CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor.

Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.

Transportin-serine/arginine-rich (Tnpo-SR) proteins are necessary for proper lipid storage in the Drosophila fat body.

After a meal, excess nutrients are stored within adipose tissue as triglycerides in structures called lipid droplets. Previous genome-wide RNAi screens have identified that mRNA splicing factor genes are required for normal lipid droplet formation in Drosophila cells. We have previously shown that mRNA splicing factors called serine/arginine-rich (SR) proteins are important for triglyceride storage in the Drosophila fat body. SR proteins shuttle in and out of the nucleus with the help of proteins called Transportins (Tnpo-SR); however, whether this transport is important for SR protein-mediated regulation of lipid storage is unknown. The purpose of this study is to characterize the role of Tnpo-SR proteins in regulating lipid storage in the Drosophila fat body. Decreasing Tnpo-SR in the adult fat body resulted in an increase in triglyceride storage and consistent with this phenotype, Tnpo-SR-RNAi flies also have increased starvation resistance. In addition, the lipid accumulation in Tnpo-SR-RNAi flies is the result of increased triglyceride stored in each fat body cell and not due to increased food consumption. Interestingly, the splicing of CPT1, an enzyme important for the ß-oxidation of fatty acids, is altered in Tnpo-SR-RNAi fat bodies. The isoform that produces the less catalytically active form of CPT1 accumulates in fat bodies where Tnpo-SR levels are decreased, suggesting a decrease in lipid breakdown, potentially causing the excess triglyceride storage observed in these flies. Together, these data suggest that the transport of splicing proteins in and out of the nucleus is important for proper triglyceride storage in the Drosophila fat body.

Heliothis virescens ascovirus 3h blocks the cell cycle of Spodoptera exigua fat body cells at G2 /M phase by downregulating cyclin B1 and cyclin-dependent kinase 1.

Ascoviruses are double-stranded DNA viruses that are pathogenic to noctuid larvae. In vitro infection causes the cells to fail to replicate and proliferate normally. However, the molecular mechanisms are unclear. In this study, the transmission electron microscopy data of infected-Spodoptera exigua (Hübner) fat body cells (SeFB, IOZCAS-SpexII-A cells) showed that virions were internalized in phagocytic vesicles, but not in the nucleus. FACS of cell-cycle progression was performed in SeFB cells infected with Heliothis virescens ascovirus 3h (HvAV-3h). The cell cycle phase distributions of the SeFB cells were G1 = 29.52 ± 1.10%, S = 30.33 ± 1.19%, and G2 /M = 40.06 ± 0.75%. The cell culture doubling time was approximately 24 h. The G1 , S, and G2 /M phases were each approximately 8 h. The unsynchronized or synchronized cells were arrested at G2 /M phase after infection with HvAV-3h. Our data also showed that cells with more than 4N DNA content appeared in the HvAV-3h-treated group. While the mRNA levels of cyclin B1 , cyclin H, and cyclin-dependent kinase 1 (CDK1) were downregulated after HvAV-3h infection, the mRNA expression levels of cyclin A, cyclin D, and cyclin B2 were not significantly changed. Western blotting results showed that the expression of cyclin B1 and CDK1 in infected SeFB cells within 24 h postinfection (hpi), and HvAV-3h infection inhibited the expression of cyclin B1 and CDK1 at 12-24 hpi. Overall, these data implied that HvAV-3h infection leads to an accumulation of cells in the G2 /M phases by downregulating the expression of cyclin B1 and CDK1.

Fear-of-intimacy-mediated zinc transport controls fat body cell dissociation through modulating Mmp activity in Drosophila.

