Larval Fat Body Research Articles

고온에서 배추좀나방 유충 지방체의 유전자 발현 변화 분석

고온에서 배추좀나방 유충 지방체의 유전자 발현 변화 분석

MRT, Functioning with NURF Complex, Regulates Lipid Droplet Size

Lipid droplets (LDs) are highly dynamic organelles that store neutral lipids. Through a gene overexpression screen in the Drosophila larval fat body, we have identified that MRT, an Myb/switching-defective protein 3 (Swi3), Adaptor 2 (Ada2), Nuclear receptor co-repressor (N-CoR), Transcription factor (TF)IIIB (SANT)-like DNA-binding domain-containing protein, regulates LD size and lipid storage. MRT directly interacts with, and is functionally dependent on, the PZG and NURF chromatin-remodeling complex components. MRT binds to the promoter of plin1, the gene encoding the LD-resident protein perilipin, and inhibits the transcription of plin1. Invitro LD coalescence assays suggest that mrt overexpression or loss of plin1 function facilitates LD coalescence. Our findings suggest that MRT functions together with chromatin-remodeling factors to regulate LD size, likely through the transcriptional repression of plin1.

Dynamic changes in ORC localization and replication fork progression during tissue differentiation

BackgroundGenomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role.ResultsWe exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, we found that sites of ORC binding show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited.ConclusionsThese studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation.

The lysosomal membrane protein LAMP2A promotes autophagic flux and prevents SNCA-induced Parkinson disease-like symptoms in the Drosophila brain

ABSTRACT The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerative conditions. Alterations in autophagy processes, including macroautophagy and chaperone-mediated autophagy (CMA), have been described in Parkinson disease (PD). CMA is a selective autophagic process that depends on LAMP2A (lysosomal-associated membrane protein 2A), a mammal and bird-specific membrane glycoprotein that translocates cytosolic proteins containing a KFERQ-like peptide motif across the lysosomal membrane. Drosophila reportedly lack CMA and use endosomal microautophagy (eMI) as an alternative selective autophagic process. Here we report that neuronal expression of human LAMP2A protected Drosophila against starvation and oxidative stress, and delayed locomotor decline in aging flies without extending their lifespan. LAMP2A also prevented the progressive locomotor and oxidative defects induced by neuronal expression of PD-associated human SNCA (synuclein alpha) with alanine-to-proline mutation at position 30 (SNCAA30P). Using KFERQ-tagged fluorescent biosensors, we observed that LAMP2A expression stimulated selective autophagy in the adult brain and not in the larval fat body, but did not increase this process under starvation conditions. Noteworthy, we found that neurally expressed LAMP2A markedly upregulated levels of Drosophila Atg5, a key macroautophagy initiation protein, and that it increased the density of Atg8a/LC3-positive puncta, which reflects the formation of autophagosomes. Furthermore, LAMP2A efficiently prevented accumulation of the autophagy defect marker Ref(2)P/p62 in the adult brain under acute oxidative stress. These results indicate that LAMP2A can potentiate autophagic flux in the Drosophila brain, leading to enhanced stress resistance and neuroprotection. Abbreviations: Act5C: actin 5C; a.E.: after eclosion; Atg5: autophagy-related 5; Atg8a/LC3: autophagy-related 8a; CMA: chaperone-mediated autophagy; DHE: dihydroethidium; elav: embryonic lethal abnormal vision; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; GABARAP: GABA typeA receptor-associated protein; Hsc70-4: heat shock protein cognate 4; HSPA8/Hsc70: heat shock protein family A (Hsp70) member 8; LAMP2: lysosomal associated membrane protein 2; MDA: malondialdehyde; PA-mCherry: photoactivable mCherry; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PD: Parkinson disease; Ref(2)P/p62: refractory to sigma P; ROS: reactive oxygen species; RpL32/rp49: ribosomal protein L32; RT-PCR: reverse transcription polymerase chain reaction; SING: startle-induced negative geotaxis; SNCA/α-synuclein: synuclein alpha; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; UAS: upstream activating sequence.

20-Hydroxyecdysone-upregulated proteases involved in Bombyx larval fat body destruction.

