Insect Fat Body Research Articles

Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli

Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD-intein-pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48h at 4°C, under which 12.3mg of recombinant pxCECA1 could be recovered from 1l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.

Vitellogenin content in fat body and ovary homogenates of workers and queens of Melipona quadrifasciata anthidioides during vitellogenesis

Abstract Vitellogenin (Vg) is an egg yolk protein that is produced primarily in the fat body of most female insects. In the advanced social structure of eusocial honeybees, the presence of the queen inhibits egg maturation in the workers’ ovaries. However in the stingless bee Melipona quadrifasciata, the workers always develop ovaries and lay a certain amount of eggs while provisioning the brood cells with larval food during what is known as the worker nurse phase. The present work is a comparative study of the presence of Vg in homogenates of the fat bodies and ovaries of the nurse workers, and the virgin and physogastric queens of M. quadrifasciata. The presence of Vg was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using Apis mellifera anti‐egg antibody. Vg was not detected in the fat bodies or ovaries of the workers, but it was found in the ovaries of virgin and physogastric queens and in the fat body of physogastric queens. The results are discussed, taking into account the reproductive state of the individuals and the other possible roles of Vg, such as a storage protein for metoabolism of other organs.

A comparative study of fat body morphology in five mosquito species

The insect fat body plays major roles in the intermediary metabolism, in the storage and transport of haemolymph compounds and in the innate immunity. Here, the overall structure of the fat body of five species of mosquitoes (Aedes albopictus, Aedes fluviatilis, Culex quinquefasciatus, Anopheles aquasalis and Anopheles darlingi) was compared through light, scanning and transmission electron microscopy. Generally for mosquitoes, the fat body consists of lobes projecting into the haemocoel and is formed by great cell masses consisting of trophocytes and oenocytes. Trophocytes are rich in lipid droplets and protein granules. Interestingly, brown pigment granules, likely ommochromes, were found exclusively in the trophocytes located within the thorax and near the dorsal integument of Anopheles, which is suggestive of the role these cells play in detoxification via ommochrome storage. This study provides a detailed comparative analysis of the fat body in five different mosquito species and represents a significant contribution towards the understanding of the structural-functional relationships associated with this organ.

Tissue-Specific Transcriptomics of the Exotic Invasive Insect Pest Emerald Ash Borer (Agrilus planipennis)

BackgroundThe insect midgut and fat body represent major tissue interfaces that deal with several important physiological functions including digestion, detoxification and immune response. The emerald ash borer (Agrilus planipennis), is an exotic invasive insect pest that has killed millions of ash trees (Fraxinus spp.) primarily in the Midwestern United States and Ontario, Canada. However, despite its high impact status little knowledge exists for A. planipennis at the molecular level.Methodology and Principal FindingsNewer-generation Roche-454 pyrosequencing was used to obtain 126,185 reads for the midgut and 240,848 reads for the fat body, which were assembled into 25,173 and 37,661 high quality expressed sequence tags (ESTs) for the midgut and the fat body of A. planipennis larvae, respectively. Among these ESTs, 36% of the midgut and 38% of the fat body sequences showed similarity to proteins in the GenBank nr database. A high number of the midgut sequences contained chitin-binding peritrophin (248)and trypsin (98) domains; while the fat body sequences showed high occurrence of cytochrome P450s (85) and protein kinase (123) domains. Further, the midgut transcriptome of A. planipennis revealed putative microbial transcripts encoding for cell-wall degrading enzymes such as polygalacturonases and endoglucanases. A significant number of SNPs (137 in midgut and 347 in fat body) and microsatellite loci (317 in midgut and 571 in fat body) were predicted in the A. planipennis transcripts. An initial assessment of cytochrome P450s belonging to various CYP clades revealed distinct expression patterns at the tissue level.Conclusions and SignificanceTo our knowledge this study is one of the first to illuminate tissue-specific gene expression in an invasive insect of high ecological and economic consequence. These findings will lay the foundation for future gene expression and functional studies in A. planipennis.