Matrix metalloproteinases (Mmps) are pivotal extracellular proteinases that have been implicated in tumour invasion and metastasis. Drosophila fat body is important for energy storage and utilization, as well as biosynthetic and metabolic activities. The fat body undergoes remodelling during metamorphosis which is characterized by the dissociation of the fat body into individual cells. Mmps play important roles in the regulation of fat body cell dissociation. Here we show that a zinc transporter fear-of-intimacy (foi) is necessary for the cell dissociation of fat body in Drosophila. The progression of fat body cell dissociation was delayed by fat body-specific foi knockdown while it was accelerated by foi overexpression (OE). In essence, these phenotypes are closely associated with intracellular zinc homeostasis, which can be modulated by dietary zinc intervention or genetic modulation of other zinc transporters. Further study indicated that Mmp1 and Mmp2 levels could be transcriptionally regulated by zinc in vivo. Consistently, the retarded fat body cell dissociation caused by Mmp1 or Mmp2 RNAi could be regulated by modulating the expression of foi. Further, by using Drosophila models of malignant tumour RafGOFscrib-/- and RasV12lgl-/-, we showed that the tumour growth, invasion and migration could be markedly inhibited by foi knockdown. These findings demonstrate a close connection between zinc levels and cell dissociation in vivo, and also suggest that manipulation of zinc levels may provide a novel therapeutic strategy for cancer.

Segmentation of the subcuticular fat body in Apis mellifera females with different reproductive potentials.

Evolution has created different castes of females in eusocial haplodiploids. The difference between them lies in their functions and vulnerability but above all in their reproductive potentials. Honeybee queens are highly fertile. On the other hand, the workers are facultatively sterile. However, rebel workers, i.e. workers that develop in a queenless colony, reproduce more often than normal workers. As a result, the fat body of these bees, which apart from acting as the energy reserve, is also the site of numerous metabolic processes, had to specialize in different functions perfected over millions of years of eusocial evolution. Assuming that the variety of functions manifests itself in the pleomorphic structure of the fat body cells, we predicted that also different parts of the fat body, e.g. from different segments of the abdomen, contain different sets of cells. Such differences could be expected between queens, rebels and normal workers, i.e. females with dramatically different reproductive potentials. We confirmed all these expectations. Although all bees had the same types of cells, their proportion and segmental character corresponded with the caste reproductive potential and physiological characteristics shaped in the evolutionary process. The females with an increased reproductive potential were characterized by the presence of oenocytes in the third tergite and high concentrations of compounds responsible for energy reserves, like glucose, glycogen and triglycerides. Queens had very large trophocytes, especially in the third tergite. Only in workers did we observe intercellular spaces in all the segments of the fat body, as well as high protein concentrations-especially in the sternite. As expected, the rebels combined many features of the queens and normal workers, what with other findings can help understand the ways that led to the origin of different castes in females of eusocial Hymenoptera.

Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and lipid-independent mechanisms.

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1-CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1-CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1-CTDNEP1 downregulation also restores nuclear pore membrane fusion in post-mitotic dTorsinKO fat body cells. However, dTorsin-associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel-like structures in place of mature nuclear pores. We conclude that the NEP1R1-CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid-dependent and lipid-independent mechanisms, explaining some of the pleiotropic effects of Torsins.

Differential metabolic sensitivity of insulin-like-response- and TORC1-dependent overgrowth in Drosophila fat cells.

Glycolysis and fatty acid (FA) synthesis directs the production of energy-carrying molecules and building blocks necessary to support cell growth, although the absolute requirement of these metabolic pathways must be deeply investigated. Here, we used Drosophila genetics and focus on the TOR (Target of Rapamycin) signaling network that controls cell growth and homeostasis. In mammals, mTOR (mechanistic-TOR) is present in two distinct complexes, mTORC1 and mTORC2; the former directly responds to amino acids and energy levels, whereas the latter sustains insulin-like-peptide (Ilp) response. The TORC1 and Ilp signaling branches can be independently modulated in most Drosophila tissues. We show that TORC1 and Ilp-dependent overgrowth can operate independently in fat cells and that ubiquitous over-activation of TORC1 or Ilp signaling affects basal metabolism, supporting the use of Drosophila as a powerful model to study the link between growth and metabolism. We show that cell-autonomous restriction of glycolysis or FA synthesis in fat cells retrains overgrowth dependent on Ilp signaling but not TORC1 signaling. Additionally, the mutation of FASN (Fatty acid synthase) results in a drop in TORC1 but not Ilp signaling, whereas, at the cell-autonomous level, this mutation affects none of these signals in fat cells. These findings thus reveal differential metabolic sensitivity of TORC1- and Ilp-dependent growth and suggest that cell-autonomous metabolic defects might elicit local compensatory pathways. Conversely, enzyme knockdown in the whole organism results in animal death. Importantly, our study weakens the use of single inhibitors to fight mTOR-related diseases and strengthens the use of drug combination and selective tissue-targeting.

