INTRODUCTION: The midgut epithelium functions as tissue for nutrient uptake as well as physical barrier against pathogens. Additionally, it responds to pathogen contact by production and release of various factors including antimicrobial peptides, similar to the systemic innate immune response. However, if such a response is restricted to a local stimulus or if it appears in response to a systemic infection, too is a rather underexplored topic in insect immunity. We addressed the role of the midgut and the role of systemic immune tissues in the defense against gut-borne and systemic infections, respectively. METHODS: Manduca sexta larvae were challenged with DAP-type peptidoglycan bacteria - Bacillus thuringiensis for local gut infection and Escherichia coli for systemic stimulation. We compared the immune response to both infection models by measuring mRNA levels of four selected immunity-related genes in midgut, fat body, hematopoietic organs (HOs), and hemocytes, and determined hemolymph antimicrobial activity. Hemocytes and HOs were tested for presence and distribution of lysozyme mRNA and protein. RESULTS: The midgut and circulating hemocytes exhibited a significantly increased level of lysozyme mRNA in response to gut infection but did not significantly alter expression in response to a systemic infection. Conversely, fat body and HOs responded to both infection models by altered mRNA levels of at least one gene monitored. Most, but not all hemocytes and HO cells contain lysozyme mRNA and protein. DISCUSSION: These data suggest that the gut recruits immune-related tissues in response to gut infection whereas systemic infections do not induce a response in the midgut. The experimental approach implies a skewed cross-talk: An intestinal infection triggers immune activity in systemic immune organs, while a systemic infection does not elicit any or only a restricted immune response in the midgut. The HOs, which form and release hemocytes in larval M. sexta, i) synthesize lysozyme, and ii) respond to immune challenges by increased immune gene expression. These findings strongly suggest that they not only provide phagocytes for the cellular immune response but also synthesize humoral immune components.

Applied Zoology Department of Biology Technische Universität Dresden Dresden Germany

Institute of Zoology and Developmental Biology Justus Liebig University Gießen Gießen Germany

Laboratory of Cellular and Molecular Immunology Institute of Microbiology of the Czech Academy of Sciences Prague Czechia

Zobrazit více v PubMed

Abraham C., Medzhitov R. (2011). Interactions between the host innate immune system and microbes in inflammatory bowel disease. Gastroenterology 140 (6), 1729–1737. doi: 10.1053/j.gastro.2011.02.012 PubMed DOI PMC

Adamo S. A., Fidler T. L., Forestell C. A. (2007). Illness-induced anorexia and its possible function in the caterpillar, Manduca sexta . Brain Behavior Immun. 21, 292–300. doi: 10.1016/j.bbi.2006.10.006 PubMed DOI

Anderson R. S., Cook M. L. (1979). Induction of lysozyme-like activity in the hemolymph and hemocytes of an insect, Spodoptera eridania . J. Invertebrate Pathol. 33 (2), 197–203. doi: 10.1016/0022-2011(79)90153-8 DOI

Basset A., Khush R. S., Braun A., Gardan L., Boccard F., Hoffmann J. A., et al. . (2000). The phytopathogenic bacteria Erwinia carotovora infects Drosophila and activates an immune response. Proc. Natl. Acad. Sci. 97 (7), 3376–3381. doi: 10.1073/pnas.97.7.3376 PubMed DOI PMC

Beetz S., Brinkmann M., Trenczek T. (2004). Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation. J. Insect Physiol. 50 (9), 805–819. doi: 10.1016/j.jinsphys.2004.06.003 PubMed DOI

Bergman P., Esfahani S. S., Engström Y. (2017). Drosophila as a model for human diseases—focus on innate immunity in barrier epithelia. Curr. topics Dev. Biol. 121, 29–81. doi: 10.1016/bs.ctdb.2016.07.002 PubMed DOI

Broderick N. A., Raffa K. F., Handelsman J. (2006). Midgut bacteria required for Bacillus thuringiensis insecticidal activity. Proc. Natl. Acad. Sci. 103 (41), 15196–15199. doi: 10.1073/pnas.0604865103 PubMed DOI PMC

Caccia S., Di Lelio I., La Storia A., Marinelli A., Varricchio P., Franzetti E., et al. . (2016). Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism. Proc. Natl. Acad. Sci. 113 (34), 9486–9491. doi: 10.1073/pnas.1521741113 PubMed DOI PMC

