Isolation and Molecular Characterization of Microflora from the Midgut of Armigeres Subalbatus Species Collected from Buldhana District, India.
Main Article Content
Abstract
Mosquitoes are of great importance to human health. A number of studies have shown that midgut and salivary gland microflora have an impact on malaria parasite burden through colonization mechanisms, involving either direct Plasmodium microbiota interaction or bacterial-mediated induction of mosquito immune response. The objective of this study was to isolate and identify the microflora from the midgut of Armigeres subalbatus species. A total of 10 field-collected adult Armigeres mosquitoes were anesthetized by chloroform and dissected. 70% of ethanol was used for surface sterilization of mosquitoes and laboratory equipment, followed by rinsing Armigeres mosquitoes two times with 1X PBS. Individually dissected midguts were transferred to 1.5ml Eppendorf tube containing 100µl of phosphate buffered saline (PBS) and homogenized. Gut homogenate was serially diluted (10 folds) in PBS and was directly pour plated on sterile nutrient agar media for 24 h at 35 ± 2 °C. From all field collected adult Armigeres subalbatus mosquitoes, total 26 bacterial isolates were obtained. From a total of 26 identified microflora, Bacillus cereus was the most abundant microbiota comprised 34.61% of the total species isolated. Pseudomonas aeruginosa was the second most abundant microbiota comprised 23.07% of the total species isolated. Staphyllococcus epidermidis was found to comprise 19.23% of total isolates. Pseudomonas geniculate & Serratia marcescens were found to constitute 11.53% each of the total identified microflora. The results of present investigation are quite encouraging & it can be used as a baseline for studying the relationship between microbiota and mosquitoes, and for the development of a new biological control.
Downloads
Article Details
This work is licensed under a Creative Commons Attribution 4.0 International License.
References
[1] A. N. Clements, “The biology of mosquitoes,” in Development, nutrition, and reproduction, vol. 1, Chapman & Hall, New York, 1992
[2] E. H. Hinman,“A study of the food of mosquito larvae,” American Journal of Hygiene, vol. 12, pp. 238–270, 1930.
[3] J. Chao and G. A. Wistreich, “Microbial isolations from the mid-gut of Culex tarsalis coquillet,”Journal of Insect Pathology, vol. 1, pp. 311–318, 1959.
[4] R. S. Michael, “Composition and functional roles of the gut microbiota in mosquitoes,” Current Opinion in Insect Science, vol. 28, pp. 59–65, 2018.
[5] L. Gonzalez-Ceron, F. Santillan, M. H. Rodriguez, D. Mendez, and J. E. Hernandez-Avila, “Bacteria in midguts of fieldcollected Anopheles albimanus block plasmodium vivax sporogonic development,” Journal of Medical Entomology, vol. 40, no. 3, pp. 371–374, 2003.
[6] Y. Dong, F. Manfredini, and G. Dimopoulos, “Implication of the mosquito midgut microbiota in the defense against malaria parasites,” PLoS Pathogens, vol. 5, no. 5, article e1000423, 2009.
[7] C. A. M. Finney, S. Kamhawi, and J. D. Wasmuth, “Does the arthropod microbiota impact the establishment of vectorborne diseases in mammalian hosts,” PLoS Pathogens, vol. 11, no. 4, article e1004646, 2015.
[8] K. K. Yadav, A. Naglot, K. Chandel et al., “Molecular characterization of midgut bacteria of Aedes albopictus exhibiting swarming motility property,” African Journal of Microbiology Research, vol. 10, no. 25, pp. 949–959, 2016.
[9] X. Ren, E. Hoiczyk, and J. L. Rasgon,“Viral paratransgenesis in the malaria vector Anopheles gambiae,” PLoS Pathogens, vol. 4, no. 8, article e1000135, 2008.
[10] J. L. Rasgon, “Using infections to fight infections: Paratransgenic fungi can block malaria transmission in mosquitoes,” Future Microbiology, vol. 6, no. 8, pp. 851–853, 2011.