Effects of short- and long-term exposure to cadmium on salivary glands and fat body of soil centipede Lithobius forficatus (Myriapoda, Chilopoda): Histology and ultrastructure.

Cadmium (Cd) is the most widely studied heavy metal in terms of food-chain accumulation and contamination because it can strongly affect all environments (e.g., soil, water, air). It can accumulate in different tissues and organs and can affect the organism at different levels of organization: from organs, tissues and cells though cell organelles and structures to activation of mechanisms of survival and cell death. In soil-dwelling organisms heavy metals gather in all tissues with accumulation properties: midgut, salivary glands, fat body. The aim of this study was to describe the effects of cadmium on the soil species Lithobius forficatus, mainly on two organs responsible for gathering different substances, the fat body and salivary glands, at the ultrastructural level. Changes caused by cadmium short- and long-term intoxication, connected with cell death (autophagy, apoptosis, necrosis), and the crosstalk between them, were analyzed. Adult specimens of L. forficatus were collected in a natural environment and divided into three experimental groups: C (the control group), Cd1 (cultured in soil with 80 mg/kg of CdCl2 for 12 days) and Cd2 (cultured in soil with 80 mg/kg of CdCl2 for 45 days). Transmission electron microscopy revealed ultrastructural alterations in both of the organs analyzed (reduction in the amount of reserve material, the appearance of vacuoles, etc.). Qualitative analysis using TUNEL assay revealed distinct crosstalk between autophagy and necrosis in the fat body adipocytes, while crosstalk between autophagy, apoptosis and necrosis in the salivary glands was detected in salivary glands of the centipedes examined here. We conclude that different organs in the body can react differently to the same stressor, as well as to the same concentration and time of exposure. Different mechanisms at the ultrastructural level activate different types of cell death and with different dynamics.

The predicted collagen-binding domains of Drosophila SPARC are essential for survival and for collagen IV distribution and assembly into basement membranes.

The assembly of basement membranes (BMs) into tissue-specific morphoregulatory structures requires non-core BM components. Work in Drosophila indicates a principal role of collagen-binding matricellular glycoprotein SPARC (Secreted Protein, Acidic, Rich in Cysteine) in larval fat body BM assembly. We report that SPARC and collagen IV (Col(IV)) first colocalize in the trans-Golgi of hemocyte-like cell lines. Mutating the collagen-binding domains of Drosophila SPARC led to the loss of colocalization with Col(IV), a fibrotic-like BM, and 2nd instar larval lethality, indicating that SPARC binding to Col(IV) is essential for survival. Analysis of this mutant at 2nd instar reveals increased Col(IV) puncta within adipocytes, reflecting a disruption in the intracellular chaperone-like activity of SPARC. Removal of the disulfide bridge in the C-terminal EF-hand2 of SPARC, which is known to enhance Col(IV) binding, did not lead to larval lethality; however, a less intense fat body phenotype was observed. Additionally, both SPARC mutants exhibited altered fat body BM pore topography. Wing imaginal disc-derived SPARC did not localize within Col(IV)-rich matrices. This raises the possibility that SPARC interaction with Col(IV) requires initial intracellular interaction to colocalize at the BM or that wing-derived SPARC undergoes differential post-translational modifications that impacts its function. Collectively, these data provide evidence that the chaperone-like activity of SPARC on Col(IV) begins just prior to their co-secretion and demonstrate for the first time that the Col(IV) chaperone-like activity of SPARC is necessary for Drosophila development beyond the 2nd instar.

The role of the heterogeneous nuclear ribonucleoprotein (hnRNP) Hrb27C in regulating lipid storage in the Drosophila fat body.