Cao X., He Y., Hu Y., Wang Y., Chen Y.-R., Bryant B., et al. . (2015). The immune signaling pathways of Manduca sexta. Insect Biochem. Mol. Biol. 62, 64–74. doi: 10.1016/j.ibmb.2015.03.006 PubMed DOI PMC

Costechareyre D., Capo F., Fabre A., Chaduli D., Kellenberger C., Roussel A., et al. . (2016). Tissue-specific regulation of Drosophila NF-κB pathway activation by peptidoglycan recognition protein SC. J. innate Immun. 8 (1), 67–80. doi: 10.1159/000437368 PubMed DOI PMC

Das De T., Sharma P., Thomas T., Singla D., Tevatiya S., Kumari S., et al. . (2018). Interorgan molecular communication strategies of “Local” and “Systemic” innate immune responses in mosquito Anopheles stephensi . Frontiers in immunology 9, 1–17. doi: 10.3389/fimmu.2018.00148 PubMed DOI PMC

Dorland (2011).

Duressa T. F., Vanlaer R., Huybrechts R. (2015). Locust cellular defense against infections: sites of pathogen clearance and hemocyte proliferation. Dev. Comp. Immunol. 48 (1), 244–253. doi: 10.1016/j.dci.2014.09.005 PubMed DOI

Dziarski R., Gupta D. (2006). The peptidoglycan recognition proteins (PGRPs). Genome Biol. 7 (8), 232. doi: 10.1186/gb-2006-7-8-232 PubMed DOI PMC

Dziarski R., Gupta D. (2010). Mammalian peptidoglycan recognition proteins (PGRPs) in innate immunity. Innate Immun. 16 (3), 168–174. doi: 10.1177/1753425910366059 PubMed DOI

Eleftherianos I., Felföldi G., Ffrench-Constant R. H., Reynolds S. E. (2009). Induced nitric oxide synthesis in the gut of Manduca sexta protects against oral infection by the bacterial pathogen Photorhabdus luminescens . Insect Mol. Biol. 18 (4), 507–516. doi: 10.1111/j.1365-2583.2009.00899.x PubMed DOI

Engel P., Moran N. ,. A. (2013). The gut microbiota of insects – diversity in structure and function. FEMS Microbiol. Rev. 37), 699–673. doi: 10.1111/1574-6976.12025 PubMed DOI

Foley E., O'Farrell P. H. (2003). Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila . Genes Dev. 17 (1), 115–125. doi: 10.1101/gad.1018503 PubMed DOI PMC

Garcia-Garcia E., Galindo-Villegas J., Mulero V. (2013). Mucosal immunity in the gut: the non-vertebrate perspective. Dev. Comp. Immunol. 40 (3-4), 278–288. doi: 10.1016/j.dci.2013.03.009 PubMed DOI

Gillespie J. P., Kanost M. R., Trenczek T. (1997). Biological mediators of insect immunity. Annu. Rev. entomology 42 (1), 611–643. doi: 10.1146/annurev.ento.42.1.611 PubMed DOI

Hoffmann J. A. (1970). Les organes hématopoïétiques de deux insectes orthoptères: Locusta migratoria et Gryllus bimaculatus . Z. für Zellforschung und mikroskopische Anatomie 106 (3), 451–472. doi: 10.1007/BF00335786 PubMed DOI

Horohov D. W., Dunn P. E. (1983). Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa . J. Invertebrate Pathol. 41 (2), 203–213. doi: 10.1016/0022-2011(83)90220-3 DOI

Hothorn T., Hornik K., van de Wiel M. A., Zeileis A. (2008). Implementing a class of permutation tests: the coin package. J. Stat. Software 28 (8), 1–23. doi: 10.18637/jss.v028.i08 DOI

Hultmark D., Engström Å., Bennich H., Kapur R., Boman H. G. (1982). Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae. Eur. J. Biochem. 127, 207–217. doi: 10.1111/j.1432-1033.1982.tb06857.x PubMed DOI

Jiao Y., Wu L., Huntington N. D., Zhang X. (2020). Crosstalk between gut microbiota and innate immunity and its implication in autoimmune diseases. Front. Immunol. 11. doi: 10.3389/fimmu.2020.00282 PubMed DOI PMC