Discovery of a Novel Insect Neuropeptide Signaling System Closely Related to the Insect Adipokinetic Hormone and Corazonin Hormonal Systems

Neuropeptides and their G protein-coupled receptors (GPCRs) play a central role in the physiology of insects. One large family of insect neuropeptides are the adipokinetic hormones (AKHs), which mobilize lipids and carbohydrates from the insect fat body. Other peptides are the corazonins that are structurally related to the AKHs but represent a different neuropeptide signaling system. We have previously cloned an orphan GPCR from the malaria mosquito Anopheles gambiae that was structurally intermediate between the A. gambiae AKH and corazonin GPCRs. Using functional expression of the receptor in cells in cell culture, we have now identified the ligand for this orphan receptor as being pQVTFSRDWNAamide, a neuropeptide that is structurally intermediate between AKH and corazonin and that we therefore named ACP (AKH/corazonin-related peptide). ACP does not activate the A. gambiae AKH and corazonin receptors and, vice versa, AKH and corazonin do not activate the ACP receptor, showing that the ACP/receptor couple is an independent and so far unknown peptidergic signaling system. Because ACP is structurally intermediate between AKH and corazonin and the ACP receptor between the AKH and corazonin receptors, this is a prominent example of receptor/ligand co-evolution, probably originating from receptor and ligand gene duplications followed by mutations and evolutionary selection, thereby yielding three independent hormonal systems. The ACP signaling system occurs in the mosquitoes A. gambiae, Aedes aegypti, and Culex pipiens (Diptera), the silkworm Bombyx mori (Lepidoptera), the red flour beetle Tribolium castaneum (Coleoptera), the parasitic wasp Nasonia vitripennis (Hymenoptera), and the bug Rhodnius prolixus (Hemiptera). However, the ACP system is not present in 12 Drosophila species (Diptera), the honeybee Apis mellifera (Hymenoptera), the pea aphid Acyrthosiphon pisum (Hemiptera), the body louse Pediculus humanus (Phthiraptera), and the crustacean Daphnia pulex, indicating that it has been lost several times during arthropod evolution. In particular, this frequent loss of hormonal systems is unique for arthropods compared with vertebrates.

Cloning and expression of fat body hexamerin receptor and its identification in other hexamerin sequestering tissue of rice moth, Corcyra cephalonica

Selective receptor mediated uptake is a widely prevalent mechanism in insects by which important macromolecules are acquired. Among the various proteins sequestered by the insect fat body, the larval hexamerins form the major group. In the present work full length cDNA (2.6 kb) of hexamerin receptor with an ORF of 2.4 kb was cloned from the larval fat body of rice moth, Corcyra cephalonica. This was followed by the recombinant expression of truncated N-terminal sequence of putative hexamerin receptor and the confirmation of the expressed recombinant protein as the truncated hexamerin receptor by ligand blot analysis. Apart from this we also analyzed other hexamerin sequestering tissues like salivary gland, male accessory reproductive gland and ovary for the presence of hexamerin receptor. We found that the receptor in these tissues was similar in size and mode of activation to that of fat body hexamerin receptor, thus cementing the fact that identical hexamerin receptors are present in all the hexamerin sequestering tissues in the rice moth.

Coordination of Hepatocyte Nuclear Factor 4 and Seven-up Controls Insect Counter-defense Cathepsin B Expression

CmCatB, a cathepsin B-type cysteine protease, is insensitive to inhibition by the soybean cysteine protease inhibitor (scN). Cowpea bruchids dramatically induce CmCatB expression when major digestive proteases are inactivated by dietary scN, which is presumably an adaptive strategy that insects use to minimize effects of nutrient deficiency. In this study, we cloned the cowpea bruchid hepatocyte nuclear factor 4 (CmHNF-4) and demonstrated its involvement in transcriptional activation of CmCatB in the digestive tract of scN-adapted bruchids. Electrophoretic mobility shift assays demonstrated that CmHNF-4 binds to a CmCatB promoter region containing two tandem chicken ovalbumin upstream promoter (COUP) sites, which is also the cis-element for Seven-up (CmSvp), a previously identified transcriptional repressor of CmCatB. Although CmSvp is predominantly expressed in unadapted insect midgut, CmHNF-4 is more abundant in adapted bruchids. When transiently expressed in Drosophila S2 cells, CmHNF-4 substantially increased CmCatB expression through COUP binding. CmSvp inhibited CmHNF-4-mediated transcriptional activation even in the absence of its DNA-binding domain. Thus antagonism resulted, at least in part, from protein-protein interactions between CmSvp and CmHNF-4. Association of the two transcription factors was subsequently confirmed by glutathione S-transferase pulldown assays. Interestingly, anti-CmHNF-4 serum caused a supershift not only with nuclear extracts of scN-adapted insect midgut but with that of unadapted control insects as well. The presence of CmHNF-4 in unadapted insects further supported the idea that interplay between CmSvp and CmHNF-4 controls CmCatB transcription activation. Together, these results suggest that coordination between CmHNF-4 and CmSvp is important in counter-defense gene regulation in insects.

