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1.
Insect Biochem Mol Biol ; 116: 103261, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31698082

RESUMO

A network of serine proteases (SPs) and their non-catalytic homologs (SPHs) activates prophenoloxidase (proPO), Toll pathway, and other insect immune responses. However, integration and conservation of the network and its control mechanisms have not yet been fully understood. Here we present evidence that these responses are initiated through a conserved serine protease and negatively regulated by serpins in two species, Manduca sexta and Anopheles gambiae. We have shown that M. sexta serpin-12 reduces the proteolytic activation of HP6, HP8, proPO activating proteases (PAPs), SPHs, and POs in larval hemolymph, and we hypothesized that these effects are due to the inhibition of the immune pathway-initiating protease HP14. To test whether these changes are due to HP14 inhibition, we isolated a covalent complex of HP14 with serpin-12 from plasma using polyclonal antibodies against the HP14 protease domain or against serpin-12, and confirmed formation of the complex by 2D-electrophoresis, immunoblotting, and mass spectrometry. Upon recognition of bacterial peptidoglycans or fungal ß-1,3-glucan, the zymogen proHP14 became active HP14, which formed an SDS-stable complex with serpin-12 in vitro. Activation of proHP21 by HP14 was suppressed by serpin-12, consistent with the decrease in steps downstream of HP21, proteolytic activation of proPAP3, proSPH1/2 and proPO in hemolymph. Guided by the results of phylogenetic analysis, we cloned and expressed A. gambiae proSP217 (an ortholog of HP14) and core domains of A. gambiae serpin-11 and -17. The recombinant SP217 zymogen became active during expression, with cleavage between Tyr394 and Ile395. Both MsHP14 and AgSP217 cleaved MsSerpin-12 and AgSRPN11 at Leu*Ser (P1*P1') and formed complexes in vitro. ProPO activation in M. sexta plasma increased after recombinant AgSP217 had been added, indicating that it may function in a similar manner as the endogenous initiating protease HP14. Based on these data, we propose that inhibition of an initiating modular protease by a serpin may be a common mechanism in holometabolous insects to regulate proPO activation and other protease-induced immune responses.


Assuntos
Anopheles/imunologia , Manduca/imunologia , Serpinas/metabolismo , Animais , Anopheles/metabolismo , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Hemolinfa/enzimologia , Proteínas de Insetos/metabolismo , Larva/genética , Larva/imunologia , Larva/metabolismo , Manduca/genética , Manduca/metabolismo , Peptidoglicano/farmacologia , Filogenia , Serina Proteases/genética , Serina Proteases/metabolismo , beta-Glucanas/farmacologia
2.
Insect Mol Biol ; 29(1): 66-76, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31301266

RESUMO

Storage proteins are haemolymph-specific proteins in insects, mainly synthesized in the fat body, released into the haemolymph, and then selectively reabsorbed by the fat body before pupation. These storage proteins play an important role in insect metamorphosis and egg development. Some of these storage proteins are responsive to pathogen infection and can even suppress pathogen multiplication. However, the mechanisms of the physiological, biochemical and immune-responsive functions of storage proteins remain unclear. In this study, the expression patterns of Bombyx mori storage protein 1 (BmSP1) during the larval stage were analysed. Then, BmSP1 protein fused with enhanced green fluorescent protein (EGFP) was successfully expressed in a B. mori baculovirus vector expression system. Quantitative real-time PCR showed that the expression level of BmSP1 increased with the advance of instars and reached the highest level in the fifth instar, especially in the fat body. Recombinant BmSP1 expressed in silkworm larvae inhibited haemolymph melanization. Then, proteins that interact with BmSP1 were identified with EGFP used as an antigenic determinant by co-immunoprecipitation. A 30 kDa low molecular weight lipoprotein PBMHP-6 precursor (BmLP6) was shown to interact with BmSP1. Yeast two-hybrid experiments confirmed the interaction between BmSP1 and BmLP6. The results obtained in this study will be helpful for further study of the functions of BmSP1 and BmLP6 in the regulatory network of silkworm development and innate immunity.


Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Animais , Bombyx/genética , Bombyx/imunologia , Linhagem Celular , Corpo Adiposo/metabolismo , Proteínas de Fluorescência Verde , Hemolinfa/imunologia , Imunidade Inata , Proteínas de Insetos/genética , Larva/genética , Larva/imunologia , Larva/metabolismo , Proteínas Recombinantes
3.
Exp Parasitol ; 208: 107802, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31730782

RESUMO

In insects, diet plays an important role in growth and development. Insects can vary their diet composition based on their physiological needs. In this study we tested the influence of diet composition involving varying concentrations of macronutrients and zinc on the immune-tolerance following parasite and pathogen exposure in Spodoptera litura larvae. We also tested the insecticidal potential of Mesorhabditis belari, Enterobacter hormaechei and its secondary metabolites on Spodoptera litura larvae. The results shows macronutrient composition does not directly affect the larval tolerance to nematode infection. However, Zinc supplemented diet improved the immune tolerance. While larvae exposed to bacterial infection performed better on carbohydrate rich diet. Secondary metabolites from bacteria produced an immune response in dose dependent mortality. The study shows that the larvae maintained on different diet composition show varied immune tolerance which is based on the type of infection.


