Larval Serum Protein Research Articles

Drosophila Gut Immune Pathway Suppresses Host Development-Promoting Effects of Acetic Acid Bacteria.

The physiology of most organisms, including Drosophila, is heavily influenced by their interactions with certain types of commensal bacteria. Acetobacter and Lactobacillus, two of the most representative Drosophila commensal bacteria, have stimulatory effects on host larval development and growth. However, how these effects are related to host immune activity remains largely unknown. Here, we show that the Drosophila development-promoting effects of commensal bacteria are suppressed by host immune activity. Mono-association of germ-free Drosophila larvae with Acetobacter pomorum stimulated larval development, which was accelerated when host immune deficiency (IMD) pathway genes were mutated. This phenomenon was not observed in the case of mono-association with Lactobacillus plantarum. Moreover, the mutation of Toll pathway, which constitutes the other branch of the Drosophila immune pathway, did not accelerate A. pomorum-stimulated larval development. The mechanism of action of the IMD pathway-dependent effects of A. pomorum did not appear to involve previously known host mechanisms and bacterial metabolites such as gut peptidase expression, acetic acid, and thiamine, but appeared to involve larval serum proteins. These findings may shed light on the interaction between the beneficial effects of commensal bacteria and host immune activity.

A Blueprint of Microstructures and Stage-Specific Transcriptome Dynamics of Cuticle Formation in Bombyx mori.

Insect cuticle is critical for the environmental adaptability and insecticide resistance of insects. However, there is no clear understanding of the structure and protein components of the cuticle during each developmental stage of holometabolous insects, and knowledge about the protein components within each layer is vague. We conducted serial sectioning, cuticular structure analysis, and transcriptome sequencing of the larval, pupal, and adult cuticles of Bombyx mori. The deposition processes of epicuticle, exocuticle, and endocuticle during larval, pupal, and adult cuticle formation were similar. Transcriptome analysis showed that these cuticle formations share 74% of the expressed cuticular protein (CP) genes and 20 other structural protein genes, such as larval serum protein and prisilkin. There are seven, six, and eleven stage-specific expressed CP genes in larval, pupal, and adult cuticles, respectively. The types and levels of CP genes may be the key determinants of the properties of each cuticular layer. For example, the CPs of the RR-2 protein family with high contents of histidine (His) are more essential for the exocuticle. Functional analysis suggested that BmorCPAP1-H is involved in cuticle formation. This study not only offers an in-depth understanding of cuticle morphology and protein components but also facilitates the elucidation of molecular mechanisms underlying cuticle formation in future studies.

Protein Stores Regulate When Reproductive Displays Begin in the Male Caribbean Fruit Fly.

Many animals exhibit reproductive behavior that requires expenditure of valuable nutrients. In males of many species, competitive energetically demanding displays and the development of sexual ornaments require prior accumulation of nutrient stores. Males must coordinate nutrient stores with ornament development and reproductive displays or they risk depleting their resources mid-development or mid-display, reducing their chance of mating. Males may use nutrient stores to regulate their reproductive behavior. Amino acid reserves may be important for reproduction, but the roles of amino acid stores in initiating maturation and reproductive behavior are less studied than fat stores. Insects store amino acids as hexamerin storage proteins. Many fly species use a specific hexamerin, larval serum protein 2 (LSP-2), as both a juvenile storage medium and to store protein consumed after adult eclosion. Protein stored as LSP-2 has previously been suggested to regulate reproduction in females, but no role has been proposed for LSP-2 in regulating male maturation. We use males of the Caribbean fruit fly, Anastrepha suspensa, a species with nutrient-intensive male sexual displays to test whether LSP-2 stores regulate male reproductive displays. We fed adult A. suspensa males a diet with or without protein, then assayed these males for lsp-2 transcript abundance via qRT-PCR, LSP-2 protein abundance via Western blot, and reproductive display behavior via observation. We found that adult males with ad libitum dietary protein had greater lsp-2 transcript and protein abundance, earlier sexual display behavior, and were more likely to exhibit sexual display behavior than protein-deprived adult males. We show that lsp-2 knockdown via RNAi decreases the proportion of males exhibiting reproductive displays, particularly early in the onset of reproductive behavior. Our results suggest circulating LSP-2 protein stores regulate reproductive behavior in A. suspensa males, consistent with protein stores modulating reproduction in males with expensive reproductive strategies. Our results are consistent with hexamerin storage proteins performing dual roles of protein storage and protein signaling. Our work also has substantial practical applications because tephritid flies are a pest group and the timing and expression of male reproductive displays in this group are important for control efforts using the sterile insect technique.