Insect Fat Body: Energy, Metabolism, and Regulation

The fat body plays major roles in the life of insects. It is a dynamic tissue involved in multiple metabolic functions. One of these functions is to store and release energy in response to the energy demands of the insect. Insects store energy reserves in the form of glycogen and triglycerides in the adipocytes, the main fat body cell. Insect adipocytes can store a great amount of lipid reserves as cytoplasmic lipid droplets. Lipid metabolism is essential for growth and reproduction and provides energy needed during extended nonfeeding periods. This review focuses on energy storage and release and summarizes current understanding of the mechanisms underlying these processes in insects.

First insight into the lipid uptake, storage and mobilization in arachnids: Role of midgut diverticula and lipoproteins

The importance of midgut diverticula (M-diverticula) and hemolymph lipoproteins in the lipid homeostasis of Polybetes phythagoricus was studied. Radioactivity distribution in tissues and hemolymph was analyzed either after feeding or injecting [1- 14C]-palmitate. In both experiments, radioactivity was mostly taken up by M-diverticula that synthesized diacylglycerols, triacylglycerols and phospholipids in a ratio close to its lipid class composition. M-diverticula total lipids represent 8.08% (by wt), mostly triacylglycerols (74%) and phosphatidylcholine (13%). Major fatty acids were (in decreasing order of abundance) 18:1 n − 9, 18:2 n − 6, 16:0, 16:1 n − 7, 18:0, 18:3 n − 3. Spider hemocyanin-containing lipoprotein (VHDL) transported 83% of the circulating label at short incubation times. After 24 h, VHDL and HDL-1 (comparable to insect lipophorin) were found to be involved in the lipid uptake and release from M-diverticula, HDL-2 playing a negligible role. Lipoprotein's labelled lipid changed with time, phospholipids becoming the main circulating lipid after 24 h. These results indicate that arachnid M-diverticula play a central role in lipid synthesis, storage and movilization, analogous to insect fat body or crustacean midgut gland. The relative contribution of HDL-1 and VHDL to lipid dynamics indicated that, unlike insects, spider VHDL significantly contributes to the lipid exchange between M-diverticula and hemolymph.

Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection

Insect hemocytes (blood cells) are a central part of the insect's cellular response to bacterial pathogens, and these specialist cells can both recognize and engulf bacteria. During this process, hemocytes undergo poorly characterized changes in adhesiveness. Previously, a peptide termed plasmatocyte-spreading peptide (PSP), which induces the adhesion and spreading of plasmatocytes on foreign surfaces, has been identified in lepidopteran insects. Here, we investigate the function of this peptide in the moth Manduca sexta using RNA interference (RNAi) to prevent expression of the precursor protein proPSP. We show that infection with the insect-specific bacterial pathogen Photorhabdus luminescens and non-pathogenic Escherichia coli induces proPSP mRNA transcription in the insect fat body but not in hemocytes; subsequently, proPSP protein can be detected in cell-free hemolymph. We used RNAi to silence this upregulation of proPSP and found that the knock-down insects succumbed faster to infection with P. luminescens, but not E. coli. RNAi-treated insects infected with E. coli showed a reduction in the number of circulating hemocytes and higher bacterial growth in hemolymph as well as a reduction in overall cellular immune function compared with infected controls. Interestingly, RNAi-mediated depletion of proPSP adversely affected the formation of melanotic nodules but had no additional effect on other cellular responses when insects were infected with P. luminescens, indicating that this pathogen employs mechanisms that suppress key cellular immune functions in M. sexta. Our results provide evidence for the central role of PSP in M. sexta cellular defenses against bacterial infections.