Assuntos
Enterobacter/fisiologia , Controle Biológico de Vetores , Rhabditoidea/fisiologia , Spodoptera/imunologia , Análise de Variância , Animais , Bioensaio , Carboidratos/administração & dosagem , Cromatografia Líquida de Alta Pressão , Dieta , Enterobacter/imunologia , Enterobacter/patogenicidade , Cromatografia Gasosa-Espectrometria de Massas , Tolerância Imunológica , Larva/imunologia , Dose Letal Mediana , Proteínas/administração & dosagem , Rhabditoidea/imunologia , Rhabditoidea/patogenicidade , Espectroscopia de Infravermelho com Transformada de Fourier , Spodoptera/fisiologia , Simbiose , Virulência , Zinco/administração & dosagem
4.
Insect Sci ; 27(2): 239-255, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30328680

RESUMO

Larval Galleria mellonella (L.) hemocytes form microaggregates in response to stimulation by Gram-positive bacteria. Hemocyte adhesion to foreign materials is mediated by the cAMP/ protein kinase A pathway and the ß-subunit of cholera toxin using a cAMP-independent mechanism. Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide, implying integrins may be part of the mechanism. Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses α5 , αv , ß1 , and ß3 subunits occurred on both granular cell and plasmatocyte hemocyte subtypes. A fluorescent band representing the binding of rabbit α5 -integrin subunit antibodies occurred between adhering heterotypic hemocytes. The frequency of the bands was increased by cholera toxin. The α5 and ß1 rabbit integrin subunit antibodies inhibited removal of Bacillus subtilis (Cohn) from the hemolymph in vivo. A α5 ß1 -specific synthetic peptide blocker similarly diminished hemocyte function whereas the αv ß3 -specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities. Western blots revealed several proteins reacting with a given integrin-antibody subtype. Thus integrin-antibody reactive proteins (which may include integrins) with possible α5 and ß1 epitopes modulate immediate hemocyte function. Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followed by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.


Assuntos
Hemócitos/imunologia , Integrinas/imunologia , Mariposas/imunologia , Animais , Larva/imunologia
5.
PLoS Negl Trop Dis ; 13(9): e0007730, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525197

RESUMO

BACKGROUND: The current strategy for the elimination of onchocerciasis is based on annual or bi-annual mass drug administration with ivermectin. However, due to several limiting factors there is a growing concern that elimination of onchocerciasis cannot be achieved solely through the current strategy. Additional tools are critically needed including a prophylactic vaccine. Presently Ov-103 and Ov-RAL-2 are the most promising vaccine candidates against an Onchocerca volvulus infection. METHODOLOGY/PRINCIPAL FINDINGS: Protection induced by immunization of mice with the alum-adjuvanted Ov-103 or Ov-RAL-2 vaccines appeared to be antibody dependent since AID-/- mice that could not mount antigen-specific IgG antibody responses were not protected from an Onchocerca volvulus challenge. To determine a possible association between antigen-specific antibody responses and anti-larvae protective immunity in humans, we analyzed the presence of anti-Ov-103 and anti-Ov-RAL-2 cytophilic antibody responses (IgG1 and IgG3) in individuals classified as putatively immune, and in infected individuals who developed concomitant immunity with age. It was determined that 86% of putatively immune individuals and 95% individuals with concomitant immunity had elevated IgG1 and IgG3 responses to Ov-103 and Ov-RAL-2. Based on the elevated chemokine levels associated with protection in the Ov-103 or Ov-RAL-2 immunized mice, the profile of these chemokines was also analyzed in putatively immune and infected individuals; both groups contained significantly higher levels of KC, IP-10, MCP-1 and MIP-1ß in comparison to normal human sera. Moreover, human monospecific anti-Ov-103 antibodies but not anti-Ov-RAL-2 significantly inhibited the molting of third-stage larvae (L3) in vitro by 46% in the presence of naïve human neutrophils, while both anti-Ov-103 and anti-Ov-RAL-2 antibodies significantly inhibited the molting by 70-80% when cultured in the presence of naive human monocytes. Interestingly, inhibition of molting by Ov-103 antibodies and monocytes was only in part dependent on contact with the cells, while inhibition of molting with Ov-RAL-2 antibodies was completely dependent on contact with the monocytes. In comparison, significant levels of parasite killing in Ov-103 and Ov-RAL-2 vaccinated mice only occurred when cells enter the parasite microenvironment. Taken together, antibodies to Ov-103 and Ov-RAL-2 and cells are required for protection in mice as well as for the development of immunity in humans. CONCLUSIONS/SIGNIFICANCE: Alum-adjuvanted Ov-103 and Ov-RAL-2 vaccines have the potential of reducing infection and thus morbidity associated with onchocerciasis in humans. The development of cytophilic antibodies, that function in antibody-dependent cellular cytotoxicity, is essential for a successful prophylactic vaccine against this infection.