Identification of the larval serum proteins as major fruit fly (Drosophila melanogaster) occupational allergens

Identification of the larval serum proteins as major fruit fly (Drosophila melanogaster) occupational allergens

A Common Suite of Coagulation Proteins Function in Drosophila Muscle Attachment.

The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. We have made the surprising discovery that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes Thrombospondin (Tsp) and Tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. We further demonstrate that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, we propose that shared extracellular protein-protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment.

Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization

Vinclozolin, a dicarboximide fungicide, is an endocrine disrupting chemical that competes with an androgenic endocrine disruptor compound. Most research has focused on the epigenetic effect of vinclozolin in humans. In terms of ecotoxicology, understanding the effect of vinclozolin on non-target organisms is important. The expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin was examined. The expressed sequence tags in low-dose vinclozolin-treated and -untreated amphipods were isolated and identified by suppression subtractive hybridization. DNA dot blotting was used to confirm the results and establish a subtracted cDNA library for comparing all differentially expressed sequences with and without vinclozolin treatment. In total, 494 differentially expressed genes, including hemocyanin, heatshock protein, cytochrome, cytochrome oxidase and NADH dehydrogenase were detected. Hemocyanin was the most abundant gene. DNA dot blotting revealed 55 genes with significant differential expression. These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc-finger–containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein. Vinclozolin appears to upregulate stress-related genes and hemocyanin, related to immunity. Moreover, vinclozolin downregulated NADH dehydrogenase, related to respiration. Thus, even a non-lethal concentration of vinclozolin still has an effect at the genetic level in H. azteca and presents a potential risk, especially as it would affect non-target organism hormone metabolism.

CDNA cloning and transcriptional expression profiles of a hexamerin in the oriental fruit fly, Bactrocera dorsalis.

A Bactrocera dorsalis hexamerin (BdAr) cDNA was cloned (GenBank accession no. KF815528), and its transcriptional expression profiles were determined. The complete 2,530-bp cDNA encodes a 780-amino acid protein with a predicted molecular mass of 94.01 kDa. The proportions of phenylalanine (7.8%), tyrosine (11.2%), and methionine (2.6%) in BdAr as well as all other amino acids are reported. BdAr transcripts were detected in the brain, flight muscle, foregut, Malpighian tubules, and fat body. In the larval stage, BdAr transcripts were expressed in the early third instar and increased in the late third instar. In pupae, the highest expression of BdAr mRNA was present on day 1, then declined and persisted through day 2 to day 8. In adult females, the relative expression of BdAr was significantly higher on day 0 and day 1 compared to day 6 to day 10 while it was highest in newly eclosed adult males. The comparison of the BdAr expression between 8-10-day-old males and females showed a higher level in females. Our phylogenetic analysis results suggest to us that BdAr is similar to Drosophila larval serum protein 1γ.

Over‐transcription of genes in a parathion‐resistant strain of mosquito Culex pipiens quinquefasciatus

Insecticide resistance is an evolutionary adaptation that develops quite quickly in mosquitoes because of the high selection pressure of chemical insecticides, rapid generation time and large population size. Identification of genes associated with insecticide resistance is fundamental to understand the complex processes responsible for resistance. We compared the gene transcriptional profiles of parathion-resistant and -susceptible Culex pipiens quinquefasciatus using a combination of suppression subtractive hybridization and complementary DNA (cDNA) microarray techniques. A total of 278colonies were selected from the resistant-susceptible mosquito subtractive library, 38 of which showed more than two fold stronger immunoblotting signals in the resistant strain than in the susceptible strain using cDNA microarray selection. The sequencing results showed that the 38colonies can be matched to 12 genes of C. p. quinquefasciatus. Eight genes were confirmed to be overexpressed by more than two fold in the resistant strain. These genes encode chymotrypsin-1, theta glutathione S-transferase, lipase 3, larval serum protein 1β chain, cytochrome b, mitochondrial ribosomal large subunit, 28S rRNA, and a protein with unknown function. This study serves as a preliminary attempt to identify new genes associated with organophosphate resistance in this mosquito species and provides insights into the complicated physiological phenomenon of insecticide resistance.