The storage of excess nutrients as triglycerides is essential for all organisms to survive when food is scarce; however, the mechanisms by which triglycerides are stored are not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing that are important in the regulation of triglyceride storage. Our lab has identified a number of splicing factors important for regulating lipid metabolism; however, the full complement of splicing proteins involved in achieving metabolic homeostasis is unknown. Heterogeneous nuclear ribonucleoproteins (hnRNPs), RNA binding proteins that inhibit the splicing of introns by preventing the assembly of splicing complexes, have no established metabolic functions. To assess any metabolic functions of hnRNPs, we used the GAL4/UAS system to induce RNAi to six hnRNP's: hnRNP-K, rumpelstiltskin (rump), smooth (sm), Hrb27C (also referred to as Hrp48), Hrb98DE, and Hrb87F in the Drosophila fat body. Decreasing the levels of hnRNP-K and rump resulted in a decrease in triglyceride storage, whereas decreasing the levels of sm, Hrb27C, and Hrb98DE resulted in an increase in triglyceride storage. The excess triglyceride phenotype in Hrb27C-RNAi flies resulted from both an increase in the number of fat body cells and the amount of fat stored per cell. In addition, both the splicing of the ß-oxidation gene, CPT1, and the expression of the lipase brummer (bmm) was altered in flies with decreased Hrb27C, providing insight into the lipid storage phenotype in these flies. Together, these results suggest that the hnRNP family of splicing factors have varying metabolic functions and may act on specific metabolic genes to control their expression and processing.

Autophagic paintings: In the frontier of art and science.

As a PhD student I explored macroautophagy/autophagy induced by starvation in Drosophila melanogaster using different microscopy techniques. The beauty and complexity of this process impressed me so deeply that I felt the need to paint it. Thus, I made 2 oil paintings based on my own scientific work, representing the autophagy mechanism at different scales with diverse artistic resources. The first painting, called Autophagy 1, is inspired by fluorescence confocal microscopy images. Therefore, saturated colors predominate in the composition. The second one is an oil on canvas titled Autophagy 2 which reflects autophagy at a smaller scale. This painting depicts this process as revealed by transmission electron microscopy, employing mainly a gray scale of colors. I performed these works with the intention to catch the essence of this biological process, conveying scientific ideas through art. My paintings are not intended only for the scientific community but also for the general public, as an instrument of enjoyment and popularization of science.

The Fat Body of the Hematophagous Insect, Panstrongylus megistus (Hemiptera: Reduviidae): Histological Features and Participation of the ß-Chain of ATP Synthase in the Lipophorin-Mediated Lipid Transfer.

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.

Characterization of fat body cells at different developmental stages of Culex pipiens.

The fat body, originates from mesoderm, has many metabolic functions which changes as the embryonic development of the insect progresses. It plays an important role in the intermediate metabolism and in the metabolism of proteins, lipids and carbohydrates. It has roles in synthesis, absorption and storage of nutrients from hemolymph. It is also responsible for the production of immunological system components, antibacterial compounds and blood clotting proteins. The most common type of fat body cells are trophocytes (the basic cells of the fat body) and oenocytes are found associated with the fat body. In this study, it is aimed at determining the cell types contained in the fat body of Culex pipiens at different developmental stages as well as identifying the molecules such as carbohydrate, protein and lipid contained in each of these cells. Knowing the regional distribution of the fat body cells and the concentration of its content at each developmental stage is important in understanding the process related to its physiology and it may help in fighting against the pest C. pipiens, which is a vector species for many contagious diseases observed in humans and other species. To achieve our goal, we have employed different histochemical techniques (fixatives and staining methods) for staining C. pipiens preparates of different developmental stages and analyzed the structure of the fat body, its distribution, its cell types and the macromolecular contents of the cells. We only observed trophocytes and oenocytes as fat body components in C. pipiens. The trophocytes had all the three macromolecules (lipids, proteins, carbohydrates) in the cytoplasm varying in concentration between the different regions and different stages. The oenocytes were observed below the integument as well as between the muscles in the larvae of Culex pipiens. They were present either as single cells or in clusters and also varied in size. Their cytoplasm was stained strongly for proteins when bromophenol blue staining was applied, but it was rather heterogeneous due to the lipid inclusions. On the contrary, oenocytes were not observed among the adult C. pipiens preparations.

Novel host immune evasion strategy of the endoparasitoid Drino inconspicuoides.

The tachinid fly Drino inconspicuoides (Diptera: Tachinidae) is an ovolarviparous endoparasitoid whose larvae develop in the host haemocoel and avoids the host immune system. In this study, we investigated the immune evasion mechanisms of this species during infestation in the host Mythimna separata (Lepidoptera: Noctuidae). We discovered a unique 'cloak' that surrounded D. inconspicuoides larvae that penetrated into the host and determined through genomic polymerase chain reaction analysis that this structure originated from the host rather than the tachinid. The 'cloak' contained both haemocytes and fat body cells from the host, with the haemocytes assembling around the larvae first and the fat body cells then covering the haemocyte layer, following which the two mixed. Living D. inconspicuoides larvae that were wrapped in the 'cloak' were not melanized whereas encapsulated dead larvae were melanized, suggesting that this structure contributes to the avoidance of host immune reactions.