[11] Ngo CTN, Aujoulant F, Veas F, Jumas-Bilak E, Manguin S. Bacterial diversity associated with wild caught Anopheles mosquitoes from Dak Nong Province, Vietnam using culture and DNA fingerprint. PLoS One. 2015;10(3): e0118634.
[12] Gusmao DS, Santos VA, Marinic DC, Jr MB, Berbert-Molina MA, Lemos FJA. Culture-dependent and culture-independent characterization of microorganisms associated with Aedesaegypti (Diptera: Culicidae) (L) and dynamics of bacterial colonization in the midgut. Acta Trop. 2010;115(1): 275–81.
[13] Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC. Bacterial population dynamics in three Anopheles species: the impact on Plasmodium sporogonic development. Am J TropMed Hyg1996; 54 (2): 214–218
[14] Straif SC, Mbogo CN, Toure AM, Walker ED, Kaufman M, Toure YT Beier JC. Mid gut bacteria in Anopheles gambiae and An. Funestus (Diptera: culicidae) from Kenya and Mali. JMed Entomol. 1998;35(3):222–6.
[15] V. J. Pidiyar, K. Jangid, M. S. Patole, and Y. S. Shouche, “Studies on cultured and uncultured microbiota of wild Culex quinquefasciatus mosquito midgut based on 16s ribosomal RNA gene analysis,” The American Journal of Tropical Medicine and Hygiene, vol. 70, no. 6, pp. 597–603, 2004.
[16] Y. Wang, T. M. Gilbreath, P. Kukutla, G. Yan, and J. Xu, “Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya,” PLoS One, vol. 6, no. 9, article e24767, 2011.
[17] K. Zouache, F. N. Raharimalala, V. Raquin et al., “Bacterial diversity of field-caught mosquitoes, Aedes albopictus and Aedes aegypti, from different geographic regions of Madagascar,” FEMS Microbiology Ecology, vol. 75, pp. 377–389, 2011.
[18] A. Boissière, M. T. Tchioffo, D. Bachar et al., “Midgut microbiota of the malaria mosquito vector Anopheles gambiae and interactions with Plasmodium falciparum infection,” PLoS Pathogens, vol. 8, no. 5, article e1002742, 2012.
[19] A. R. Chavshin, M. A. Oshaghi, H. Vatandoost et al., “Identification of bacterial microflora in the midgut of the larvae and adult of wild caught Anopheles stephensi: a step toward finding suitable paratransgenesis candidates,” Acta Tropica, vol. 121, pp. 129–134, 2011.
[20] J. Osei-Poku, C. M. Mbogo, W. J. Palmer, and F. M. Jiggins, “Deep sequencing reveals extensive variation in the gut microbiota of wild mosquitoes from Kenya,” Molecular Ecology, vol. 21, no. 20, pp. 5138–5150, 2012.
[21] G. Minard, P. Mavingui, and C. V. Moro, “Diversity and function of bacterial microbiota in the mosquito holobiont,” Parasites & Vectors, vol. 6, no. 1, p. 146, 2013.
[22] J. Demaio, C. B. Pumpuni, M. Kent, and J. C. Beier, “The midgut bacterial flora of wild Aedes triseriatus, Culex pipiens, and Psorophora columbiae mosquitoes,” The American Journal of Tropical Medicine and Hygiene, vol. 54, no. 2, pp. 219–223, 1996.
[23] K. Chandel, M. J. Mendki, R. Y. Parikh et al., “Midgut microbial community of Culex quinquefasciatus mosquito populations from India,” PLoS One, vol. 8, no. 11, article e80453, 2013
[24] Dennison NJ, Jupatanakul N, Dimopoulos G. The mosquito microbiota influences vector competence for human pathogens. Curr Opin Insect Sci. 2014;1(3):6–13.
[25] Minard G, Mavingui P, Moro CV. Diversity and function of bacterial microbiota in the mosquito holobiont. Parasit Vectors. 2013;6:146.
[26] Sharma P, Sharma S, Maurya RK, De TD, Thomas T, Lata S, et al. Salivary glands harbor more diverse microbial communities than gut in Anopheles culicifacies. Parasit Vectors. 2014;7(1):230–5.