Assuntos
Imunogenicidade da Vacina , Onchocerca volvulus/imunologia , Oncocercose/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Quimiocinas/sangue , Imunoglobulina G/sangue , Larva/crescimento & desenvolvimento , Larva/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos , Onchocerca volvulus/crescimento & desenvolvimento , Oncocercose/parasitologia , Oncocercose/prevenção & controle , Vacinação , Vacinas/administração & dosagem
6.
Exp Parasitol ; 206: 107755, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31493393

RESUMO

The aim of the present study was to assess the expression of cytokines and FCεR1A receptor stimulated by Haemonchus placei larval excretory and secretory (ES) products associated with the pathogenesis in calves. Bovine peripheral blood mononuclear cells (PBMC) were stimulated in in vitro assays with H. placei L4 ES product at 8, 12, 16 and 24 h. ES products were collected in in vitro assays at 48 h with molecular weight of 72/60 kDa and isoelectric point of 7.2 pI. Specific IgG for infected and control calves, positive and negative, were employed to recognise H. placei larval ES products by indirect ELISA, showing a mean of 1.8, 0.83 and 0.28 OD, respectively, (p ≤ 0.001). The quantification of relative gene expression was performed using a set of cytokines (IL-2, IFNγ, TGFß, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13), FCεR1A receptor and housekeeping (GAPDH, ß-actin and ß-2-microglobulin) by RT-qPCR. An early increased expression, 2.2- to 3.4-fold change, of IL-2 (p ≤ 0.001), IL-5 and TGFß (p ≥ 0.05) was determined, followed by TGFß (30.7 and 14.14), IL-8 (102.8 and 1504.4) and IL-10 (60.4 and 1.7) (p ≤ 0.05) after 12 and 16 h, respectively, and reducing the expression level at 24 h. In addition, IL-6, IL-13 and FCεR1A receptor also displayed mild expression level, 2.1 - to 7.60-fold change, at 24 h (p ≥ 0.05). We conclude that ES products of 72/60 kDa collected in vitro from H. placei larvae are recognised by infected hosts and have the ability to induce diverse immune factors to modulate the nematode damage.


Assuntos
Citocinas/metabolismo , Haemonchus/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Receptores de IgE/metabolismo , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Haemonchus/genética , Haemonchus/imunologia , Imunoglobulina G/metabolismo , Larva/genética , Larva/imunologia , Larva/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , RNA Mensageiro/metabolismo , Regulação para Cima
7.
Insect Biochem Mol Biol ; 114: 103231, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31479697

RESUMO

Mycoplasmas, the smallest self-replicating organisms, are unique in that they lack cell walls but possess distinctive plasma membranes containing sterol acquired from their growth environment. Although mycoplasmas are known to be successful pathogens in a wide range of animal hosts, including humans, the molecular basis for their virulence and interaction with the host immune systems remains largely unknown. This study was conducted to elucidate the biochemical relationship between mycoplasma and the insect immune system. We investigated defense reactions of Tenebrio molitor that were activated in response to infection with Mycoplasma pulmonis. The results revealed that T. molitor larvae were more resistant to mycoplasma infection than normal bacteria equipped with cell walls. Intruding M. pulmonis cells were effectively killed by toxins generated from activation of the proPO cascade in hemolymph, but not by cellular reactions or antimicrobial peptides. It was determined that these different anti-mycoplasma effects of T. molitor immune components were primarily attributable to surface molecules of M. pulmonis such as phospholipids occurring in the outer leaflet of the membrane lipid bilayer. While phosphatidylcholine, a phospholipid derived from the growth environment, contributed to the resistance of M. pulmonis against antimicrobial peptides produced by T. molitor, phosphatidylglycerol was responsible for triggering activation of the proPO cascade.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Mycoplasma pulmonis/fisiologia , Tenebrio/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/sangue , Catecol Oxidase/metabolismo , Precursores Enzimáticos/metabolismo , Larva/imunologia , Larva/microbiologia , Fagocitose , Fosfolipídeos/imunologia , Tenebrio/microbiologia
8.
Cell Host Microbe ; 26(3): 412-425.e5, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31492656