Modulation of antiviral immunity by the ichnovirus HdIV in Spodoptera frugiperda.

Polydnaviruses (PDVs) are obligatory symbionts found in thousands of endoparasitoid species and essential for successful parasitism. The two genera of PDVs, ichnovirus (IV) and bracovirus (BV), use different sets of virulence factors to ensure successful parasitization of the host. Previous studies have shown that PDVs target apoptosis, one of the innate antiviral responses in many host organisms. However, IV and BV have been shown to have opposite effects on this process. BV induces apoptosis in host cells, whereas some IV proteins have been shown to have anti-apoptotic activity. The different biological contexts in which the assays were performed may account for this difference. In this study, we evaluated the interplay between apoptosis and the ichnovirus HdIV from the parasitoid Hyposoter didymator, in the HdIV-infected hemocytes and fat bodies of S. frugiperda larvae, and in the Sf9 insect cell line challenged with HdIV. We found that HdIV induced cell death in hemocytes and fat bodies, whereas anti-apoptotic activity was observed in HdIV-infected Sf9 cells, with and without stimulation with viral PAMPs or chemical inducers. We also used an RT-qPCR approach to determine the expression profiles of a set of genes known to encode key components of the other main antiviral immune pathways described in insects. The analysis of immune gene transcription highlighted differences in antiviral responses to HdIV as a function of host cell type. However, all these antiviral pathways appeared to be neutralized by low levels of expression for the genes encoding the key components of these pathways, in all biological contexts. Finally, we investigated the effect of HdIV on the general antiviral defenses of the lepidopteran larvae in more detail, by studying the survival of S. frugiperda co-infected with HdIV and the entomopathogenic densovirus JcDV. Coinfected S. frugiperda larvae have increased resistance to JcDV at an early phase of infection, whereas HdIV effects enhance the virulence of the virus at later stages of infection. Overall, these results reveal complex interactions between HdIV and its cellular environment.

Pest and natural enemy: how the fat bodies of both the southern armyworm Spodoptera eridania and the predator Ceraeochrysa claveri react to azadirachtin exposure.

The effects of biopesticides on insects can be demonstrated by morphological and ultrastructural tools in ecotoxicological analysis. Azadirachtin-based products are widely used as biopesticides, affecting numerous insect populations. Through morphological biomarkers, this study aimed to characterize the fat bodies of both the southern armyworm Spodoptera eridania and the predator Ceraeochrysa claveri after chronic exposure to azadirachtin. Larvae of S. eridania and C. claveri were fed with fresh purple lettuce leaves (Lactuca sativa) and egg clusters of Diatraea saccharalis treated with azadirachtin solution of 6 mg active ingredient (a.i.)/L and 18 mg a.i./L for 7 days, respectively. The biological data showed a significant reduction in survival and body mass in S. eridania and cytotoxic effects in the parietal and perivisceral fat bodies in both species. Ultrastructural cell damage was observed in the trophocytes of both species such as dilated cisternae of the rough endoplasmic reticulum and swollen mitochondria. Trophocytes of S. eridania and C. claveri of the parietal and perivisceral layers responded to those injuries by different cytoprotective and detoxification means such as an increase in the amount of cytoplasmic granules containing calcium, expression of heat shock protein (HSP)70/HSP90, and development of the smooth endoplasmic reticulum. Despite all the different means of cytoprotection and detoxification, they were not sufficient to recover from all the cellular damages. Azadirachtin exhibited an excellent performance for the control of S. eridania and a moderate selectivity for the predator C. claveri, which presents better biological and cytoprotective responses to chronic exposure to azadirachtin.

Matrix metalloproteinases promote fat body cell dissociation and ovary development in Bombyx mori.