During insect larval-pupal metamorphosis, the obsolete larval organs and tissues undergo histolysis and programmed cell death to recycle cellular materials. It has been demonstrated that some cathepsins are essential for histolysis in larval tissues, but the process of tissue destruction is not well documented. Fat body, the homologous organ to mammalian liver and adipose tissue, goes through a distinct destruction process during larval-pupal transition. Herein, we found that most of the Bombyx proteases - including Bombyx cathepsin B (BmCatB) (BmCatLL-2), Bombyx cathepsin D (BmCatD), Bombyx cathepsin L like-1 (BmCatLL-1) and -2(BmCatLL-2), Bombyx fibroinase (BmBcp), Bombyx matrix metalloprotease (BmMmp), Bombyx A disintegrin and metalloproteinase with thrombospondin motifs 1 (BmAdamTS-1), Bombyx A disintegrin and metalloproteinase with thrombospondin motifs like (BmAdamTS L) and Bombyx cysteine protease inhibitor (Bmbcpi)- were expressed highly in fat body during feeding and metamorphosis, with a peak occurring during the nonfeeding moulting or prepupal stage, as well as being responsive to 20-hydroxyecdysone (20E). The aforementioned protease genes expression was upregulated by injection of 20E into the feeding larvae, while blocking 20E signalling transduction led to downregulation. Western blotting and immunofluorescent staining of BmCatB and BmBcp confirmed the coincident variation of their messenger RNA (mRNA) and protein level during the development and after the treatments. Moreover, BmCatB, BmBcp, BmMmp and BmAdamTS-1 RNA interference all led to blockage of larval fat body destruction. Taken together, we conclude that 20E regulates larval fat body destruction by upregulating related protease gene expression and protein levels during larval-pupal transition.

Pleiotropy of the Drosophila melanogaster foraging gene on larval feeding-related traits

Little is known about the molecular underpinning of behavioral pleiotropy. The Drosophila melanogaster foraging gene is highly pleiotropic, affecting many independent larval and adult phenotypes. Included in foraging’s multiple phenotypes are larval foraging path length, triglyceride levels, and food intake. foraging has a complex structure with four promoters and 21 transcripts that encode nine protein isoforms of a cGMP dependent protein kinase (PKG). We examined if foraging’s complex molecular structure underlies the behavioral pleiotropy associated with this gene. Using a promotor analysis strategy, we cloned DNA fragments upstream of each of foraging’s transcription start sites and generated four separate forpr-Gal4s. Supporting our hypothesis of modular function, they had discrete, restricted expression patterns throughout the larva. In the CNS, forpr1-Gal4 and forpr4-Gal4 were expressed in neurons while forpr2-Gal4 and forpr3-Gal4 were expressed in glia cells. In the gastric system, forpr1-Gal4 and forpr3-Gal4 were expressed in enteroendocrine cells of the midgut while forpr2-Gal4 was expressed in the stem cells of the midgut. forpr3-Gal4 was expressed in the midgut enterocytes, and midgut and hindgut visceral muscle. forpr4-Gal4’s gastric system expression was restricted to the hindgut. We also found promoter specific expression in the larval fat body, salivary glands, and body muscle. The modularity of foraging’s molecular structure was also apparent in the phenotypic rescues. We rescued larval path length, triglyceride levels (bordered on significance), and food intake of for0 null larvae using different forpr-Gal4s to drive UAS-forcDNA. In a foraging null genetic background, forpr1-Gal4 was the only promoter driven Gal4 to rescue larval path length, forpr3-Gal4 altered triglyceride levels, and forpr4-Gal4 rescued food intake. Our results refine the spatial expression responsible for foraging’s associated phenotypes, as well as the sub-regions of the locus responsible for their expression. foraging’s pleiotropy arises at least in part from the individual contributions of its four promoters.

Manduca sexta serpin-12 controls the prophenoloxidase activation system in larval hemolymph

Insect prophenoloxidase activation is coordinated by a serine protease network, which is regulated by serine protease inhibitors of the serpin superfamily. The enzyme system also leads to proteolytic processing of a Spätzle precursor. Binding of Spätzle to a Toll receptor turns on a signaling pathway to induce the synthesis of defense proteins. Previous studies of the tobacco hornworm Manduca sexta have revealed key members of the protease cascade, which generates phenoloxidase for melanogenesis and Spätzle to induce immunity-related genes. Here we provide evidence that M. sexta serpin-12 regulates hemolymph protease-14 (HP14), an initiating protease of the cascade. This inhibitor, unlike the other serpins characterized in M. sexta, has an amino-terminal extension rich in hydrophilic residues and an unusual P1 residue (Leu429) right before the scissile bond cleaved by a target protease. Serpins with similarities to serpin-12, including Drosophila Necrotic, were identified in a wide range of insects including flies, moths, wasps, beetles, and two hemimetabolous species. The serpin-12 mRNA is present at low, constitutive levels in larval fat body and hemocytes and becomes more abundant after an immune challenge. We produced the serpin-12 core domain (serpin-12ΔN) in insect cells and in Escherichia coli and demonstrated its inhibition of human cathepsin G, bovine α-chymotrypsin, and porcine pancreatic elastase. MALDI-TOF analysis of the reaction mixtures confirmed the predicted P1 residue of Leu429. Supplementation of larval plasma samples with the serpin-12ΔN decreased prophenoloxidase activation elicited by microbial cells and reduced the proteolytic activation of the protease precursors of HP6, HP8, PAPs, and other serine protease-related proteins. After incubation of plasma stimulated with peptidoglycan, a 72 kDa protein appeared, which was recognized by polyclonal antibodies against both serpin-12 and HP14, suggesting that a covalent serpin-protease complex formed when serpin-12 inhibited HP14. Together, these data suggest that M. sexta serpin-12 inhibits HP14 to regulate melanization and antimicrobial peptide induction.

Drosophila larvae fed palm fruit juice (PFJ) delay pupation via expression regulation of hormetic stress response genes linked to ageing and longevity

Palm fruit juice (PFJ) containing oil palm phenolics is obtained as a by-product from oil palm (Elaeis guineensis) fruit milling. It contains shikimic acid, soluble fibre and various phenolic acids including p-hydroxybenzoic acid and three caffeoylshikimic acid isomers. PFJ has also demonstrated beneficial health properties in various biological models. Increasing concentrations of PFJ and different PFJ fractions were used to assess growth dynamics and possible anti-ageing properties in fruit flies (Drosophila melanogaster) genotype w1118. Microarray gene expression analysis was performed on whole fruit fly larvae and their fat bodies, after the larvae were fed a control Standard Brandeis Diet (SBD) with or without PFJ. Transcripts from Affymetrix GeneChips were utilised to identify the possible mechanisms involved, with genes having fold changes > |1.30| and p < 0.05 considered differentially expressed. PFJ dose-dependently delayed larval growth and pupation, but not percent eclosion from pupae. Eclosed male fruit flies fed PFJ or its fractions during the larval stage tended to have 20–40% improved survival ratings over controls when allowed to age on the control diet (SBD). Microarray analysis of whole fruit fly larvae revealed that 127 genes were up-regulated, while 67 were down-regulated by PFJ. Functional analysis revealed transport and metabolic processes were up-regulated, while development and morphogenesis processes, including the nutrient-sensing Tor gene, were down-regulated by PFJ, whereas microarray analysis of larval fat bodies found 161 genes were up-regulated, while 84 genes were down-regulated. Genes involved in defence response and determination of adult lifespan, including those encoding various heat shock proteins and the antioxidant enzyme Sod2, were up-regulated, while cell cycle and growth genes were down-regulated. Thus, PFJ supplementation lengthened the growth stages in fruit fly larvae that was reflected in extended ageing of adult flies, suggesting that larval expression of hormetic stress response genes was linked to subsequent ageing and longevity.

Innate Immune Signaling in Drosophila Blocks Insulin Signaling by Uncoupling PI(3,4,5)P3 Production and Akt Activation

SUMMARYIn obese adipose tissue, Toll-like receptor signaling in macrophages leads to insulin resistance in adipocytes. Similarly, Toll signaling in the Drosophila larval fat body blocks insulin-dependent growth and nutrient storage. We find that Toll acts cell autonomously to block growth but not PI(3,4,5)P3 production in fat body cells expressing constitutively active PI3K. Fat body Toll signaling blocks whole-animal growth in rictor mutants lacking TORC2 activity, but not in larvae lacking Pdk1. Phosphorylation of Akt on the Pdk1 site, Thr342, is significantly reduced by Toll signaling, and expression of mutant AktT342D rescues cell and animal growth, nutrient storage, and viability in animals with active Toll signaling. Altogether, these data show that innate immune signaling blocks insulin signaling at a more distal level than previously appreciated, and they suggest that manipulations affecting the Pdk1 arm of the pathway may have profound effects on insulin sensitivity in inflamed tissues.

The MTT-formazan assay: Complementary technical approaches and in vivo validation in Drosophila larvae

The MTT assay was the first widely accepted method to assess cytotoxicity and cell viability. However, there is controversy on whether this indicator is a useful tool. In this work we intend to expand the interpretability of the MTT study by its combination with widely used cellular biology techniques. We propose complementary approaches to the colorimetric assay, based on the use of measurements in three different settings: confocal microscopy, multi-well plate assay and flow cytometry. Using confocal microscopy, we confirmed that MTT uptake and reduction by cells is a time-dependent process, and that formazan accumulates in round-shaped organelles. Quantitative measurements with a multi-well fluorimeter combined with nuclear staining result in a useful method, yielding a ratio between formazan production and cell number that informs about the average cell metabolic state. We also found that flow cytometry is a suitable technique to measure MTT reduction in large cell populations. When assaying the effect of an oxidizing agent such as paraquat (PQ), this approach allows for the distinction of subpopulations of cells with different reducing power. Finally, we prove that it is feasible to monitor MTT reduction in an in vivo model, the Drosophila larvae, without affecting its survival rate. Formazan accumulates exclusively in the larval fat body, confirming its lipid solubility. The methods explored in this work expand the MTT potential as a useful tool to provide information of the physiological state of cells and organisms.

Mechanical stress regulates insulin sensitivity through integrin-dependent control of insulin receptor localization.

Insulin resistance, the failure to activate insulin signaling in the presence of ligand, leads to metabolic diseases, including type 2 diabetes. Physical activity and mechanical stress have been shown to protect against insulin resistance, but the molecular mechanisms remain unclear. Here, we address this relationship in the Drosophila larval fat body, an insulin-sensitive organ analogous to vertebrate adipose tissue and livers. We found that insulin signaling in Drosophila fat body cells is abolished in the absence of physical activity and mechanical stress even when excess insulin is present. Physical movement is required for insulin sensitivity in both intact larvae and fat bodies cultured ex vivo. Interestingly, the insulin receptor and other downstream components are recruited to the plasma membrane in response to mechanical stress, and this membrane localization is rapidly lost upon disruption of larval or tissue movement. Sensing of mechanical stimuli is mediated in part by integrins, whose activation is necessary and sufficient for mechanical stress-dependent insulin signaling. Insulin resistance develops naturally during the transition from the active larval stage to the immotile pupal stage, suggesting that regulation of insulin sensitivity by mechanical stress may help coordinate developmental programming with metabolism.

A Complex Relationship between Immunity and Metabolism in Drosophila Diet-Induced Insulin Resistance.

Both systemic insulin resistance and tissue-specific insulin resistance have been described in Drosophila and are accompanied by many indicators of metabolic disease. The downstream mediators of insulin-resistant pathophysiology remain unclear. We analyzed insulin signaling in the fat body studying loss and gain of function. When expression of the sole Drosophila insulin receptor (InR) was reduced in larval fat bodies, animals exhibited developmental delay and reduced size in a diet-dependent manner. Fat body InR knockdown also led to reduced survival on high-sugar diets. To look downstream of InR at potential mediators of insulin resistance, transcriptome sequencing (RNA-seq) studies in insulin-resistant fat bodies revealed differential expression of genes, including those involved in innate immunity. Obesity-associated insulin resistance led to increased susceptibility of flies to infection, as in humans. Reduced innate immunity was dependent on fat body InR expression. The peptidoglycan recognition proteins (PGRPs) PGRP-SB2 and PGRP-SC2 were selected for further study based on differential expression studies. Downregulating PGRP-SB2 selectively in the fat body protected animals from the deleterious effects of overnutrition, whereas downregulating PGRP-SC2 produced InR-like phenotypes. These studies extend earlier work linking the immune and insulin signaling pathways and identify new targets of insulin signaling that could serve as potential drug targets to treat type 2 diabetes.

Expression Profiles of the Trehalose-6-Phosphate Synthase Gene Associated With Thermal Stress in Ostrinia furnacalis (Lepidoptera: Crambidae)

Trehalose is the major blood sugar in insects. Physiological significance of this compound has been extensively reported. Trehalose-6-phosphate synthase (TPS) is an important enzyme in the trehalose biosynthesis pathway. Full-length cDNAs of TPS (Of tps) and its alternative splicing isoform (Of tps_isoformI) were cloned from the Asian corn borer (ACB), Ostrinia furnacalis (Guenée; Lepidoptera: Crambidae) larvae. The Of tps and Of tps_isoformI transcripts were 2913 and 1689 bp long, contained 2529 and 1293 bp open reading frames encoding proteins of 842 and 430 amino acids with a molecular mass of 94.4 and 48.6 kDa, respectively. Transcriptional profiling and response to thermal stress of Of tps gene were determined by quantitative real-time PCR showing that the Of tps was predominantly expressed in the larval fat body, significantly enhanced during molting and transformation; and thermal stress also induced Of tps expression. Gene structure analysis is indicating that one TPS domain and one trehalose-6-phosphate phosphatase (TPP) domain were located at the N- and C-termini of Of TPS, respectively, while only the TPS domain was detected in OfTPS_isoformI. Three-dimensional modeling and heterologous expression were developed to predict the putative functions of OfTPS and Of TPS_isoformI. We infer that the expression of Of tps gene is thermally induced and might be crucial for larvae survival.

Characterization of the Bombyx mori homolog of islet homeostasis protein (IHoP) cDNA: BmIHoP controls insulin secretion from the β-cells

Islet homeostasis protein (IHoP) was firstly characterized as a novel protein that regulates glucagon synthesis in alpha-cells of pancreatic islet. Here, we report a cDNA designed BmIHoP encoding an IHop homolog cloned from the fat body of the silkworm, Bombyx mori, using conserved domain homology search method. The resultant BmIHoP translated into a protein that encodes 693 amino acids, with a theoretical isoelectric point of 5.15 and a molecular weight of 77.5 kDa. Homology analysis revealed that BmIHoP has higher similarity at the amino acid level with those of other species, 52% identity with H. sapiens, 52% with M. musculus, 51% with D. rerio, and 73% with D. melanogaster. The expression of BmIHoP was detected in all tissues tested with 4 fold higher expression in the midgut, Malpighian, ovary and testis, and stronger expression in the larval fat body at 5th-3rd instar. The overexpression of BmIHoP in the β-cells reduced insulin secretion, which is not associated with SOX17 regulation. To the best of our knowledge, this is the first report of B. mori IHoP cDNA, BmIHoP, and its associated role in controlling insulin secretion from the β-cells.

Comparison of Gene Expression in Larval Fat Body of Helicoverpa assulta in Different Temperature Conditions

Comparison of Gene Expression in Larval Fat Body of Helicoverpa assulta in Different Temperature Conditions

E2F/DP Prevents Cell-Cycle Progression in Endocycling Fat Body Cells by Suppressing dATM Expression

To understand the consequences of the complete elimination of E2F regulation, we profiled the proteome of Drosophila dDP mutants that lack functional E2F/DP complexes. The results uncovered changes in the larval fat body, a differentiated tissue that grows via endocycles. We report an unexpected mechanism of E2F/DP action that promotes quiescence in this tissue. In the fat body, dE2F/dDP limits cell-cycle progression by suppressing DNA damage responses. Loss of dDP upregulates dATM, allowing cells to sense and repair DNA damage and increasing replication of loci that are normally under-replicated in wild-type tissues. Genetic experiments show that ectopic dATM is sufficient to promote DNA synthesis in wild-type fat body cells. Strikingly, reducing dATM levels in dDP-deficient fat bodies restores cell-cycle control, improves tissue morphology, and extends animal development. These results show that, in some cellular contexts, dE2F/dDP-dependent suppression of DNA damage signaling is key for cell-cycle control and needed for normal development.