Hormonal and nutritional regulation of insect fat body development and function

The insect fat body is an organ analogue to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. Similar to other larval organs, fat body undergoes a developmental "remodeling" process during the period of insect metamorphosis, with the massive destruction of obsolete larval tissues by programmed cell death and the simultaneous growth and differentiation of adult tissues from small clusters of progenitor cells. Genetic ablation of Drosophila fat body cells during larval-pupal transition results in lethality at the late pupal stage and changes sizes of other larval organs indicating that fat body is the center for pupal development and adult formation. Fat body development and function are largely regulated by several hormonal (i.e. insulin and ecdysteroids) and nutritional signals, including oncogenes and tumor suppressors in these pathways. Combining silkworm physiology with fruitfly genetics might provide a valuable system to understand the mystery of hormonal regulation of insect fat body development and function.

Hormonal and nutritional regulation of insect fat body development and function

Hormonal and nutritional regulation of insect fat body development and function

Effects of methamidophos and deltamethrin on in vitro protein phosphorylation in Monochamus alternatus

Abstract Monochamus alternatus Hope (Coleoptera: Cerambycidae) is not only a serious pest insect to pine trees but also the main vector of pine wood nemadote Bursaphelenchus xylophilus, which causes pine wilt disease. To explore the insecticidal mechanism of insecticides to M. alternatus, we chose methamidophos and deltamethrin as the representatives of two groups of insecticides (organophosphates and pyrethroids), which are widely used for pest control in China and investigated their effects on phosphorylation of proteins from the insect. Phosphorylation of proteins from the insect fat body and head was determined by in vitro32P‐labelling. In the fat body, deltamethrin obviously reduced basal phosphorylation levels of proteins at 111, 95, 77, and 44 kDa, but enhanced the basal phosphorylation level of a protein at 138 kDa. However, in the presence of calmodulin but not cyclic adenosine monophosphate (cAMP), deltamethrin increased phosphorylation of the protein at 111 kDa. In the head, deltamethrin inhibited basal phosphorylation levels of proteins at 113, 98, and 51 kDa, but potentiated phosphorylation of a protein at 167 kDa activated by cAMP. Methamidophos inhibited phosphorylation of a protein at 44 kDa in the fat body. Although methamidophos did not impact basal phosphorylation levels of any proteins in the head, it inhibited calcium/calmodulin (Ca2+/CaM)‐stimulated phosphorylation of a protein at 51 kDa. Together, our data indicate that methamidophos and deltamethrin altered phosphorylation levels of various proteins in the head and fat body of the pine insect and these two kinds of insecticides acted on the proteins that can be phosphorylated in the tissues respectively, which is possibly related to their toxicity.

Delipidation of insect lipoprotein, lipophorin, affects its binding to the lipophorin receptor, LpR: Implications for the role of LpR-mediated endocytosis

The insect lipophorin receptor (LpR), an LDL receptor (LDLR) homologue that is expressed during restricted periods of insect development, binds and endocytoses high-density lipophorin (HDLp). However, in contrast to LDL, HDLp is not lysosomally degraded, but recycled in a transferrin-like manner, leaving a function of receptor-mediated uptake of HDLp to be uncovered. Since a hallmark of circulatory HDLp is its ability to function as a reusable shuttle that selectively loads and unloads lipids at target tissues without being endocytosed or degraded, circulatory HDLp can exist in several forms with respect to lipid loading. To investigate whether lipid content of the lipoprotein affects binding and subsequent endocytosis by LpR, HDLp was partially delipidated in vitro by incubation with α-cyclodextrin, yielding a particle of buoyant density 1.17 g/mL (HDLp-1.17). Binding experiments demonstrated that LpR bound HDLp-1.17 with a substantially higher affinity than HDLp both in LpR-transfected Chinese hamster ovary (CHO) cells and isolated insect fat body tissue endogenously expressing LpR. Similar to HDLp, HDLp-1.17 was targeted to the endocytic recycling compartment after endocytosis in CHO(LpR) cells. The complex of HDLp-1.17 and LpR appeared to be resistant to endosomal pH, as was recently demonstrated for the LpR–HDLp complex, corroborating that HDLp-1.17 is recycled similar to HDLp. This conclusion was further supported by the observation of a significant decrease with time of HDLp-1.17-containing vesicles after endocytosis of HDLp-1.17 in LpR-expressing insect fat body tissue. Collectively, our results indicate that LpR favors the binding and subsequent endocytosis of HDLp-1.17 over HDLp, suggesting a physiological role for LpR in selective endocytosis of relatively lipid-unloaded HDLp particles, while lipid reloading during their intracellular itinerary might result in decreased affinity for LpR and thus allows recycling.