Johnston P. R., Crickmore N. (2009). Gut bacteria are not required for the insecticidal activity of Bacillus thuringiensis toward the tobacco hornworm, Manduca sexta . Appl. Environ. Microbiol. 75 (15), 5094–5099. doi: 10.1128/AEM.00966-09 PubMed DOI PMC

Keehnen N. L., Rolff J., Theopold U., Wheat C. W. (2017). “Insect antimicrobial defences: a brief history, recent findings, biases, and a way forward in evolutionary studies,” in Advances in insect physiology, vol. 52. (London: Academic Press; ), 1–33. doi: 10.1016/bs.aiip.2017.02.003 DOI

Kingan S. L., Ensign J. C. (1968). Isolation and characterization of three autolytic enzymes associated with sporulation of Bacillus thuringiensis var. thuringiensis . J. bacteriology 96 (3), 629–638. doi: 10.1128/jb.96.3.629-638.1968 PubMed DOI PMC

Kyriakides T. R., Bedoyan J. K., Patil C. S., Spence K. D. (1993). In vivo distribution of immune protein scolexin in bacteria-injected Manduca sexta larvae. Tissue Cell 25 (3), 423–434. doi: 10.1016/0040-8166(93)90082-V PubMed DOI

Lanot R., Zachary D., Holder F., Meister M. (2001). Postembryonic hematopoiesis in Drosophila . Dev. Biol. 230 (2), 243–257. doi: 10.1006/dbio.2000.0123 PubMed DOI

Lee K. A., Kim S. H., Kim E. K., Ha E. M., You H., Kim B., et al. . (2013). Bacterial-derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila . Cell 153 (4), 797–811. doi: 10.1016/j.cell.2013.04.009 PubMed DOI

Levin D. M., Breuer L. N., Zhuang S., Anderson S. A., Nardi J. B., Kanost M. R. (2005). A hemocyte-specific integrin required for hemocytic encapsulation in the tobacco hornworm, Manduca sexta . Insect Biochem. Mol. Biol. 35 (5), 369–380. doi: 10.1016/j.ibmb.2005.01.003 PubMed DOI

Ling E., Yu X. Q. (2006). Hemocytes from the tobacco hornworm Manduca sexta have distinct functions in phagocytosis of foreign particles and self dead cells. Dev. Comp. Immunol. 30 (3), 301–309. doi: 10.1016/j.dci.2005.05.006 PubMed DOI

Livak K. J., Schmittgen T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 (4), 402–408. doi: 10.1006/meth.2001.1262 PubMed DOI

Mason K. L., Stepien T. A., Blum J. E., Holt J. F., Labbe N. H., Rush J. S., et al. . (2011). From Commensals to Pathogen: Translocation of Enterococcus faecalis from the Midgut to the Hemocoel of. Manduca sexta. mBio 2 (3), e00065–e00011. doi: 10.1128/mBio.00065-11 PubMed DOI PMC

McMillan L. E., Adamo S. A. (2020). Friend or foe? Effects of host immune activation on the gut microbiome in the caterpillar Manduca sexta . J. Exp. Biol. 223 (19), jeb226662. doi: 10.1242/jeb.226662 PubMed DOI

Meraj S., Mohr E., Ketabchi N., Bogdanovic A., Lowenberger C., Gries G. (2021). Time-and tissue-specific antimicrobial activity of the common bed bug in response to blood feeding and immune activation by bacterial injection. J. Insect Physiol. 135, 104322. doi: 10.1016/j.jinsphys.2021.104322 PubMed DOI

Minnick M. F., Rupp R. A., Spence K. D. (1986). A bacterial-induced lectin which triggers hemocyte coagulation in Manduca sexta. Biochem. Biophys. Res. Commun. 137 (2), 729–735. doi: 10.1016/0006-291X(86)91139-3 PubMed DOI

Mohrig W., Messner B. (1968). Immunreaktionen bei Insekten: II. Lysozym als antimikrobielles Agens im Darmtrakt von Insekten. Biologisches Zentralblatt 87, 705–718.