[27] Alvarez C, Kukutla P, Jiang J, Yu W, Xu J. Draft genome sequence of Pseudomonas sp. strain Ag1, isolated from the midgut of the malaria mosquito Anopheles gambiae. J Bacteriol. 2012;194(19):5442–9.
[28] N. Gunathilaka, S. Siriwardana, L. Wijesooriya, G. Gunaratne, and N. Perera, “Subcutaneous dirofilariasis caused by Dirofilaria (Nochtiella) repens in Sri Lanka: a potential risk of transmitting human dirofilariasis,” SAGE Open Medical Case Reports, vol. 5, pp. 1–4, 2017.
[29] F. P. Amerasinghe, “Illustrated keys to the genera of mosquitoes (Diptera: Culicidae) in Sri Lanka,” Journal of National Science Council of Sri Lanka, vol. 23, no. 4, pp. 183–211, 1995.
[30] R. V. Chelliah, “Keys and illustrations to the genera of mosquitoes of Sri Lanka (Diptera: Culicidae),” Contributions of the American Entomological Institute, vol. 7, no. 4, pp. 1–84, 1984.
[31] R. Rattanarithikul, B. A. Harrison, P. Panthusiri, and R. E. Coleman, “Illustrated keys to the mosquitoes of Thailand 1, background; geographic distribution; list of genera, subgenera and species; and a key to the genera,” The Southeast Asian Journal of Tropical Medicine and Public Health, vol. 36, no. 1, pp. 1–80, 2005.
[32] K. Chandel, M. J. Mendki, R. Y. Parikh et al., “Midgut microbial community of Culex quinquefasciatus mosquito populations from India,” PLoS One, vol. 8, no. 11, article e80453, 2013.
[33] S. Srivastava and V. Singh, “Identification of regulatory elements in 16S rRNA gene of Acinetobacter species isolated from water sample,” Bioinformation, vol. 3, no. 4, pp. 173– 176, 2008.
[34] National Center for Biotechnology Information, “Basic Local Alignment Search Tool,” 2020, https://www.ncbi.nlm.nih .gov/BLAST.
[35] I. Eleftherianos, J. Atri, J. Accetta, and J. C. Castillo, “Endosymbiotic bacteria in insects: guardians of the immune system,” Frontiers in Physiology, vol. 4, p. 46, 2013.
[36] P. Azambuja, E. S. Garcia, and N. A. Ratcliffe, “Gut microbiota and parasite transmission by insect vectors,” Trends in Parasitology, vol. 21, no. 12, pp. 568–572, 2005.
[37] O. Terenius, J. M. Lindh, K. Eriksson-Gonzales et al., “Midgut bacterial dynamics in Aedes aegypti,” FEMS Microbiology Ecology, vol. 80, no. 3, pp. 556–565, 2012.
[38] C. Valiente Moro, F. H. Tran, F. N. Raharimalala, P. Ravelonandro, and P. Mavingui, “Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus,” BMC Microbiology, vol. 13, no. 1, p. 70, 2013.
[39] B. Chouaia, P. Rossi, M. Montagna et al., “Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species,” Applied and Environmental Microbiology, vol. 76, no. 22, pp. 7444–7450, 2010.
[40] N. J. Dennison, N. Jupatanakul, and G. Dimopoulos, “The mosquito microbiota influences vector competence for human pathogens,” Current Opinion in Insect Science, vol. 3, pp. 6–13, 2014.
[41] Kang X, Wang Y, Li S, Sun X, Lu X, Rajaofera MJN, Lu Y, Kang L, Zheng A, Zou Z, Xia Q. Comparative Analysis of the Gut Microbiota of Adult Mosquitoes From Eight Locations in Hainan, China. Front Cell Infect Microbiol. 2020 Dec 15;10:596750. doi: 10.3389/fcimb.2020.596750. PMID: 33384969; PMCID: PMC7769952.
[42] K. K. Yadav, A. Bora, S. Datta et al., “Molecular characterization of midgut microbiota of Aedes albopictus and Aedes aegypti from Arunachal Pradesh, India,” Parasites & Vectors, vol. 18, no. 8, p. 641, 2015.