The main triglyceride-lipase from the insect fat body is an active phospholipase A1: identification and characterization

The main triglyceride-lipase (TG-lipase) from the fat body of Manduca sexta has been identified as the homolog of Drosophila melanogaster CG8552. This protein is conserved among insects and also shares significant sequence similarity with vertebrate phospholipases (PLs) from the phosphatidic acid preferring-phospholipase A1 (PA-PLA(1)) family. It is shown here that the TG-lipase is also a PL. TG-lipase and PL activities copurify and are inhibited by, or resistant to, the same lipase inhibitors, indicating that both activities are catalyzed by the same enzyme and active site. The PL activity of TG-lipase corresponded to PL type A(1). The concentration dependence of lipase activity with TG and PL micellar substrates showed saturation kinetics, with apparent K(m) values of 152 +/- 11 and 7.8 +/- 1.1 muM, respectively. TG-lipase was able to hydrolyze the major phospholipid components of the lipid droplets, phosphatidylcholine and phosphatidylethanolamine. The enzyme hydrolyzes 77 molecules of TG for every molecule of PL contained in the lipid droplets. It was observed that the activation of lipolysis in vivo is accompanied by activation of the hydrolysis of phospholipids of the lipid droplets. These results suggest that the PL activity of the insect TG-lipase could be required to allow access of the lipase to TG molecules contained in the core of the lipid droplets.

Adipokinetic hormone‐induced mobilization of fat body triglyceride stores in Manduca sexta: Role of TG‐lipase and lipid droplets

Triglycerides (TG) stores build up in the insect fat body as lipid droplets at times of excess of food. The mobilization of fat body triglyceride (TG) is stimulated by adipokinetic hormones (AKH). The action of AKH involves a rapid activation of cAMP-dependent protein kinase (PKA). Recent in vitro studies have shown that PKA phosphorylates and activates the TG-lipase substrate, the lipid droplets. Conversely, purified TG-lipase from Manduca sexta fat body is phosphorylated by PKA in vitro but is not activated. This study was directed to learn whether or not AKH promotes a change in the state of phosphorylation of the lipase in vivo, and what are the relative contributions of cytosol and lipid droplets to the overall increase of lipolysis triggered by AKH. TG-lipase activity of fat body cytosols isolated from control and AKH-treated insects was determined against the native substrate, in vivo [3H]-TG radiolabeled lipid droplets, obtained from control and AKH-treated insects. The lipase activity of the system composed of AKH-cytosol and AKH-lipid droplets (11.1 +/- 2.1 nmol TG/min-mg) was 3.1-fold higher than that determined with control cytosol and lipid droplets (3.6 +/- 0.5 nmol TG/min-mg). Evaluation of the role of AKH-induced changes in the lipid droplets on lipolysis showed that changes in the lipid droplets are responsible for 70% of the lipolytic response to AKH. The remaining 30% appears to be due to AKH-dependent changes in the cytosol. However, the phosphorylation level of the TG-lipase was unchanged by AKH, indicating that phosphorylation of the TG-lipase plays no role in the activation of lipolysis induced by AKH.

Lysosomes as the target of yessotoxin in invertebrate and vertebrate cell lines

The toxic effects of the algal polyether phycotoxin yessotoxin (YTX) are studied in the insect fat body IPLB-LdFB and the mouse fibroblast NIH3T3 cell lines. Our experiments confirm the cytotoxic action exerted by the toxin in both insect and mammalian cells, but morphological observations, TUNEL experiments and electrophoretic evalution of DNA integrity failed to evidence a clear pro-apoptotic role for YTX. In both IPLB-LdFB and NIH3T3 cell lines, neutral red and acridine orange stainings, together with evaluation of acid phosphatase activity demonstrate that YTX first damages lysosomal vesicles. This is then followed by a progressive depolymerization of actin microfilaments, as shown by phalloidin fluorescent immunostaining. Overall, our data identify in early lysosomal damage and the subsequent cytoskeletal disruption two common steps related to YTX toxicity towards metazoan cells.

