Regulation of thermoregulatory behavior by commensal bacteria in Drosophila.

Commensal bacteria affect many aspects of host physiology. In this study, we focused on the role of commensal bacteria in the thermoregulatory behavior of Drosophila melanogaster. We demonstrated that the elimination of commensal bacteria caused an increase in the preferred temperature of Drosophila third-instar larvae without affecting the activity of transient receptor potential ankyrin 1 (TRPA1)-expressing thermosensitive neurons. We isolated eight bacterial strains from the gut and culture medium of conventionally reared larvae and found that the preferred temperature of the larvae was decreased by mono-association with Lactobacillus plantarum or Corynebacterium nuruki. Mono-association with these bacteria did not affect the indices of energy metabolism such as ATP and glucose levels of larvae, which are closely linked to thermoregulation in animals. Thus, we show a novel role for commensal bacteria in host thermoregulation and identify two bacterial species that affect thermoregulatory behavior in Drosophila.

Extreme deformability of insect cell membranes is governed by phospholipid scrambling.

Organization of dynamic cellular structure is crucial for a variety of cellular functions. In this study, we report that Drosophila and Aedes have highly elastic cell membranes with extremely low membrane tension and high resistance to mechanical stress. In contrast to other eukaryotic cells, phospholipids are symmetrically distributed between the bilayer leaflets of the insect plasma membrane, where phospholipid scramblase (XKR) that disrupts the lipid asymmetry is constitutively active. We also demonstrate that XKR-facilitated phospholipid scrambling promotes the deformability of cell membranes by regulating both actin cortex dynamics and mechanical properties of the phospholipid bilayer. Moreover, XKR-mediated construction of elastic cell membranes is essential for hemocyte circulation in the Drosophila cardiovascular system. Deformation of mammalian cells is also enhanced by the expression of Aedes XKR, and thus phospholipid scrambling may contribute to formation of highly deformable cell membranes in a variety of living eukaryotic cells.

Functional expression of Δ12 fatty acid desaturase modulates thermoregulatory behaviour in Drosophila.

Polyunsaturated fatty acids (PUFAs) play crucial roles in adaptation to cold environments in a wide variety of animals and plants. However, the mechanisms by which PUFAs affect thermoregulatory behaviour remain elusive. Thus, we investigated the roles of PUFAs in thermoregulatory behaviour of Drosophila melanogaster. To this end, we generated transgenic flies expressing Caenorhabditis elegans Δ12 fatty acid desaturase (FAT-2), which converts mono-unsaturated fatty acids to PUFAs such as linoleic acid [C18:2 (n-6)] and linolenic acid [C18:3 (n-3)]. Neuron-specific expression of FAT-2 using the GAL4/UAS expression system led to increased contents of C18:2 (n-6)-containing phospholipids in central nerve system (CNS) and caused significant decreases in preferred temperature of third instar larvae. In genetic screening and calcium imaging analyses of thermoreceptor-expressing neurons, we demonstrated that ectopic expression of FAT-2 in TRPA1-expressing neurons led to decreases in preferred temperature by modulating neuronal activity. We conclude that functional expression of FAT-2 in a subset of neurons changes the thermoregulatory behaviour of D. melanogaster, likely by modulating quantities of PUFA-containing phospholipids in neuronal cell membranes.

Different mechanisms for selective transport of fatty acids using a single class of lipoprotein in Drosophila.