RESUMO

Surviving infection requires immune and repair mechanisms. Developing organisms face the additional challenge of integrating these mechanisms with tightly controlled developmental processes. The larval Drosophila midgut lacks dedicated intestinal stem cells. We show that, upon infection, larvae perform limited repair using adult midgut precursors (AMPs). AMPs differentiate in response to damage to generate new enterocytes, transiently depleting their pool. Developmental delay allows for AMP reconstitution, ensuring the completion of metamorphosis. Notch signaling is required for the differentiation of AMPs into the encasing, niche-like peripheral cells (PCs), but not to differentiate PCs into enterocytes. Dpp (TGF-ß) signaling is sufficient, but not necessary, to induce PC differentiation into enterocytes. Infection-induced JAK-STAT pathway is both required and sufficient for differentiation of AMPs and PCs into new enterocytes. Altogether, this work highlights the constraints imposed by development on an organism's response to infection and demonstrates the transient use of adult precursors for tissue repair.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Trato Gastrointestinal/metabolismo , Larva/metabolismo , Animais , Diferenciação Celular , Modelos Animais de Doenças , Drosophila/microbiologia , Drosophila/fisiologia , Proteínas de Drosophila/genética , Enterócitos/metabolismo , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/patologia , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/patologia , Janus Quinases/metabolismo , Larva/imunologia , Larva/microbiologia , Metamorfose Biológica , Pectobacterium carotovorum/patogenicidade , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma
9.
Cell Tissue Res ; 377(3): 469-474, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31463705

RESUMO

The embryo of the purple sea urchin has been a fruitful model for the study of developmental gene regulatory networks. For similar reasons, the feeding sea urchin larva provides a gene regulatory model to investigate immune interactions at the gut epithelium. Here we describe what is known of the gut structure and immune cells of the sea urchin larva, and the cellular and gene expression response of the larva to gut-associated immune challenge. As a focused example of how the sea urchin larva can be compared with vertebrate systems, we discuss the expression and function of the IL-17 signalling system in the course of the larval immune response.


Assuntos
Sistema Digestório/imunologia , Epitélio/imunologia , Interleucina-17/imunologia , Larva/imunologia , Strongylocentrotus purpuratus/imunologia , Animais , Regulação da Expressão Gênica/imunologia
10.
J Therm Biol ; 84: 136-145, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31466746

RESUMO

Temperature is one of the important environmental elements affecting ecological fitness of insects through alterations in physiological systems. In the current study, a comparison was made on the cellular and humoral immune responses of the Chilo suppressalis larvae exposed to thermal stress (34 °C) and optimal rearing temperature (24 °C). Although total hemocyte count increased in the injected larvae by Beauveria bassiana, elevation of hemocyte numbers was significantly different in the larvae exposed to 34 °C for a short-time period compared to long-term exposure and control. A similar trend was observed in plasmatocyte and granulocyte counts as well as phenoloxidase activity. Gene expression of some antimicrobial peptides, including attacin1, attacin2, cecropin1, cecropin2, defensin, gallerimycin, lysozyme and prophenoloxidase-activating proteinase-3, was compared in the larvae exposed to thermal regimes and injection challenges. In all cases, expression of the target genes was relatively higher in the larvae injected by B. bassiana and short-term exposure at 34 °C. The present results confirmed that C. suppressalis could modulate the immune system in response to different thermal stress conditions mainly over a short period.


Assuntos
Beauveria , Resposta ao Choque Térmico , Interações Hospedeiro-Patógeno/imunologia , Larva/imunologia , Lepidópteros/imunologia , Animais , Contagem de Células Sanguíneas , Granulócitos/imunologia , Hemócitos/imunologia , Proteínas de Insetos/imunologia , Irã (Geográfico) , Larva/microbiologia , Lepidópteros/microbiologia , Monofenol Mono-Oxigenase/imunologia
11.
Parasit Vectors ; 12(1): 339, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292008

RESUMO

BACKGROUND: The primary cause of parasitic gastroenteritis in small ruminants in temperate regions is the brown stomach worm, Teladorsagia circumcincta. Host immunity to this parasite is slow to develop, consistent with the ability of T. circumcincta to suppress the host immune response. Previous studies have shown that infective fourth-stage T. circumcincta larvae produce excretory-secretory products that are able to modulate the host immune response. The objective of this study was to identify immune modulatory excretory-secretory proteins from populations of fourth-stage T. circumcincta larvae present in two different host-niches: those associated with the gastric glands (mucosal-dwelling larvae) and those either loosely associated with the mucosa or free-living in the lumen (lumen-dwelling larvae). RESULTS: In this study excretory-secretory proteins from mucosal-dwelling and lumen-dwelling T. circumcincta fourth stage larvae were analysed using comparative 2-dimensional gel electrophoresis. A total of 17 proteins were identified as differentially expressed, with 14 proteins unique to, or enriched in, the excretory-secretory proteins of mucosal-dwelling larvae. One of the identified proteins, unique to mucosal-dwelling larvae, was a putative peroxiredoxin (T. circumcincta peroxiredoxin 1, Tci-Prx1). Peroxiredoxin orthologs from the trematode parasites Schistosoma mansoni and Fasciola hepatica have previously been shown to alternatively activate macrophages and play a key role in promoting parasite induced Th2 type immunity. Here we demonstrate that Tci-Prx1 is expressed in all infective T. circumcincta life-stages and, when produced as a recombinant protein, has peroxidase activity, whereby hydrogen peroxide (H2O2) is reduced and detoxified. Furthermore, we use an in vitro macrophage stimulation assay to demonstrate that, unlike peroxiredoxins from trematode parasites Schistosoma mansoni and Fasciola hepatica, Tci-Prx1 is unable to alternatively activate murine macrophage cells. CONCLUSIONS: In this study, we identified differences in the excretory-secretory proteome of mucosal-dwelling and lumen-dwelling infective fourth-stage T. circumcincta larvae, and demonstrated the utility of this comparative proteomic approach to identify excretory-secretory proteins of potential importance for parasite survival and/or host immune modulation.