Monner D. A., Jonsson S., Boman H. G. (1971). Ampicillin-resistant mutants of Escherichia coli K-12 with lipopolysaccharide alterations affecting mating ability and susceptibility to sex-specific bacteriophages. J. Bacteriology 107 (2), 420–432. doi: 10.1128/jb.107.2.420-432.1971 PubMed DOI PMC

Mulnix A. B., Dunn P. E. (1994). Structure and induction of a lysozyme gene from the tabacco hornworm, Manduca sexta . Insect Biochem. Mol. Biol. 24 (3), 271–281. doi: 10.1016/0965-1748(94)90007-8 PubMed DOI

Nardi J. B., Pilas B., Ujhelyi E., Garsha K., Kanost M. R. (2003). Hematopoietic organs of Manduca sexta and hemocyte lineages. Dev. Genes Evol. 213 (10), 477–491. doi: 10.1007/s00427-003-0352-6 PubMed DOI

Nutting W. L. (1951). A comparative anatomical study of the heart and accessory structures of the orthopteroid insects. J. Morphology 89 (3), 501–597. doi: 10.1002/jmor.1050890306 DOI

Paredes J. C., Welchman D. P., Poidevin M., Lemaitre B. (2011). Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection. Immunity 35 (5), 770–779. doi: 10.1016/j.immuni.2011.09.018 PubMed DOI

Pauchet Y., Wilkinson P., Vogel H., Nelson D. R., Reynolds S. E., Heckel D. G., et al. . (2010). Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence. Insect Mol. Biol. 19 (1), 61–75. doi: 10.1111/j.1365-2583.2009.00936.x PubMed DOI

R Development Core Team (2008). R: A language and environment for statistical computing (Vienna, Austria: R Foundation for Statistical Computing; ). Available at: http://www.R-project.org, ISBN: ISBN 3-900051-07-0.

Reynolds S. E., Yeomans M. R., Timmins W. A. (1986). The feeding behaviour of caterpillars (Manduca sexta) on tobacco and on artificial diet. Physiol. Entomology 11 (1), 39–51. doi: 10.1111/j.1365-3032.1986.tb00389.x DOI

Rozen S., Skaletsky H. (2000). “Primer3 on the WWW for general users and for biologist programmers,” in Bioinformatics methods and protocols (Totowa, NJ: Humana Press; ), 365–386. doi: 10.1385/1-59259-192-2:365 PubMed DOI

Rupp R. A., Spence K. D. (1985). Protein alterations in Manduca sexta midgut and haemolymph following treatment with a sublethal dose of Bacillus thuringiensis crystal endotoxin. Insect Biochem. 15 (2), 147–154. doi: 10.1016/0020-1790(85)90002-2 DOI

Russell V., Dunn P. E. (1996). Antibacterial proteins in the midgut of Manduca sexta during metamorphosis. J. Insect Physiol. 42 (1), 65–71. doi: 10.1016/0022-1910(95)00083-6 PubMed DOI

Russell V. W., Dunn P. E. (1991). Lysozyme in the midgut of Manduca sexta during metamorphosis. Arch. Insect Biochem. Physiol. 17 (2-3), 67–80. doi: 10.1002/arch.940170202 PubMed DOI

Ryu J. H., Kim S. H., Lee H. Y., Bai J. Y., Nam Y. D., Bae J. W., et al. . (2008). Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science 319 (5864), 777–782. doi: 10.1126/science.1149357 PubMed DOI

Sampson M. N., Gooday G. W. (1998). Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects. Microbiology 144 (8), 2189–2194. doi: 10.1099/00221287-144-8-2189 PubMed DOI

Siva-Jothy M. T., Moret Y., Rolff J. (2005). Insect immunity: an evolutionary ecology perspective. Adv. Insect Physiol. 32, 1–48. doi: 10.1016/S0065-2806(05)32001-7 DOI

Sumathipala N., Jiang H. (2010). Involvement of Manduca sexta peptidoglycan recognition protein-1 in the recognition of bacteria and activation of prophenoloxidase system. Insect Biochem. Mol. Biol. 40 (6), 487–495. doi: 10.1016/j.ibmb.2010.04.008 PubMed DOI PMC

Tamez-Guerra P., Valadez-Lira J. A., Alcocer-González J. M., Oppert B., Gomez-Flores R., Tamez-Guerra R., et al. . (2008). Detection of genes encoding antimicrobial peptides in Mexican strains of Trichoplusia ni (Hübner) exposed to Bacillus thuringiensis . J. Invertebrate Pathol. 98 (2), 218–227. doi: 10.1016/j.jip.2008.02.008 PubMed DOI