AP-1/Fos-TGase2 Axis Mediates Wounding-induced Plasmodium falciparum Killing in Anopheles gambiae

Anopheline mosquitoes are the only vectors of human malaria worldwide. It is now widely accepted that mosquito immune responses play a crucial role in restricting Plasmodium development within the vector; therefore, further dissection of the molecular mechanisms underlying these processes should inform new vector control strategies urgently needed to roll back the disease. Here, using genome-wide transcriptional profiling, bioinformatics, and functional gene analysis, we identify a new axis of mosquito resistance to monoclonal Plasmodium falciparum infections that includes the AP-1 transcription factor Fos and the transglutaminase 2 (TGase2), a cross-linking enzyme with known roles in wound responses. We demonstrate that Fos regulates induction of TGase2 expression after wounding but does not affect expression of the components of the well characterized complement-like system. Silencing of Fos or of TGase2 aborts the wounding-induced mosquito killing of P. falciparum. These results reveal multiple signaling pathways that are required for efficient Plasmodium killing in Anopheles gambiae.

Cloning, Characterization and Bioinformatic Analysis of the Gene Encoding the Larval Serum Protein 2 in Diapause of the Onion Maggot, Delia Antiqua

The full-length cDNA encoding Larval serum protein 2 (LSp-2) in the onion maggot, Delia antiqua, was cloned and sequenced by rapid amplification of cDNA ends methods. The result showed that the cDNA was 2203 bp long and the open reading frame (ORF) of 2106 bp encoded 701 amino acid with a calculated molecular weight of 80.5 kDa and an isoelectric point of 5.87. The onion maggot LSp-2 shows highest homology (83%) to that of Calliphora vicina at amino acid level. Its signal peptides, domains and structures were predicted and analyzed by using bioinformatic methods. The amino acid sequence of LSP-2 suggests that it would be a typical hexamerin.

Temperature treatments during larval development reveal extensive heritable and plastic variation in gene expression and life history traits

Little is known about variation in gene expression that affects life history traits in wild populations of outcrossing species. Here, we analyse heritability of larval development traits and associated variation in gene expression in the Glanville fritillary butterfly (Melitaea cinxia) across three ecologically relevant temperatures. We studied the development of final-instar larvae, which is greatly affected by temperature, and during which stage larvae build up most of the resources for adult life. Larval development time and weight gain varied significantly among families sampled from hundreds of local populations, indicating substantial heritable variation segregating in the large metapopulation. Global gene expression analysis using common garden-reared F2 families revealed that 42% of the >8000 genes surveyed exhibited significant variation among families, 39% of the genes showed significant variation between the temperature treatments, and 18% showed a significant genotype-by-environment interaction. Genes with large family and temperature effects included larval serum protein and cuticle-binding protein genes, and the expression of these genes was closely correlated with the rate of larval development. Significant expression variation in these same categories of genes has previously been reported among adult butterflies originating from newly established versus old local populations, supporting the notion of a life history syndrome put forward based on ecological studies and involving larval development and adult dispersal capacity. These findings suggest that metapopulation dynamics in heterogeneous environments maintain heritable gene expression variation that affects the regulation of life history traits.

Protein Crosslinking by Transglutaminase Controls Cuticle Morphogenesis in Drosophila

Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins—two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin—as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.

Insights into aging through measurements of the Drosophila proteome as a function of temperature

Drosophila melanogaster is used as a model system to investigate protein changes associated with the aging process under conditions that alter organism lifespan. Changes in the proteome are assessed at various ages in populations of Oregon-R adult males that have mean lifetimes of 47 and 111 days at 28 and 18 °C, respectively. Peptide hits detected from strong-cation-exchange and reversed-phase liquid chromatography coupled to tandem mass spectrometry analysis are employed to examine patterns in relative protein expression. Thirty-three proteins were identified as having similar patterns of expression at both temperatures investigated when scaling the organism age to lifespan. In addition, the proteins ferritin 2 light chain homologue and larval serum protein 1β were identified in relatively high abundance and displayed distinctly different patterns of expression between the two temperatures. Overall, the results support the notion that aspects of the aging process may be preprogrammed at the protein level.