Assuntos
Proteínas de Helminto/metabolismo , Peroxirredoxinas/metabolismo , Trichostrongyloidea/metabolismo , Animais , Eletroforese em Gel Bidimensional , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita , Peróxido de Hidrogênio/metabolismo , Enteropatias Parasitárias , Larva/imunologia , Larva/metabolismo , Camundongos , Membrana Mucosa/parasitologia , Peroxirredoxinas/genética , Proteoma/análise , Proteômica , Ovinos/parasitologia , Doenças dos Ovinos/parasitologia , Trichostrongyloidea/imunologia
12.
Insect Biochem Mol Biol ; 111: 103179, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31255640

RESUMO

Eicosanoids play crucial roles in mediating insect immune responses. In vertebrates, phospholipase A2 (PLA2) releases arachidonic acid (AA) from phospholipids (PLs) for biosynthesis of various eicosanoids. However, little AA is found in PLs of lepidopteran insects. Spodoptera exigua, a lepidopteran insect, is known to use eicosanoids to mediate immunity. Although AA was not detected in PLs of hemocytes and fat body (two immune tissues) of naïve larvae, it was detected at small but significant level after bacterial infection, suggesting induction of AA biosynthesis for immunity. Based on a mammalian AA biosynthetic pathway, this study hypothesizes that AA is synthesized from C18 polyunsaturated fatty acid (PUFA) precursor by subsequent desaturation and elongation reactions because PLs of S. exigua larvae are rich in linoleic acid. After inhibiting PLA2 activity to prevent release of free fatty acids, different PUFA precursors were injected to S. exigua larvae followed by assessment of eicosanoid-mediated cellular immune response. ω-6 PUFAs were effective in inducing immune response whereas α-linolenic acid (an ω-3 PUFA) was not. Several fatty acyl desaturases (SeDESs) have been predicted from S. exigua transcriptomes. Specific inhibitors against Δ5 or Δ6 DESs inhibited eicosanoid-mediated immune responses. Furthermore, RNA interference (RNAi) specific to Δ5 or Δ6 DES genes significantly suppressed eicosanoid-mediated immune responses. Four very long chain fatty acid elongase genes (SeEloV-A ∼ SeEloV-D) were predicted. Among respective RNAi treatments of these genes, only one RNAi treatment specific to type 5 elongase (SeEloV-B) suppressed eicosanoid-mediated immune response. These results suggest that S. exigua larvae can synthesize AA from linoleic acid via Δ5- and Δ6-desaturations by SeDESs along with chain elongation by SeEloV-B. Finally, this study showed significant fitness cost of uncontrolled AA biosynthesis. AA injection alone without bacterial challenge significantly induced both cellular and humoral immune responses. This unnecessary energy expense due to free AA resulted in reduced pupal size and decreased adult egg production. The detrimental effect of free AA explains physiological significance of little AA content in lepidopteran insects except for life-or-death situation such as pathogen infection.


Assuntos
Ácido Araquidônico/biossíntese , Spodoptera/metabolismo , Animais , Vias Biossintéticas , Eicosanoides/metabolismo , Escherichia coli , Ácidos Graxos Insaturados/metabolismo , Larva/genética , Larva/imunologia , Larva/metabolismo , Larva/microbiologia , Interferência de RNA , Spodoptera/genética , Spodoptera/imunologia , Spodoptera/microbiologia
13.
Parasitol Res ; 118(9): 2621-2633, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300888

RESUMO

Little information is available on the effects of neonicotinoid insecticides on vertebrates. Previous work using amphibians found chronic exposure to some neonicotinoids had no detrimental effects on fitness-relevant traits. However, there is some evidence of more subtle effects of neonicotinoids on immune traits and evidence that other pesticides can suppress tadpole immunity resulting in elevated levels of parasitism in the exposed tadpoles. The objective of our study was to assess whether neonicotinoid exposure affected tadpole immunometrics and susceptibility to parasitic helminths. We assessed northern leopard frog tadpole (Lithobates pipiens) levels of parasitism and leukocyte profiles following exposure to environmentally relevant concentrations of clothianidin and free-living infective cercariae of a helminth parasite, an Echinostoma sp. trematode. When comparing tadpoles from controls to either 1 or 100 µg/L clothianidin treatments, we found similar measures of parasitism (i.e. prevalence, abundance and intensity of echinostome cysts) and similar leukocyte profiles. We also confirmed that clothianidin was not lethal for cercariae; however, slight reductions in swimming activity were detected at the lowest exposure concentration of 0.23 µg/L. Our results show that exposure to clothianidin during the larval amphibian stage does not affect leukocyte profiles or susceptibility to parasitism by larval trematodes in northern leopard frogs although other aspects such as length of host exposure require further study.