In mammals, lipids are selectively transported to specific sites using multiple classes of lipoproteins. However, in Drosophila, a single class of lipoproteins, lipophorin, carries more than 95% of the lipids in the hemolymph. Although a unique ability of the insect lipoprotein system for cargo transport has been demonstrated, it remains unclear how this single class of lipoproteins selectively transports lipids. In this study, we carried out a comparative analysis of the fatty-acid composition among lipophorin, the CNS, and CNS-derived cell lines and investigated the transport mechanism of fatty acids, particularly focusing on the transport of PUFAs in Drosophila We showed that PUFAs are selectively incorporated into the acyl chains of lipophorin phospholipids and effectively transported to CNS through lipophorin receptor-mediated endocytosis of lipophorin. In addition, we demonstrated that C14 fatty acids are selectively incorporated into the diacylglycerols (DAGs) of lipophorin and that C14 fatty-acid-containing DAGs are spontaneously transferred from lipophorin to the phospholipid bilayer. These results suggest that PUFA-containing phospholipids and C14 fatty-acid-containing DAGs in lipophorin could be transferred to different sites by different mechanisms to selectively transport fatty acids using a single class of lipoproteins.

Severe Fertility Effects of sheepish Sperm Caused by Failure To Enter Female Sperm Storage Organs in Drosophila melanogaster.

In Drosophila, mature sperm are transferred from males to females during copulation, stored in the sperm storage organs of females, and then utilized for fertilization. Here, we report a gene named sheepish (shps) of Drosophila melanogaster that is essential for sperm storage in females. shps mutant males, although producing morphologically normal and motile sperm that are effectively transferred to females, produce very few offspring. Direct counts of sperm indicated that the primary defect was correlated to failure of shps sperm to migrate into the female sperm storage organs. Increased sperm motion parameters were seen in the control after transfer to females, whereas sperm from shps males have characteristics of the motion parameters different from the control. The few sperm that occasionally entered the female sperm storage organs showed no obvious defects in fertilization and early embryo development. The female postmating responses after copulation with shps males appeared normal, at least with respect to conformational changes of uterus, mating plug formation, and female remating rates. The shps gene encodes a protein with homology to amine oxidases, including as observed in mammals, with a transmembrane region at the C-terminal end. The shps mutation was characterized by a nonsense replacement in the third exon of CG13611, and shps was rescued by transformants of the wild-type copy of CG13611 Thus, shps may define a new class of gene responsible for sperm storage.

An N-terminal di-proline motif is essential for fatty acid-dependent degradation of Δ9-desaturase in Drosophila.

The Δ9-fatty acid desaturase introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how Δ9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole Δ9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid-dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid-dependent degradation. Furthermore, we also found that the Ca2+-dependent cysteine protease calpain is involved in the sequential proline-dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a "di-proline motif," which plays a crucial role in the regulation of Δ9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids.

Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates.

Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family­pleurotolysin A2 (PlyA2; K(D) = 12 nM), ostreolysin (Oly; K(D) = 1.3 nM), and erylysin A (EryA; K(D) = 1.3 nM)­strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K(D) = 41 µM), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.

Genetic analysis of chaste, a new mutation of Drosophila melanogaster characterized by extremely low female sexual receptivity.

By screening about 2,000 P-element-insertion lines of Drosophila melanogaster, we isolated a new behavioral mutant line, chaste (chst), the females of which display extraordinarily strong rejection behavior against courting males. The chst mutation is mapped to the muscleblind (mbl) locus at 54B on the right arm of chromosome 2. The reduced sexual receptivity in chst mutant females is reversed to the wild-type level by introducing a transgene, which expresses either the mblB (+) or mblC (+) isoform, demonstrating that chst is an allele of mbl. Among the P-elements inserted upstream of the mbl gene, those inserted in the same orientation as that of mbl express the chst phenotype, whereas a P-element inserted in the opposite orientation does not. This finding implies that the former P-elements induce the mutant phenotype by a mechanism that is sensitive to the direction of transcription (e.g., transcriptional interference). The mbl alleles, with deletions near the transcription start site and/or in part of the exons, complement the chst mutation in the sexual receptivity phenotype, but not in the lethality phenotype of mbl mutations. Such interallelic complementation of the sexual receptivity phenotype in the mbl locus disappears in the presence of a mutant copy of zeste (z), a gene encoding a protein that mediates transvection. We suggest that the mbl gene function is required for the normal development of neural substrates that regulate female sexual receptivity.

Expression analysis of the lingerer gene in the larval central nervous system of Drosophila melanogaster.

