Critical Roles of Spätzle5 in Antimicrobial Peptide Production Against Escherichia coli in Tenebrio molitor Malpighian Tubules.

The dimeric cytokine ligand Spätzle (Spz) is responsible for Toll pathway activation and antimicrobial peptide (AMP) production upon pathogen challenge in Tenebrio molitor. Here, we indicated that TmSpz5 has a functional role in response to bacterial infections. We showed that the highest expression of TmSpz5 is induced by Candida albicans. However, TmSpz5 knockdown reduced larval survival against Escherichia coli and Staphylococcus aureus. To evaluate the molecular mechanism underlying the observed survival differences, the role of TmSpz5 in AMP production was examined by RNA interference and microbial injection. T. molitor AMPs that are active against Gram-negative and -positive bacteria, including Tmtenecins, Tmattacins, Tmcoleoptericins, Tmtaumatin-like-proteins, and Tmcecropin-2, were significantly downregulated by TmSpz-5 RNAi in the Malpighian tubules (MTs) following a challenge with E. coli and S. aureus. However, upon infection with C. albicans the mRNA levels of most AMPs in the dsTmSpz5-injected group were similar to those in the control groups. Likewise, the expression of the transcription factors NF-κB, TmDorX2, and TmRelish were noticeably suppressed in the MTs of TmSpz5-silenced larvae. Moreover, E. coli-infected TmSpz5 knockdown larvae showed decreased antimicrobial activity in the MTs and hindgut compared with the control group. These results demonstrate that TmSpz5 has a defined role in T. molitor innate immunity by regulating AMP expression in MTs in response to E. coli.

Renal Purge of Hemolymphatic Lipids Prevents the Accumulation of ROS-Induced Inflammatory Oxidized Lipids and Protects Drosophila from Tissue Damage.

Animals require complex metabolic and physiological adaptations to maintain the function of vital organs in response to environmental stresses and infection. Here, we found that infection or injury in Drosophila induced the excretion of hemolymphatic lipids by Malpighian tubules, the insect kidney. This lipid purge was mediated by a stress-induced lipid-binding protein, Materazzi, which was enriched in Malpighian tubules. Flies lacking materazzi had higher hemolymph concentrations of reactive oxygen species (ROS) and increased lipid peroxidation. These flies also displayed Malpighian tubule dysfunction and were susceptible to infections and environmental stress. Feeding flies with antioxidants rescued the materazzi phenotype, indicating that the main role of Materazzi is to protect the organism from damage caused by stress-induced ROS. Our findings suggest that purging hemolymphatic lipids presents a physiological adaptation to protect host tissues from excessive ROS during immune and stress responses, a process that is likely to apply to other organisms.

TmDorX2 positively regulates antimicrobial peptides in Tenebrio molitor gut, fat body, and hemocytes in response to bacterial and fungal infection.

Dorsal, a member of the nuclear factor-kappa B (NF-κB) family of transcription factors, is a critical downstream component of the Toll pathway that regulates the expression of antimicrobial peptides (AMPs) against pathogen invasion. In this study, the full-length ORF of Dorsal was identified from the RNA-seq database of the mealworm beetle Tenebrio molitor (TmDorX2). The ORF of TmDorX2 was 1,482 bp in length, encoding a polypeptide of 493 amino acid residues. TmDorX2 contains a conserved Rel homology domain (RHD) and an immunoglobulin-like, plexins, and transcription factors (IPT) domain. TmDorX2 mRNA was detected in all developmental stages, with the highest levels observed in 3-day-old adults. TmDorX2 transcripts were highly expressed in the adult Malpighian tubules (MT) and the larval fat body and MT tissues. After challenging the larvae with Staphylococcus aureus and Escherichia coli, the TmDorX2 mRNA levels were upregulated 6 and 9 h post infection in the whole body, fat body, and hemocytes. Upon Candida albicans challenge, the TmDorX2 mRNA expression were found highest at 9 h post-infection in the fat body. In addition, TmDorX2-knockdown larvae exposed to E. coli, S. aureus, or C. albicans challenge showed a significantly increased mortality rate. Furthermore, the expression of 11 AMP genes was downregulated in the gut and fat body of dsTmDorX2-injected larvae upon E. coli challenge. After C. albicans and S. aureus challenge of dsTmDorX2-injected larvae, the expression of 11 and 10 AMPs was downregulated in the gut and fat body, respectively. Intriguingly, the expression of antifungal transcripts TmTenecin-3 and TmThaumatin-like protein-1 and -2 was greatly decreased in TmDorX2-silenced larvae in response to C. albicans challenge, suggesting that TmDorX2 regulates antifungal AMPs in the gut in response to C. albicans infection. The AMP expression profiles in the fat body, hemocytes, gut, and MTs suggest that TmDorX2 might have an important role in promoting the survival of T. molitor larvae against all mentioned pathogens.

Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells.

BACKGROUND: Drosophila is a powerful model for the study of factors modulating innate immunity. This study examines the effect of water-loss dehydration on innate immune responsiveness in the Drosophila renal system (Malpighian tubules; MTs), and how this leads to elevated host defense and contributes to immunosenescence. RESULTS: A short period of desiccation-elevated peptidoglycan recognition protein-LC (PGRP-LC) expression in MTs, increased antimicrobial peptide (AMP) gene induction, and protected animals from bacterial infection. We show that desiccation increased ecdysone synthesis in MTs, while inhibition of ecdysone synthesis or ecdysone receptor expression, specifically within MTs, prevented induction of PGRP-LC and reduced protection from bacterial infection. Additionally, aged flies are constitutively water-stressed and have elevated levels of ecdysone and PGRP-LC. Conversely, adults aged at high relative humidity show less water loss and have reduced expression of PGRP-LC and AMPs. CONCLUSIONS: The Drosophila renal system is an important contributor to host defense and can modulate immune responses in an organ autonomous manner, responding to environmental changes such as desiccation. Desiccation primes immune responsiveness by elevating PGRP-LC expression specifically in MTs. In response to desiccation, ecdysone is produced in MTs and acts in a paracrine fashion to increase PGRP-LC expression, immune responsiveness, and improve host defense. This activity of the renal system may contribute to the immunosenescence observed in Drosophila.

Characterization of a novel Cry8Ea3-binding V-ATPase Subunit A in Holotrichia parallela.

Several receptor proteins of Cry toxin have been previously identified, including cadherin-like, aminopeptidase N, and alkaline phosphatase. In the present work, a novel binding protein, V-ATPase subunit A (HpVAA), was identified in Holotricia parallela larvae and characterized. We performed reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends technology to obtain the cDNA of the full-length hpvaa. Sequencing analysis showed that the open reading frame of hpvaa (GenBank accession No. KU497557) is 1845 bp long, encoding 614 amino acid residues. The predicted molecular weight and isoelectric point of HpVAA were 67.85 kDa and 4.9, respectively. The HpVAA protein, which includes two putative conserved domains, ATP-synt_ab_N and ATP-synt_ab_C, and a Walker A (GAFGCGKT) motif and a Walker B (SMMAD) motif, possesses the same structural characteristics as V-ATPase subunit A from other insects. The protein was successfully expressed in Escherichia coli, and a ligand blot assay showed binding of the protein with Cry8Ea3 toxin. Transcriptional analysis of hpvaa in different tissues of H. parallela larvae was performed by qRT-PCR, which showed that the relative expression of hpvaa in the Malpighian tubules is higher than that in other tissues.

Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster.

In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. Here we provide evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. We show that early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. We also show that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down regulated and activation and translocation of transcription factor Relish is also affected.

Immune response and anti-microbial peptides expression in Malpighian tubules of Drosophila melanogaster is under developmental regulation.

Malpighian tubules (MT) of Drosophila melanogaster are osmoregulatory organs that maintain the ionic balance and remove toxic substances from the body. Additionally they act as autonomous immune sensing organs, which secrete antimicrobial peptides in response to invading microbial pathogens. We show that the antimicrobial peptides (AMP) diptericin, cecropinA, drosocin and attacinA are constitutively expressed and are regulated in developmental stage specific manner. Their developmental expression begins from 3(rd) instar larval stage and an immune challenge increases the expression several folds. Spatial variations in the level of expression along the MT tissue are observed. The mortality of 3(rd) instar larvae fed on bacterial food is much less than that of the earlier larval stages, coinciding with the onset of innate immunity response in MT. Ectopic expression of AMP imparts better resistance to infection while, loss of function of one of the AMP through directed RNAi reduces host survival after immune challenge. The AMP secreted from the MT exhibit bactericidal activity. Expression of the NF-κB transcription factor, Relish, also coincides with activation of immune responsive genes in MT, demonstrating that immune regulation in MT is under developmental control and is governed by the Imd pathway.

Insights into the Malpighian tubule from functional genomics.

Classical physiological study of the Malpighian tubule has led to a detailed understanding of fluid transport and its control across several species. With the sequencing of the Drosophila genome, and the concurrent development of post-genomic technologies such as microarrays, proteomics, metabolomics and systems biology, completely unexpected roles for the insect Malpighian tubule have emerged. As the insect body plan is simpler than that of mammals, tasks analogous to those performed by multiple mammalian organ systems must be shared out among insect tissues. As well as the classical roles in osmoregulation, the Malpighian tubule is highly specialized for organic solute transport, and for metabolism and detoxification. In Drosophila, the adult Malpighian tubule is the key tissue for defence against insecticides such as DDT; and it can also detect and mount an autonomous defence against bacterial invasion. While it is vital to continue to set insights obtained in Drosophila into the context of work in other species, the combination of post-genomic technologies and physiological validation can provide insights that might not otherwise have been apparent for many years.