Assuntos
Echinostoma/fisiologia , Equinostomíase/veterinária , Guanidinas/farmacologia , Inseticidas/farmacologia , Larva/imunologia , Neonicotinoides/farmacologia , Rana pipiens/parasitologia , Tiazóis/farmacologia , Animais , Cercárias/efeitos dos fármacos , Cercárias/crescimento & desenvolvimento , Echinostoma/efeitos dos fármacos , Echinostoma/crescimento & desenvolvimento , Equinostomíase/parasitologia , Larva/efeitos dos fármacos , Larva/parasitologia , Leucócitos/imunologia , Rana pipiens/imunologia
14.
J Insect Sci ; 19(4)2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31343690

RESUMO

Bombyx mori (Lepidoptera: Bombycidae) is an important economic insect and a classic Lepidopteran model system. Although immune-related genes have been identified at a genome-wide scale in the silkworm, proteins involved in immune defense of the silkworm have not been comprehensively characterized. In this study, two types of bacteria were injected into the silkworm larvae, Gram-negative Escherichia coli (Enterobacteriales: Enterobacteriaceae), or Gram-positive Staphylococcus aureus (Bacillales: Staphylococcaceae). After injection, proteomic analyses of hemolymph were performed by liquid chromatography-tandem mass spectrometry. In total, 514 proteins were identified in the uninduced control group, 540 were identified in the E. coli-induced group, and 537 were identified in the S. aureus-induced group. Based on Uniprot annotations, 32 immunological recognition proteins, 28 immunological signaling proteins, and 21 immunological effector proteins were identified. We found that 127 proteins showed significant upregulation, including 10 immunological recognition proteins, 4 immunological signaling proteins, 11 immunological effector proteins, and 102 other proteins. Using real-time quantitative polymerase chain reaction in the fat body, we verified that immunological recognition proteins, signaling proteins, and effector proteins also showed significant increases at the transcriptional level after infection with E. coli and S. aureus. Five newly identified proteins showed upregulation at both protein and transcription levels after infection, including 30K protein, yellow-d protein, chemosensory protein, and two uncharacterized proteins. This study identified many new immune-related proteins, deepening our understanding of the immune defense system in B. mori. The data have been deposited to the iProX with identifier IPX0001337000.


Assuntos
Bombyx/genética , Bombyx/imunologia , Escherichia coli/fisiologia , Proteínas de Insetos/imunologia , Proteoma/imunologia , Staphylococcus aureus/fisiologia , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/microbiologia , Proteínas de Insetos/análise , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/microbiologia , Proteoma/análise , Proteômica
15.
J Insect Physiol ; 117: 103903, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31233768

RESUMO

Insects are able to develop enhanced resistance in response to repeated infection. This phenomenon is called immune priming. In this work, so-called "primed" Galleria mellonella larvae were re-infected with a lethal dose of Candida albicans 48 h after injection of a non-lethal dose, while "non-primed" larvae were infected only with a lethal dose. The increased resistance of the primed larvae correlated with a slower rate of body colonisation by the fungus. Changes in the protein profiles were detected in the whole hemolymph of the primed insects. The analysis of low-molecular weight proteins and peptides obtained with the use of three different organic solvents and comparative quantitative HPLC analysis thereof showed that the primed larvae did not have higher amounts of any infection-inducible polypeptides than the non-primed larvae. Moreover, electrophoresis of low-molecular weight polypeptides revealed an even lower level of immune-induced peptides in the primed larvae than in the non-primed ones. Furthermore, the defence activity of larval hemolymph, i.e. the antifungal, antibacterial, and lysozyme-type activity, was up-regulated in the primed larvae at the time of re-infection and, consequently, at the early time points after the infection with the lethal dose. Twenty four hours after the infection, these parameters were equally high in the non-primed and primed larvae. Accordingly, at the time of the injection of the lethal dose, certain immune-inducible genes were up-regulated. However, 24 h after the infection with the lethal dose, their expression in both groups was incomparably higher than at the time of the infection and, in most cases, it was as high in the primed larvae as in the non-primed ones. We found that only anti yeast-like activity was enhanced 24 h after the re-infection. This correlated with results obtained by testing the priming effect in heterologous systems: the primed animals did not exhibit higher resistance to the other pathogens tested.


Assuntos
Interações Hospedeiro-Patógeno , Imunidade Inata , Mariposas/imunologia , Animais , Candida albicans , Larva/imunologia
16.
Arch Insect Biochem Physiol ; 101(4): e21588, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31180585

RESUMO

Boric acid (BA) is widely used in various industrial process and can be accessed to nontarget organisms. This study aimed to investigate the insecticidal effects of BA and its toxic activities with respect to immunologic and genotoxic effects using Galleria mellonella larvae as a model. BA concentrations (78.125-10,000 ppm) were administrated to the larvae using the feeding method. Concentration-dependent mortality was observed in all larval groups. Probit analysis revealed LC30 , LC50 , and LC70 values to be 112.4, 320.1, and 911.4 ppm, respectively. These concentrations were used in all bioassays. Drastic reductions in total hemocyte counts along with changes in differential hemocyte counts were observed following BA treatment. Cell viability assays showed dose-dependent reductions in viable cells and an increase in the necrotic and apoptotic ratios after BA treatment. However, mitotic indices of larval hemocytes did not change at all BA concentrations. The cytotoxic effect of BA led to a significant reduction in cellular immune responses such as encapsulation, melanization, and nodulation activities of treated larvae. While BA increased micronucleus ratios at the highest concentration, comet parameters indicating DNA damage increased in G. mellonella larval hemocytes at all concentrations. These report that BA suppresses the immune system of G. mellonella and also poses risks of genotoxicity at high concentrations.


Assuntos
Ácidos Bóricos/toxicidade , Mariposas/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio Cometa , Relação Dose-Resposta a Droga , Hemócitos/efeitos dos fármacos , Hemolinfa , Inseticidas/toxicidade , Larva/efeitos dos fármacos , Larva/genética , Larva/imunologia , Testes para Micronúcleos , Mariposas/genética , Mariposas/imunologia
17.
Parasitol Int ; 72: 101933, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31128257

RESUMO

Due to the epidemiological problem of the neglected condition of human strongyloidiasis, rapid and effective diagnosis is extremely important, with the development of new diagnostic tools being essential to reduce infections and chronic cases. Avian immunoglobulin Y (IgY) technology is an alternative for antibody production that has high specificity and profitability. This study aimed to produce and fractionate IgY antibodies from the egg yolks of hens that were immunized with the total antigenic extracts of Strongyloides venezuelensis infectious filariform larvae (iL3) and parthenogenetic females (pF). IgY antibodies were then evaluated by their recognition of antigenic proteins, evolutive helminth forms, and serological diagnosis of human strongyloidiasis by the detection of immune complexes in serum samples. Egg yolks were fractionated to obtain IgY antibodies by thiophilic interaction chromatography. Immune complex detection in serum samples showed diagnostic values for anti-iL3 IgY and anti-pF IgY antibodies at 95.56% and 88.89% sensitivity and 95.56% and 91.11% specificity, respectively. Therefore, IgY technology is a promising tool for the detection of blood circulating Strongyloides antigens, with possible application as a serological diagnostic method.


Assuntos
Imunoglobulinas/imunologia , Testes Imunológicos/métodos , Strongyloides/imunologia , Estrongiloidíase/diagnóstico , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos , Galinhas , Gema de Ovo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Larva/imunologia , Sensibilidade e Especificidade , Testes Sorológicos , Estrongiloidíase/imunologia
18.
Front Biosci (Landmark Ed) ; 24: 1390-1400, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31136986

RESUMO

In the past decades, much has been learned about the protective signatures of innate immune responses during the course of infections. However, it is now evident that induction of immune effectors also commonly occurs in the absence of pathogenic cues. Such an event, termed sterile inflammation, has been linked to several debilitating acute and chronic host conditions. Using Drosophila melanogaster as a simple yet powerful model organism, identification of diverse sets of damage-associated molecular patterns and their corresponding surface and/or inside pattern recognition receptors on the cells, as well as elucidation of their significant roles in the host physiology and pathological conditions related to sterile inflammation, have been continuously reported. In addition, revelation of non-pathogenic molecular triggers leading to the orchestration of unnecessary activation of inflammatory responses has been a subject of interest. Here, we review decades of efforts to elucidate the molecular mechanisms responsible in the emergence of sterile inflammation. The characterization of the respective contributing factors, including recent demonstration of pinching as a novel sterile-stimuli in Drosophila, is also discussed.