The lingerer (lig) gene is necessary for initiation and termination of copulatory behavior in Drosophila melanogaster. The lig gene encodes cytoplasmic proteins, and is expressed in the central nervous system (CNS) during the late third-instar larval stage when the lig function is required for normal copulation to occur after adult eclosion. To characterize the lig-expressing cells in the late third-instar larval CNS, we have isolated a genomic fragment containing the promoter/enhancer region of the lig gene, and established transgenic lines in which expression of reporter genes is controlled by the lig promoter/enhancer. In the larval brain, reporter genes were expressed in all of the glial cells and in clusters of neurons that projected contralaterally. In the larval ventral ganglion, reporter genes were expressed in subperineurial glia, peripheral exit glia, and a number of interneurons, but not in motor neurons. In the cloned promoter/enhancer region, we have found the sequence motif for binding of the REPO protein, a transcription factor essential for the differentiation and maintenance of glial cells. The lig gene is thus one of the candidate target genes for the REPO transcription factor.

The ken and barbie gene encoding a putative transcription factor with a BTB domain and three zinc finger motifs functions in terminalia development of Drosophila.

Mutations in the ken and barbie locus are accompanied by the malformation of terminalia in adult Drosophila. Male and female genitalia often remain inside the body, and the same portions of genitalia and analia are missing in a fraction of homozygous flies. Rotated and/or duplicated terminalia are also observed. Terminalia phenotypes are enhanced by mutations in the gap gene tailless, the homeobox gene caudal, and the decapentaplegic gene that encodes a TGFbeta-like morphogen. The ken and barbie gene encodes a protein with three CCHH-type zinc finger motifs that are conserved in several transcription factors such as Krüppel and BCL-6. All defects in ken and barbie mutants are fully rescued by the expression of a wild-type genomic construct, which establishes the causality between phenotypes and the gene.

Enhanced phosphorylation and enzymatic activity of phosphoglucomutase by the Btk29A tyrosine kinase in Drosophila.

The Drosophila Btk29A tyrosine kinase is suggested to be involved in diverse processes, although its target proteins are unknown. In the present study, we investigated substrates of Btk29A tyrosine kinase by expressing a catalytically activated form of Btk29A-P1 (Btk-EG) in Drosophila compound eyes. Expression in eye disks led to the development of the rough-eye phenotype and increased tyrosine phosphorylation of a 65-kDa protein. Partial amino acid sequence analysis of this protein showed that it was phosphoglucomutase. Phosphoglucomutase activity in heads from Btk-EG-expressing flies was higher than that in controls, suggesting that the levels of tyrosine phosphorylation and activity of the enzyme are associated with Btk29A tyrosine kinase activity.

lingerer, a Drosophila gene involved in initiation and termination of copulation, encodes a set of novel cytoplasmic proteins.

In an effort to uncover genetic components underlying the courtship behavior of Drosophila melanogaster, we have characterized a novel gene, lingerer (lig), mutations of which result in abnormal copulation. Males carrying a hypomorphic mutation in lig fail to withdraw their genitalia upon termination of copulation, but display no overt abnormalities in their genitalia. A severe reduction in the dosage of the lig gene causes repeated attempted copulations but no successful copulations. Complete loss of lig function results in lethality during early pupal stages. lig is localized to polytene segment 44A on the second chromosome and encodes three alternatively spliced transcripts that generate two types of 150-kD proteins, Lig-A and Lig-B, differing only at the C terminus. Lig proteins show no similarity to known proteins. However, a set of homologous proteins in mammals suggest that Drosophila Lig belongs to a family of proteins that share five highly conserved domains. Lig is a cytoplasmic protein expressed in the central nervous system (CNS), imaginal discs, and gonads. Lig-A expression is selectively reduced in lig mutants and the ubiquitous supply of this protein at the beginning of metamorphosis restores the copulatory defects of the lig mutant. We propose that lig may act in the nervous system to mediate the control of copulatory organs during courtship.