salivary gland cell death, but the disintegration of the larval midgut is not affected. Electron microscopy shows that autophagy is normally induced in fat body, midgut and salivary gland cells of homozygous mutant larvae, suggesting that Hid is not required for autophagy itself. Constitutive expression of the caspase inhibitor p35 produces identical phenotypes. Our results show that the large, post-mitotic larval cells do not react or activate autophagy in response to the same strong apoptotic stimuli that trigger apoptosis in small, mitotically active imaginal disc cells.     

SNF4Agamma, the Drosophila AMPK gamma subunit is required for regulation of developmental and stress-induced autophagy

In holometabolous insects including Drosophila melanogaster a wave of autophagy triggered by 20-hydroxyecdysone is observed in the larval tissues during the third larval stage of metamorphosis. We used this model system to study the genetic regulation of autophagy. We performed a genetic screen to select P-element insertions that affect autophagy in the larval fat body. Light and electron microscopy of one of the isolated mutants (l(3)S005042) revealed the absence of autophagic vesicles in their fat body cells during the third larval stage. We show that formation of autophagic vesicles cannot be induced by 20-hydroxyecdysone in the tissues of mutant flies and represent evidence demonstrating that the failure to form autophagic vesicles is due to the insertion of a P-element into the gene coding SNF4Agamma, the Drosophila homologue of the AMPK (AMP-activated protein kinase) gamma subunit. The ability to form autophagic vesicles (wild-type phenotype) can be restored by remobilization of the P-element in the mutant. Silencing of SNF4Agamma by RNAi suppresses autophagic vesicle formation in wild-type flies. We raised an antibody against SNF4Agamma and showed that this gene product is constitutively present in the wild-type larval tissues during postembryonal development. SNF4Agamma is nearly absent from the cells of homozygous mutants. SNF4Agamma translocates into the nuclei of fat body cells at the onset of the wandering stage concurrently with the beginning of the autophagic process. Our results demonstrate that SNF4Agamma has an essential role in the regulation of autophagy in Drosophila larval fat body cells.     

Three peptidoglycan recognition protein (PGRP) genes encoding potential amidase from eri-silkworm, Samia cynthia ricini

Three cDNA clones encoding peptidoglycan recognition proteins (PGRP-B, -C and -D) were isolated from larval fat body of immunized Samia cynthia ricini. The deduced amino acid sequences show high homology to each other and also to Drosophila PGRP-LB, but rather lower homology to all of the known lepidopteran PGRPs including Samia PGRP-A, a receptor-type PGRP. The three PGRPs conserve the five amino acid residues which form the catalytic site of N-acetylmuramoyl L-alanine amidase as in Drosophila LB. The PGRP-C and -D genes were silent in naive larvae, but strongly induced in fat body by an injection of peptidoglycan. PGRP-B gene, in contrast, constitutively expressed at high levels in naive midgut, and the gene was weakly induced in fat body after injection of peptidoglycan.     

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg(2+)-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.     

Population genetics and geographic variation of alcohol dehydrogenase (Adh) paralogs and glucose-6-phosphate dehydrogenase (G6pd) in Drosophila mojavensis

Populations of Drosophila mojavensis from the deserts of the Baja California peninsula and mainland Mexico utilize different cactus hosts with different alcohol contents. The enzyme alcohol dehydrogenase (ADH) has been proposed to play an important role in the adaptation of Drosophila species to their environment. This study investigates the role of ADH in the adaptation of the cactophilic D. mojavensis to its cactus host. In D. mojavensis and its sibling species, D. arizonae, the Adh gene has duplicated, giving rise to a larval/ovarian form (Adh-1) and an adult form (Adh-2). Studies of sequence variation presented here indicate that the Adh paralogs have followed different evolutionary trajectories. Adh-1 exhibits an excess of fixed amino acid replacements, suggesting adaptive evolution, which could have been a result of several host shifts that occurred during the divergence of D. mojavensis. A 17-bp intron haplotype polymorphism segregates in Adh-2 and has markedly different frequencies in the Baja and mainland populations. The presence of the intron polymorphism suggests possible selection for the maintenance of pre-mRNA structure. Finally, this study supports the proposed Baja California origination of D. mojavensis and subsequent colonization of the mainland accompanied by a host shift.     

Activity variation in alcohol dehydrogenase paralogs is associated with adaptation to cactus host use in cactophilic Drosophila

Drosophila mojavensis and Drosophila arizonae are species of cactophilic flies that share a recent duplication of the alcohol dehydrogenase (Adh) locus. One paralog (Adh-2) is expressed in adult tissues and the other (Adh-1) in larvae and ovaries. Enzyme activity measurements of the ADH-2 amino acid polymorphism in D. mojavensis suggest that the Fast allozyme allele has a higher activity on 2-propanol than 1-propanol. The Fast allele was found at highest frequency in populations that utilize hosts with high proportions of 2-propanol, while the Slow allele is most frequent in populations that utilize hosts with high proportions of 1-propanol. This suggests that selection for ADH-2 allozyme alleles with higher activity on the most abundant alcohols is occurring in each D. mojavensis population. In the other paralog, ADH-1, significant differences between D. mojavensis and D. arizonae are associated with a previously shown pattern of adaptive protein evolution in D. mojavensis. Examination of protein sequences showed that a large number of amino acid fixations between the paralogs have occurred in catalytic residues. These changes are potentially responsible for the significant difference in substrate specificity between the paralogs. Both functional and sequence variation within and between paralogs suggests that Adh has played an important role in the adaptation of D. mojavensis and D. arizonae to their cactophilic life.     

Structure and Evolution of the Adh Genes of Drosophila Mojavensis

The nucleotide sequence of the Adh region of Drosophila mojavensis has been completed and the region found to contain a pseudogene, Adh-2 and Adh-1 arranged in that order. Comparison of the sequence divergence of these genes to one another and to the Adh region of Drosophila mulleri and other species has allowed the development of a model for the evolution of the duplication of the Adh genes. There have been two major events. An initial duplication of an Adh gene whose dual promoter structure was similar to Drosophila melanogaster, resulted in a species with two Adh genes, one of which may have had only a proximal promoter. A second duplication of this gene generated an Adh region containing three genes. It is proposed that one of these is the ancestral gene having dual promoters, while the other two possess only proximal promoters. Subsequent events have resulted in both a change in the regulation of Adh-2 such that it is expressed as if it had a "distal" type promoter and the mutational inactivation of the most upstream gene resulting in the creation of a pseudogene. The sequence of the D. mojavensis Adh region has also revealed the presence of an element which is composed of juxtaposed inverted imperfectly repeated elements. There is a surprising and not fully explainable strong similarity of the nucleotide sequence of the 5' flanking region of the pseudogene in D. mojavensis and D. mulleri.