Terra W. R., Barroso I. G., Dias R. O., Ferreira C. (2019). “Molecular physiology of insect midgut,” in Advances in insect physiology, vol. 56. (London:Academic Press; ), 117–163. doi: 10.1016/bs.aiip.2019.01.004 DOI

Thomson P. D., Smith D. J., Jr. (1994). What is infection? Am. J. Surg. 167 (1), S7–S11. doi: 10.1016/0002-9610(94)90003-5 PubMed DOI

Trenczek T., Faye I. (1988). Synthesis of immune proteins in primary cultures of fat body from Hyalophora cecropia . Insect Biochem. 18, 299–312. doi: 10.1016/0020-1790(88)90095-9 DOI

Tsakas S., Marmaras V. J. (2010). Insect immunity and its signalling: an overview. Invertebrate Survival J. 7 (2), 228–238.

Vodovar N., Vinals M., Liehl P., Basset A., Degrouard J., Spellman P., et al. . (2005). Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species. Proc. Natl. Acad. Sci. United States America 102 (32), 11414–11419. doi: 10.1073/pnas.0502240102 PubMed DOI PMC

von Bredow Y. M., von Bredow C. R., Trenczek T. E. (2020). A novel site of haematopoiesis and appearance and dispersal of distinct haemocyte types in the Manduca sexta embryo (Insecta, Lepidoptera). Dev. Comp. Immunol. 111, 103722. doi: 10.1016/j.dci.2020.103722 PubMed DOI

von Bredow C. R., von Bredow Y. M., Trenczek T. E. (2021). The larval haematopoietic organs of Manduca sexta (Insecta, Lepidoptera): An insight into plasmatocyte development and larval haematopoiesis. Dev. Comp. Immunol. 115, 103858. doi: 10.1016/j.dci.2020.103858 PubMed DOI

Willott E., Trenczek T., Thrower L. W., Kanost M. R. (1994). Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in Manduca sexta. Eur. J. Cell Biol. 65 (2), 417–423. PubMed

Wu K., Yang B., Huang W., Dobens L., Song H., Ling E. (2016). Gut immunity in Lepidopteran insects. Dev. Comp. Immunol. 64, 65–74. doi: 10.1016/j.dci.2016.02.010 PubMed DOI

Yamamoto R. (1969). Mass rearing of the tobacco hornworm. II. Larval rearing and pupation. J. Econ. Ent. 62, 1427–1431. doi: 10.1093/jee/62.6.1427 DOI

Yu X. Q., Zhu Y. F., Ma C., Fabrick J. A., Kanost M. R. (2002). Pattern recognition proteins in Manduca sexta plasma. Insect Biochem. Mol. Biol. 32 (10), 1287–1293. doi: 10.1016/S0965-1748(02)00091-7 PubMed DOI

Zachary D., Hoffmann D. (1984). Lysozyme is stored in the granules of certain haemocyte types in Locusta. J. Insect Physiol. 30 (5), 405–411. doi: 10.1016/0022-1910(84)90098-2 DOI

Zaidman-Rémy A., Hervé M., Poidevin M., Pili-Floury S., Kim M. S., Blanot D., et al. . (2006). The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 24 (4), 463–473. doi: 10.1016/j.immuni.2006.02.012 PubMed DOI

Zaidman-Rémy A., Regan J. C., Brandão A. S., Jacinto A. (2012). The Drosophila larva as a tool to study gut-associated macrophages: PI3K regulates a discrete hemocyte population at the proventriculus. Dev. Comp. Immunol. 36 (4), 638–647. doi: 10.1016/j.dci.2011.10.013 PubMed DOI

Zeng, Jaffar S., Xu Y., Qi Y. (2022). The intestinal immune defense system in insects. Y. The intestinal immune defense system in insects. Int. J. Mol. Sci. 23, 15132–15150. doi: 10.3390/ijms232315132 PubMed DOI PMC

Zhang X., Zhang F., Lu X. (2022). Diversity and functional roles of the gut microbiota in Lepidopteran insects. Microorganisms 10 (6), 1234. doi: 10.3390/microorganisms10061234 PubMed DOI PMC