Matrix metalloproteinases (Mmps) are pivotal extracellular proteinases participating in tissue remodeling. Three Mmps genes have been identified from the silkworm, Bombyx mori, and their expression levels and enzyme activity are consistent with progressive fat body cell dissociation during the early pupal stages. Using both loss-of-function and gain-of-function experiments, we have demonstrated that Mmps are functionally required for fat body cell dissociation and ovary development in female pupae. Moderate inhibition of Mmps activity via inhibitor treatments delayed fat body cell dissociation and ovary development, while severe inhibition blocked these developmental processes and eventually led to pupal lethality. Individual RNAi knockdown of each Mmp delayed fat body cell dissociation, with the strongest and weakest phenotypes occurring for Mmp3 and Mmp1, respectively. By contrast, overexpression of each Mmp promoted fat body cell dissociation and ovary development, with the strongest stimulatory effects for Mmp3 overexpression and the weakest effects for Mmp1 overexpression. This is the first time to show that Mmps induce fat body cell dissociation in Lepidoptera, and we also hypothesize that Mmps-induced fat body cell dissociation is required for ovary development in this insect species.

Role for Transferrin in Triggering Apoptosis in Helicoverpa armigera Cells Treated with 2-Tridecanone.

2-Tridecanone, a plant allelochemical present in a large range of tomato species ( Lycopersicon hirsutum f. glabratum), can induce the expression of Helicoverpa armigera transferrin ( HaTrf), which is necessary for insect growth and development. To gain further insight into the mechanism of HaTrf in response to 2-tridecanone, we measured the iron and H2O2 levels in the hemolymph during exposure to 2-tridecanone and then explored the effect of transferrin downregulation in a H. armigera fat body cell line exposed to 2-tridecanone. We found that the reduction of HaTrf levels via RNA interference caused rapid apoptotic cell death during exposure to 2-tridecanone. There have been no reports about transferrin genes related to apoptosis induced by plant allelochemicals. Our results indicate that HaTrf mediates the inhibition of apoptotic cell death during exposure to 2-tridecanone and provides insight into the importance of transferrin in the interaction between plants and insects.

Insect fat body cell morphology and response to cold stress is modulated by acclimation.

Mechanistic understanding about the nature of cellular cryoinjury and mechanisms by which some animals survive freezing while others do not is currently lacking. Here, we exploited the broadly manipulable freeze tolerance of larval malt flies (Chymomyza costata) to uncover cell and tissue morphological changes associated with freeze mortality. Diapause induction, cold acclimation and dietary proline supplementation generate malt fly variants ranging from weakly to extremely freeze tolerant. Using confocal microscopy and immunostaining of the fat body, Malpighian tubules and anterior midgut, we described tissue and cytoskeletal (F-actin and α-tubulin) morphologies among these variants after exposure to various cold stresses (from chilling at -5°C to extreme freezing at -196°C), and upon recovery from cold exposure. Fat body tissue appeared to be the most susceptible to cryoinjury: freezing caused coalescence of lipid droplets, loss of α-tubulin structure and apparent aggregation of F-actin. A combination of diapause and cold acclimation substantially lowered the temperature at which these morphological disruptions occurred. Larvae that recovered from a freezing challenge repaired F-actin aggregation but not lipid droplet coalescence or α-tubulin structure. Our observations indicate that lipid coalescence and damage to α-tubulin are non-lethal forms of freeze injury, and suggest that repair or removal (rather than protection) of actin proteins is a potential mechanism of acquired freeze tolerance.

Juvenile hormone promotes locust fat body cell polyploidization and vitellogenesis by activating the transcription of Cdk6 and E2f1.

Juvenile hormone (JH) is known to promote cell polyploidization for insect vitellogenesis and egg production, but the underlying mechanisms remain poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that the expression of cyclin-dependent kinase 6 (Cdk6) and adenovirus E2 factor-1 (E2f1), the core mediators in cell cycle progression is regulated by JH and its receptor Methoprene-tolerant (Met). JH acts through its receptor complex comprised of Met and Taiman to directly activate the transcription of Cdk6 and E2f1. Depletion of Cdk6 or E2f1 results in significantly decreased ploidy, precocious mitotic entry and increased cell numbers in the fat body, accompanied by substantial reduction of Vitellogenin gene expression, blocked ovarian growth and arrested oocyte maturation. These findings indicate a crucial role of Cdk6 and E2f1 in JH-regulated polyploidization and vitellogenesis as well as a novel regulatory machinery for endocycling in insects.