Activation of the Lipid Droplet Controls the Rate of Lipolysis of Triglycerides in the Insect Fat Body. A role for Lsdp1 (Lipid storage droplet protein 1)

The hydrolysis of triglyceride (TG) stored in the lipid droplets (LD) of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation of cAMP-dependent kinase cascade (PKA). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the LD was investigated. The activity of purified TG-lipase determined using in vivo TG-radiolabeled LD was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against LD isolated from AKH-stimulated fat bodies than unstimulated tissue. Investigation of the phosphorylation state of LD- associated proteins showed that in vivo stimulation of lipolysis promotes a rapid phosphorylation of a 42-44kDa protein. This protein was identified by mass spectrometry as Lsdp1. In vivo phosphorylation of this protein reached a peak ~10min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with LD isolated 10min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase, and LD, without additional cellular components. In vitro phosphorylation of LD catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the LD. Lsdp1 is the main targetof PKA, suggesting that this protein is a major player in theactivation of lipolysis in insects. Funding provided by OAES and NIH.

Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includens

Inducible expression of antimicrobial peptides and other humoral immune factors by the insect fat body is well documented. Hemocytes comprise the second essential arm of the insect immune system but it is unclear whether antimicrobial peptide genes are expressed by all or only some types of hemocytes. Here we report the cloning of cecropin A ( Pi-cecA), lebocin ( Pi-leb) and lysozyme ( Pi-lys) homologs from the moth Pseudoplusia includens. Relative-quantitative real-time PCR (rq-rtPCR) indicated that transcript abundance for each antimicrobial gene increased in fat body and hemocytes following immune challenge with the Gram-negative bacterium Escherichia coli. Relative transcript abundance of Pi-cecA was much higher in fat body than hemocytes. In contrast, transcript levels of Pi-leb were three-fold lower in hemocytes than fat body while transcript levels of Pi-lys were three-fold higher. Estimates for the overall contribution of the fat body and hemocytes to antimicrobial peptide expression suggested that hemocytes contribute significantly to Pi-lys transcript levels in larvae but produce much smaller amounts of Pi-cecA and Pi-leb compared to the fat body. Each antimicrobial peptide was also inducibly expressed in hemocytes following challenge with the Gram-positive bacterium Micrococcus luteus or when hemocytes formed capsules around chromatography beads. Analysis of hemocyte types indicated that granulocytes and plasmatocytes expressed all three antimicrobial peptides, whereas spherule cells and oenocytoids expressed only lysozyme. Transcriptional profiles of these antimicrobial genes were similar in granulocytes and plasmatocytes in vivo but were very different in vitro.

Activation of the Lipid Droplet Controls the Rate of Lipolysis of Triglycerides in the Insect Fat Body

The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA)). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the lipid droplet, was investigated in fat body adipocytes. The activity of purified TG-lipase determined using in vivo TG-radiolabeled lipid droplets was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against lipid droplets isolated from hormone-stimulated fat bodies than against lipid droplets isolated from unstimulated tissue. In vivo stimulation of lipolysis promotes a rapid phosphorylation of a lipid droplet protein with an apparent mass of 42-44 kDa. This protein was identified as "Lipid Storage Droplet Protein 1" (Lsdp1). In vivo phosphorylation of this protein reached a peak approximately 10 min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with lipid droplets isolated 10 min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase and lipid droplets. In vitro phosphorylation of lipid droplets catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase, demonstrating a prominent role PKA and protein phosphorylation on the activation of the lipid droplets. AKH-induced changes in the properties of the substrate do not promote a tight association of the lipase with the lipid droplets. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the lipid droplet. This activation is achieved by PKA-mediated phosphorylation of the lipid droplet. Lsdp1 is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects.