BackgroundLutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America. Sandfly immune responses are poorly understood. In previous work we showed that these vector insects respond to bacterial infections by modulating a defensin gene expression and activate the Imd pathway in response to Leishmania infection. Aspects of innate immune pathways in insects (including mosquito vectors of human diseases) have been revealed by studying insect cell lines, and we have previously demonstrated antiviral responses in the L. longipalpis embryonic cell line LL5.MethodsThe expression patterns of antimicrobial peptides (AMPs) and transcription factors were evaluated after silencing the repressors of the Toll pathway (cactus) and Imd pathway (caspar). AMPs and transcription factor expression patterns were also evaluated after challenge with heat-killed bacteria, heat-killed yeast, or live Leishmania.ResultsThese studies showed that LL5 cells have active Toll and Imd pathways, since they displayed an increased expression of AMP genes following silencing of the repressors cactus and caspar, respectively. These pathways were also activated by challenges with bacteria, yeast and Leishmania infantum chagasi.ConclusionsWe demonstrated that L. longipalpis LL5 embryonic cells respond to immune stimuli and are therefore a good model to study the immunological pathways of this important vector of leishmaniasis.
Antimicrobial peptides (AMPs) are produced to control bacteria, fungi, protozoa, and other infectious agents. Sand fly larvae develop and feed on a microbe-rich substrate, and the hematophagous females are exposed to additional pathogens. We focused on understanding the role of the AMPs attacin (Att), cecropin (Cec), and four defensins (Def1, Def2, Def3, and Def4) in Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. Larvae and adults were collected under different feeding regimens, in addition to females artificially infected by Leishmania infantum. AMPs’ gene expression was assessed by qPCR, and gene function of Att and Def2 was investigated by gene silencing. The gene knockdown effect on bacteria and parasite abundance was evaluated by qPCR, and parasite development was verified by light microscopy. We demonstrate that L. longipalpis larvae and adults trigger AMPs expression during feeding, which corresponds to an abundant presence of bacteria. Att and Def2 expression were significantly increased in Leishmania-infected females, while Att suppression favored bacteria growth. In conclusion, L. longipalpis AMPs’ expression is tuned in response to bacteria and parasites but does not seem to interfere with the Leishmania cycle.
Nanomaterials composed of natural matrices associated with biopesticides have promising applications in sustainable agriculture. In this study, the biopesticide neem Powered by Editorial Manager® and ProduXion Manager® from Aries Systems CorporationAnswer: Thank you for your comment. We have inserted the requested information throughout the manuscript. Please verify the revised version.Reviewer: Figure 5. I do not agree with the analysis. I will suggest to apply repeated measures ANOVA or survival analysis and their curves. Answer: Thank you for your comment. We would like to explain that in Figure 5, there is no need to perform repeated measurements ANOVA because our data do not represent measurements over time of the same sample. In fact, different leaves were collected on different days after the application of the treatments to perform the test, and not a single leaf was collected and analyzed 1, 6 and 12 days latter. We have modified the figure caption for better understanding. Please verify the revised version of the manuscript.Reviewer: Line 591-801. Firstly, discussion section seems like a review of literature and many of the sentenses are awkwardly placed. I will suggest to rewrite the whole sentense. Secondly, hard to get the idea due to linguistic and syntax errors. Answer: Thank you for your comment. We have abbreviated the discussion and some cited studies were deleted. Please verify the revised version of the manuscript.Reviewer: Line 592-600. The opening paragraph of the discussion section is not appropriately written. I will suggest to rewrite this section. Answer: Thank you for your comment. We have rewritten the discussion. Please verify the revised version of the manuscript.Reviewer: Line 553-554. The sentense is awkwardly placed. Answer: Thank you for your comment. We have deleted the indicated sentence.Reviewer: Line 801. I will suggest the authors to provide a comprehensive conclusion of the study. Answer: Thank you for your comment. We have rewritten the conclusion. Please verify the revised version of the manuscript. Reviewer #2: Reviewer: I have read the manuscript entitled " Nanobiopesticide based on zein nanoparticles and neem oil: a study using target and nontarget organisms". The manuscript presents a well-written and appropriately analyzed series of experiments to determine the pesticidal and biological activity of neem oil-loaded zein nanoparticles against three pests (Acanthoscelides obtectus, Bemisia tabaci, and Tetranychus urticae), in addition to the phytotoxic effects of these nanoparticles using Phaseolus vulgaris. Overall, the manuscript is generally clear and concise report of a well-executed study. The objectives are clear; the experiments are pertinent and follow a logical reasoning; the main findings of the study are convincing and the conclusion is appropriate. The paper is clearly organized and the contribution is interesting and falls within the scope of the journal. The work is generally well written, except for certain parts of the manuscript, where a good technical ...
Phlebotomine sand flies (Diptera, Psychodidae) belonging to the Lutzomyia genus transmit zoonoses in the New World. Lutzomyia longipalpis is the main vector of Leishmania infantum, which is the causative agent of visceral leishmaniasis in Brazil. To identify key molecular aspects involved in the interaction between vector and pathogens and contribute to developing disease transmission controls, we investigated the sand fly innate immunity mediated by the Janus kinase/signal transducer and activator of transcription (Jak-STAT) pathway in response to L. infantum infection. We used two study models: L. longipalpis LL5 embryonic cells co-cultured with L. infantum and sand fly females artificially infected with the parasite. We used qPCR to follow the L. longipalpis gene expression of molecules involved in the Jak-STAT pathway. Also, we modulated the Jak-STAT mediated immune response to understand its role in Leishmania parasite infection. For that, we used RNAi to silence the pathway regulators, protein inhibitor of activated STATs (PIAS) in LL5 cells, and STAT in adult females. In addition, the pathway suppression effect on parasite development within the vector was assessed by light microscopy in late-phase infection. The silencing of the repressor PIAS in LL5 cells led to a moderate increase in a protein tyrosine phosphatase 61F (PTP61F) expression. It suggests a compensatory regulation between these two repressors. L. infantum co-culture with LL5 cells upregulated repressors PIAS, suppressor of cytokine signaling (SOCS), and PTP61F. It also downmodulated virus-induced RNA-1 (VIR-1), a pathway effector, indicating that the parasite could repress the Jak-STAT pathway in LL5 cells. In Leishmania-infected L. longipalpis females, STAT and the antimicrobial peptide attacin were downregulated on the third day post-infection, suggesting a correlation that favors the parasite survival at the end of blood digestion in the sand fly. The antibiotic treatment of infected females showed that the reduction of gut bacteria had little effect on the Jak-STAT pathway regulation. STAT gene silencing mediated by RNAi reduced the expression of inducible nitric oxide synthase (iNOS) and favored Leishmania growth in sand flies on the first day post-infection. These results indicate that STAT participated in the iNOS regulation with subsequent effect on parasite survival.
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