Assuntos
Drosophila melanogaster/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Transdução de Sinais/imunologia , Animais , Proteínas de Drosophila/imunologia , Proteínas de Drosophila/metabolismo , Humanos , Inflamassomos/imunologia , Larva/imunologia , Estresse Mecânico
19.
Microb Pathog ; 132: 335-342, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31100407

RESUMO

The aim of this study was to evaluate the effects of Bacillus subtilis and Bacillus atrophaeus on Galleria mellonella immunity challenged by Candida albicans. Firstly, we analyzed the susceptibility of G. mellonella to bacilli (vegetative and sporulating forms). It was found that both vegetative and sporulating forms were not pathogenic to G. mellonella at a concentration of 1 × 104 cells/larva. Next, larvae were pretreated with two species of Bacillus, in the vegetative and sporulating forms, and then challenged with C. albicans. In addition, the gene expression of antimicrobial peptides (AMPs) such as Gallerimycin, Gloverin, Cecropin-D and Galiomicin was investigated. Survival rates increased in the Bacillus treated larvae compared with control larvae inoculated with C. albicans only. Cells and spores of Bacillus spp. upregulated Gloverin, Galiomicin and Gallerimycin genes in relation to the control group (PBS + PBS). When these larvae were infected with C. albicans, the group pretreated with spores of B. atrophaeus and B. subtilis showed a greater increase in expression of Galiomycin (49.08-fold and 13.50-fold) and Gallerimycin (27.88-fold and 68.15-fold), respectively, compared to the group infected with C. albicans only (p = 0.0001). After that, we investigated the effects of B. subtilis and B. atrophaeus on immune system of G. mellonella evaluating the number of hemocytes, quantification of melanization, cocoon formation and colony forming units (CFU) count. Hemocyte count increased in response to stimulation by Bacillus, and a higher increase was achieved when larvae were inoculated with B. subtilis spores (p = 0.0011). In the melanization assay, all groups tested demonstrated lower production of melanin compared to that in the phosphate-buffered saline (PBS) group. In addition, full cocoon formation was observed in all groups analyzed, which corresponded to a healthier wax worm. Hemolymph culture revealed higher growth of B. atrophaeus and B. subtilis in the groups inoculated with spores. We concluded that spores and cells of B. atrophaeus and B. subtilis stimulated the immune system of G. mellonella larvae and protected them of C. albicans infection.


Assuntos
Bacillus/fisiologia , Candida albicans/patogenicidade , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade , Lepidópteros/imunologia , Alcaloides/genética , Alcaloides/metabolismo , Alcaloides/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/fisiologia , Contagem de Colônia Microbiana , Defensinas/genética , Defensinas/metabolismo , Defensinas/farmacologia , Modelos Animais de Doenças , Expressão Gênica/genética , Hemócitos/imunologia , Hemócitos/metabolismo , Hemolinfa , Interações entre Hospedeiro e Microrganismos/genética , Sistema Imunitário , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Larva/imunologia , Larva/microbiologia , Lepidópteros/genética , Lepidópteros/microbiologia , Proteínas/genética , Proteínas/metabolismo , Proteínas/farmacologia , Quinolinas/metabolismo , Quinolinas/farmacologia , Esporos Bacterianos , Taxa de Sobrevida
20.
Nanoscale ; 11(17): 8343-8351, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30984949

RESUMO

European foulbrood (EFB) is an infectious disease affecting honeybee larvae caused by the bacterium Melissococcus plutonius. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for antibody-based bacteria detection, however, its sensitivity is not high enough to reveal early-stage EFB infection. Photon-upconversion nanoparticles (UCNPs) are lanthanide-doped nanomaterials that emit light of shorter wavelength under near-infrared (NIR) excitation and thus avoid optical background interference. After conjugation with specific biorecognition molecules, UCNPs can be used as ultrasensitive labels in immunoassays. Here, we introduce a method for conjugation of UCNPs with streptavidin based on copper-free click chemistry, which involves surface modification of UCNPs with alkyne-modified bovine serum albumin (BSA) that prevents the non-specific binding and provides reactive groups for conjugation with streptavidin-azide. To develop a sandwich upconversion-linked immunosorbent assay (ULISA) for M. plutonius detection, we have prepared a rabbit polyclonal anti-Melissococcus antibody. The specific capture of the bacteria was followed by binding of biotinylated antibody and UCNP-BSA-streptavidin conjugate for a highly sensitive upconversion readout. The assay yielded an LOD of 340 CFU mL-1 with a wide working range up to 109 CFU mL-1, which is 400 times better than the LOD of the conventional ELISA. The practical applicability of the ULISA was successfully demonstrated by detecting M. plutonius in spiked real samples of bees, larvae and bottom hive debris. These results show a great potential of the assay for early diagnosis of EFB, which can prevent uncontrolled spreading of the infection and losses of honeybee colonies.


Assuntos
Abelhas/microbiologia , Enterococcaceae/isolamento & purificação , Imunoensaio/métodos , Nanopartículas/química , Animais , Anticorpos Antibacterianos/imunologia , Abelhas/crescimento & desenvolvimento , Enterococcaceae/imunologia , Larva/imunologia , Larva/metabolismo , Limite de Detecção , Fótons , Dióxido de Silício/química
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