Developmental changes and hormonal regulation of mRNA of Bombyx mori larval serum protein (BmLSP)

Developmental changes and hormonal regulation of mRNA of Bombyx mori larval serum protein (BmLSP)

Stable Isotope Labeling and Label-Free Proteomics of Drosophila parkin Null Mutants

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and formation of intracytoplasmic Lewy bodies (LBs). Loss-of-function mutations in parkin which encodes an E3 ubiquitin protein ligase contribute to a predominant cause of a familial form of PD termed autosomal recessive juvenile Parkinsonism (AR-JP). Drosophila parkin null mutants display muscle degeneration and mitochondrial dysfunction, providing an animal model to study Parkin-associated molecular pathways in PD. To define protein alterations involved in Parkin pathogenesis, we performed quantitative proteomic analyses of Drosophila parkin null mutants and age-matched controls utilizing both global internal standard technology (GIST) and extracted ion chromatogram peak area (XICPA) label-free approaches. A total of 375 proteins were quantified with a minimum of two peptide identifications from the combination of the XICPA and GIST measurements applied to two independent biological replicates. Sixteen proteins exhibited significant alteration. Seven of the dysregulated proteins are involved in energy metabolism, of which six were down-regulated. All five proteins involved in transporter activity exhibited higher levels, of which larval serum protein 1alpha, larval serum protein 1beta, larval serum protein 1gamma, and fat body protein 1 showed >10-fold up-regulation and substantially higher level of fat body protein 1 was confirmed by Western blot analysis. These findings suggest that abnormalities in energy metabolism and protein transporter activity pathways may be associated with the pathogenesis of Parkin-associated AR-JP.

A novel role for the Drosophila epsin (lqf): Involvement in autophagy

Screening P-element-induced mutant collections, 52 lines were selected as potentially defected ones in endocytosis or autophagy. After excluding those which were rescued by 20-hydroxyecdosone treatment, the exact position of the inserted P-element was determined in the remaining lines. In the case of l(3)S011027 stock, that liquid facets (lqf) gene was affected which codes an epsin-homolog protein in Drosophila. We reveal that Lqf is essential to the receptor-mediated endocytosis of larval serum proteins (LSPs) in the larval fat body cells of Drosophila. In l(3)S011027 line, lack of Lqf fails the formation of autophagosomes thus leading to the arrest of destroying of trophocytes. Transgenic larvae carrying Lqf-RNAi construct were unable to generate endocytic and autophagic vacuoles and led to a prolonged larval stage. On the other hand, GFP-tagged Lqf protein showed an exclusively co-localization with the LysoTracker Red- or GFP-Atg8a labeled autophagosomes. By using the antiserum generated against the fifth exon of lqf, we demonstrated that prior to the onset of developmental autophagy the Lqf protein was present in the nucleus of fat body cell, but thereafter the protein was localized in the territory of endocytic and autophagic vacuoles. The fact that the inhibition of the target of rapamycin (TOR) did not restore the autophagic process and the normal development in the case of lqf mutant larvae points to that the Lqf is downstream to the TOR, the central kinase of the autophagy pathway.

Proteome Response to the Panneural Expression of Human Wild-Type α-Synuclein: A Drosophila Model of Parkinson’s Disease

The alpha-synuclein protein is associated with several neurodegenarative diseases, including Parkinson's disease (PD). In humans, only mutated forms of alpha-synuclein are linked to PD; however, panneural expression of human wild-type (WT) alpha-synuclein induces Parkinson's like-symptoms in Drosophila. Here, we report a quantitative proteomic analysis of WT alpha-synuclein transgenic flies with age-matched controls at the presymptomatic stage utilizing a global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry. The analysis includes two biological replicates, in which samples are isotopically labeled in forward and reverse directions. In total, 229 proteins were quantified from assignments of at least two peptide sequences. Of these, 188 (82%) proteins were detected in both forward and reverse labeling measurements. Twelve proteins were found to be differentially expressed in response to the expression of human WT alpha-synuclein; down-regulations of larval serum protein 2 and fat body protein 1 levels were confirmed by Western blot analysis. Gene Ontology analysis indicates that the dysregulated proteins are primarily associated with cellular metabolism and signaling, suggesting potential contributions of perturbed metabolic and signaling pathways to PD. An increased level of the iron (III)-binding protein, ferritin, typically found in the brains of PD patients, is also observed in presymptomatic WT alpha-synuclein expressing animals. The observed alterations in both pathology-associated and novel proteins may shed light on the pathological roles of alpha-synuclein that may lead to the development of diagnostic strategies at the presymptomatic stage.

Potential ligands of DmP29, a putative juvenile hormone esterase binding protein of Drosophila melanogaster

We previously reported the identification of a putative juvenile hormone esterase (JHE) binding protein DmP29 in Drosophila melanogaster and its primary localization to the mitochondria [Liu, Z., Ho, L., Bonning, B.C., 2007. Localization of a Drosophila melanogaster homolog of the putative juvenile hormone esterase binding protein of Manduca sexta. Insect Biochem. Mol. Biol. 37(2), 155–163]. To further characterize DmP29, we identified potential ligands of this protein. Recombinant DmP29 was shown by ligand blot and co-immunoprecipitation analyses to bind recombinant JHE as well as to larval serum proteins (LSP). The possible biological relevance of the in vitro DmP29–JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolyzed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29–LSP interactions may not be biologically relevant. Given the high abundance, and “sticky” nature of these proteins, interaction of DmP29 with LSP may result from non-specific associations. No DmP29 interactions with non-specific esterases were detected by co-immunoprecipitation analyses. The potential role of DmP29 as a chaperone of JHE is discussed.

A Role for the Adult Fat Body in Drosophila Male Courtship Behavior

Mating behavior in Drosophila depends critically on the sexual identity of specific regions in the brain, but several studies have identified courtship genes that express products only outside the nervous system. Although these genes are each active in a variety of non-neuronal cell types, they are all prominently expressed in the adult fat body, suggesting an important role for this tissue in behavior. To test its role in male courtship, fat body was feminized using the highly specific Larval serum protein promoter. We report here that the specific feminization of this tissue strongly reduces the competence of males to perform courtship. This effect is limited to the fat body of sexually mature adults as the feminization of larval fat body that normally persists in young adults does not affect mating. We propose that feminization of fat body affects the synthesis of male-specific secreted circulating proteins that influence the central nervous system. In support of this idea, we demonstrate that Takeout, a protein known to influence mating, is present in the hemolymph of adult males but not females and acts as a secreted protein.

The non-dosage compensated Lsp1α gene of Drosophila melanogaster escapes acetylation by MOF in larval fat body nuclei, but is flanked by two dosage compensated genes

BackgroundIn Drosophila melanogaster dosage compensation of most X-linked genes is mediated by the male-specific lethal (MSL) complex, which includes MOF. MOF acetylates histone H4 at lysine 16 (H4K16ac). The X-linked Larval serum protein one α (Lsp1α) gene has long been known to be not dosage compensated. Here we have examined possible explanations for why the Lsp1α gene is not dosage compensated.ResultsQuantitative RNase protection analysis showed that the genes flanking Lsp1α are expressed equally in males and females and confirmed that Lsp1α is not dosage compensated. Unlike control X-linked genes, Lsp1α was not enriched for H4K16ac in the third instar larval fat body, the tissue in which the gene is actively expressed. X-linked Lsp1α promoter-lacZ reporter transgenes are enriched for H4K16ac in third instar larval fat body. An X-linked reporter gene bracketed by Lsp1α flanking regions was dosage compensated. One of the genes flanking Lsp1α is expressed in the same tissue. This gene shows a modest enrichment for H4K16ac but only at the part of the gene most distant from Lsp1α. Phylogenetic analyses of the sequences of the genomes of 12 Drosophila species shows that Lsp1α is only present within the melanogaster subgroup of species.ConclusionLsp1α is not modified by the MSL complex but is in a region of the X chromosome that is regulated by the MSL complex. The high activity or tissue-specificity of the Lsp1α promoter does not prevent regulation by the MSL complex. The regions flanking Lsp1α do not appear to block access by the MSL complex. Lsp1α appears to have recently evolved within the melanogaster subgroup of Drosophila species. The most likely explanation for why Lsp1α is not dosage compensated is that the gene has not evolved a mechanism to independently recruit the MSL complex, possibly because of its recent evolutionary origin, and because there appears to be a low level of bound MSL complex in a nearby gene that is active in the same tissue.