Nitric Oxide Mediates Insect Cellular Immunity via Phospholipase A2 Activation

After infection or invasion is recognized, biochemical mediators act in signaling insect immune functions. These include biogenic amines, insect cytokines, eicosanoids, and nitric oxide (NO). Treating insects or isolated hemocyte populations with different mediators often leads to similar results. Separate treatments with an insect cytokine, 2 biogenic amines, and an eicosanoid lead to a single result, hemocyte spreading, understood in terms of intracellular cross-talk among these signaling systems. This study focuses on the cross-talk between NO and eicosanoid signaling in our model insect, Spodoptera exigua. Bacterial injection increased NO concentrations in the larval hemocytes and fat body, and RNA interference (RNAi) of the S. exigua NO synthase (NOS) gene suppressed NO concentrations. RNAi treatment also led to a significant reduction in hemocyte nodulation following bacterial injection. Similar RNAi treatments led to significantly reduced PLA₂ activities in the hemocytes and fat body compared to control larvae. Injection of <smlcap>L</smlcap>-NAME also prevented the induction of PLA₂ activity following bacterial challenge. An injected NO donor, S-nitroso-N-acetyl-DL-penicillamine, increased PLA₂ activity in a dose-dependent manner. However, eicosanoids did not influence NO concentrations in immune-challenged larvae. We infer that NO and eicosanoid signaling operate via cross-talk mechanisms in which the elevated NO concentrations activate PLA₂ and eicosanoid biosynthesis, which finally mediates various immune responses.

Metabolic adjustment of the larval fat body in Hermetia illucens to dietary conditions

The black soldier fly (BSF), Hermetia illucens, has great economic importance because of its ability to degrade a wide variety of organic products, including vegetable waste. The conversion of organic waste into valuable nutrients, that can be isolated from larvae and prepupae, is a widely exploited strategy to produce protein for animal feed and, consequently, comprehending the mechanisms that regulate nutrient accumulation in this insect could improve the production of insect-derived meal quantitatively and qualitatively.Since the fat body participates in metabolizing proteins, fat, and sugars in insects, detailed knowledge of this organ and of its modifications in relation to insect food intake could provide interesting clues about the nutritional value of the larvae, a fundamental aspect from an applied perspective. To this end, we performed a morphofunctional and molecular characterization of the fat body of sixth instar H. illucens larvae reared on different food substrates, focusing on markers related to nutrient accumulation.We demonstrate that a protein-poor diet affects both lipid and protein accumulation in fat body cells as well as the expression of key genes involved in these metabolic processes. Our study not only represents the first characterization of the larval fat body in this insect, but also confirms the central role of this organ in nutrient accumulation and substantiates the hypothesis of producing larvae with higher nutritional value by manipulating the diet.

Inter-adipocyte Adhesion and Signaling by Collagen IV Intercellular Concentrations in Drosophila

Sheet-forming Collagen IV is the main component of basement membranes, which are planar polymers of extracellular matrix underlying epithelia and surrounding organs in all animals. Adipocytes in both insects and mammals are mesodermal in origin and often classified as mesenchymal. However, they form true tissues where cells remain compactly associated. Neither the mechanisms providing this tissue-level organization nor its functional significance are known. Here we show that discrete Collagen IV intercellular concentrations (CIVICs), distinct from basement membranes and thicker in section, mediate inter-adipocyte adhesion in Drosophila. Loss of these Collagen-IV-containing structures in the larval fat body caused intercellular gaps and disrupted continuity of the adipose tissue layer. We also found that Integrin and Syndecan matrix receptors attach adipocytes to CIVICs and direct their formation. Finally, we show that Integrin-mediated adhesion to CIVICs promotes normal adipocyte growth and prevents autophagy through Src-Pi3K-Akt signaling. Our results evidence a surprising non-basement membrane role of Collagen IV in non-epithelial tissue morphogenesis while demonstrating adhesion and signaling functions for these structures.

Identification and expression analysis of multiple small heat shock protein genes in spruce budworm, Choristoneura fumiferana (L.).

Fifteen small heat shock protein (sHSP) genes were identified from spruce budworm, Choristoneura fumiferana (L.), an important native forest pest in North America. The transcript levels of each CfHSP were measured under non-stress conditions in all life stages from egg to adult and in five different larval tissues. CfHSP transcript levels showed variation during development, with highest levels in adults and lowest in eggs. Most CfHSP transcripts are highly expressed in larval fat body and Malpighian tubules; two CfHSPs display extremely high expression in the head and epidermis. Upon heat stress, nine CfHSP genes are significantly upregulated, increasing by 50- to 2500-fold depending on developmental stage and tissue type. Upon starvation, eight CfHSPs are upregulated or downregulated, whereas six others retain constant expression. These results suggest that CfHSPs have important and multiple roles in spruce budworm development and in response to heat stress and starvation.