Modulation of epithelial innate immunity by autocrine production of nitric oxide.

Mechanisms of innate immunity especially with relevance to epithelial tissue, are currently the focus of intense research, as epithelial immunity greatly impacts on health and disease. However, many findings regarding innate immunity signalling pathways in vertebrates stems from research using the genetic model Drosophila melanogaster. Here we discuss the central importance of epithelial tissues in innate immunity in Drosophila; the modulation of the Imd pathway via autocrine production of nitric oxide (NO); and the central importance of the Malpighian (renal) tubule in immune function of the whole animal.

PGRP-LC and PGRP-LE have essential yet distinct functions in the drosophila immune response to monomeric DAP-type peptidoglycan.

Drosophila rely entirely on an innate immune response to combat microbial infection. Diaminopimelic acid-containing peptidoglycan, produced by Gram-negative bacteria, is recognized by two receptors, PGRP-LC and PGRP-LE, and activates a homolog of transcription factor NF-kappaB through the Imd signaling pathway. Here we show that full-length PGRP-LE acted as an intracellular receptor for monomeric peptidoglycan, whereas a version of PGRP-LE containing only the PGRP domain functioned extracellularly, like the mammalian CD14 molecule, to enhance PGRP-LC-mediated peptidoglycan recognition on the cell surface. Interaction with the imd signaling protein was not required for PGRP-LC signaling. Instead, PGRP-LC and PGRP-LE signaled through a receptor-interacting protein homotypic interaction motif-like motif. These data demonstrate that like mammals, drosophila use both extracellular and intracellular receptors, which have conserved signaling mechanisms, for innate immune recognition.

The Malpighian tubule: rapid insights from post-genomic biology.

Good osmoregulation is critical to the success of insects, and the Malpighian tubules play a key role in osmoregulation. Recently, the application of genetics and genomics to the Drosophila tubule has revealed far more extensive roles than ion and water transport. Microarray analysis shows that organic solute transporters dominate the tubule transcriptome. The tubule thus has the capability to excrete actively the broadest range of organic solutes and xenobiotics. Such transporters can produce unexpected, emergent roles for the whole tissue; e.g. the tubule is highly resistant to ouabain not because the Na+, K+ ATPase is unimportant, but because it co-localises with a potent alkaloid excretory mechanism. Reinforcing this role in excretion, the tubule expresses very high levels of a particular cytochrome P450s, glutathione-S-transferases and alcohol dehydrogenases which suggest that the tubule plays a major role in metabolism and detoxification of both endogenous solutes and xenobiotics, such as insecticides. Additionally, the tubule plays a significant role in immunity; tubules are capable of sensing bacterial challenge, and mounting an effective killing response by secretion of antimicrobial peptides, entirely independent of the fat body, the canonical immune tissue. The tubule has also proved to be a good model for some human renal disease, and to act as an organotypic 'testbed' for mammalian genes. The tubule can thus bask in a greatly enhanced reputation as a key tissue for an unexpectedly wide range of functions in the insect.

Insect renal tubules constitute a cell-autonomous immune system that protects the organism against bacterial infection.

Innate immunity is a widespread and important defence against microbial attack, which in insects is thought to originate mainly in the fat body. Here we demonstrate that the fluid-transporting Malpighian (renal) tubule of Drosophila melanogaster constitutes an autonomous immune-sensing tissue utilising the nitric oxide (NO) signalling pathway. Reverse transcriptase PCR (RT-PCR) shows that tubules express those genes encoding components of the Imd pathway. Furthermore, isolated tubules bind and respond to lipopolysaccharide (LPS), by upregulating anti-microbial peptide (diptericin) gene expression and increased bacterial killing. Excised, LPS-challenged tubules, as well as tubules from LPS-infected flies, display increased NO synthase (NOS) activity upon immune challenge. Targetted expression of a Drosophila NOS (dNOS) transgene to only principal cells of the tubule main segment using the GAL4/UAS system increases diptericin expression. In live flies, such targetted over-expression of dNOS to tubule principal cells confers increased survival of the whole animal upon E. coli challenge. Thus, we describe a novel role of Malpighian tubules in immune sensing and insect survival.

An ultrastructural study on the early cellular response to Dirofilaria immitis (Nematoda) in the Malpighian tubules of Aedes aegypti (refractory strains).

Dirofilaria immitis living in Aedes aegypti refractory strains were studied in relation to ultrastructural events in primary cells of Malpighian tubules and to defense mechanisms activated by host-cells. When the microfilaria reaches the Malpighian cells, its intracellular development is blocked by defense mechanisms activated by the host, resulting in lysis of the outermost cuticle of the parasite without melanin involvement. Ultrastructural evidence suggests that lysis is brought about by Malpighian cell products.