Molecular characterization of proteolytic bacteria associated with Malaria vectors: Anopheles sundaicus and Anopheles vagus

Main Article Content

Kartika Senjarini
Antje Labes
Syubbanul Wathon
Rike Oktarianti
Tri Yudani Mardining Raras
Naura Paramitha Cindy Ardyah
Dita Paramytha Agustin
Durotun Ainiyah
Diah Ayu Utami

Abstract

BACKGROUND
Anopheles (An.) sp. transmits Plasmodium parasites that cause malaria. In its life cycle in the mosquito’s body, Plasmodium passes through 2 mosquito organs, namely the salivary glands and midgut. The bacterial community (symbiont bacteria) in these organs has been known to influence and/or inhibit the development of the Plasmodium life cycle by producing specific proteases. This research aims to isolate and characterize symbiotic bacteria with proteolytic activity from 2 important malaria vectors in Indonesia: An. sundaicus and An. vagus.


METHODS
A total of 183 bacterial originating from the salivary glands and midgut were successfully isolated. Initial screening was carried out based on morphological differences, followed by purification of the selected isolates to obtain single colonies. The selected isolates were then subjected to an initial proteolytic ability test using skim milk agar media. Only isolates with proteolytic activity were further characterized with the 16SrDNA molecular marker. The isolates were pabs5 from the salivary glands and pabs3 from the midgut of An. vagus, while pdbs3 and ecbs4 were isolates from the salivary glands and midgut of An. sundaicus.


RESULTS
Morphological and molecular characterization showed that both pabs5 and pabs3 isolates were Pseudomonas
(Ps.) aeruginosa, while ecbs4 was Enterobacter cloacae and pdbs3 was Pantoea dispersa. These species were
first discovered in Anopheles vagus and Anopheles sundaicus.


CONCLUSION
The ability of Ps. aeruginosa and Pantoea dispersa to produce proteases indicated their potential role in the exploration of new strategies to control mosquito vectors that transmit pathogens.

Article Details

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Original Articles

How to Cite

Molecular characterization of proteolytic bacteria associated with Malaria vectors: Anopheles sundaicus and Anopheles vagus. (2024). Universa Medicina, 43(2), 202-212. https://doi.org/10.18051/UnivMed.2024.v43.202-212

References

Venugopal K, Hentzschel F, Valkiūnas G, Marti M. Plasmodium asexual growth and sexual development in the haematopoietic niche of the host. Nat Rev Microbiol 2020;18:177–89. https://doi.org/10.1038/s41579-019-0306-2.

Thongsripong P, Chandler JA, Green AB, et al. Mosquito vector-associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod-borne diseases. Ecol Evol 2018;8:1352–68. https://doi.org/10.1002/ece3.3676.

Kalappa DM, Subramani PA, Basavanna SK, et al. Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections. Malar J 2018;17:385. https://doi.org/10.1186/s12936-018-2535-7.

Gabrieli P, Caccia S, Varotto-Boccazzi I, et al. Mosquito trilogy:microbiota, immunity and pathogens, and their implications for the control of disease transmission. Front Microbiol 2021;12:630438.doi: 10.3389/fmicb.2021.630438.

Berhanu A, Abera A, Nega D, et al. Isolation and identification of microflora from the midgut and salivary glands of Anopheles species in malaria endemic areas of Ethiopia. BMC Microbiol 2019;19:85. https://doi.org/10.1186/s12866-019-1456-0.

Ngo CT, Romano-Bertrand S, Manguin S, Jumas-Bilak E. Diversity of the bacterial microbiota of Anopheles mosquitoes from Binh Phuoc Province, Vietnam. Front Microbiol 2016;7:2095. https://doi.org/10.3389/fmicb.2016.02095.

Nouzova M, Clifton ME, Noriega FG. Mosquito adaptations to hematophagia impact pathogen transmission. Curr Opin Insect Sci 2019;34:21–6. https://doi.org/10.1016/j.cois.2019.02.002.

Zhang H, Dong S, Chen X, et al. Relish2 mediates bursicon homodimer-induced prophylactic immunity in the mosquito Aedes aegypti. Sci Rep 2017;7:43163. https://doi.org/10.1038/srep43163.

Arifianto RP, Masruroh D, Habib MJ, et al. Identifikasi dan analisis bionomik vektor malaria Anopheles sp. di Desa Bangsring Kecamatan Wongsorejo, Banyuwangi.[Identification and bionomic analysis of malaria vectors Anopheles sp. in Bangsring Village Wongsorejo District, Banyuwangi]. Acta Vet Indones 2018;6:44–50. Indonesian. https://doi.org/10.29244/avi.6.1.44-50

Syafruddin D, Lestari YE, Permana DH, et al. Anopheles sundaicus complex and the presence of Anopheles epiroticus in Indonesia. PLoS Negl Trop Dis 2020;14:e0008385. https://doi.org/10.1371/journal.pntd.000838.

Senjarini K, Abdullah MK, Azizah N, et al. Redesigning primer of ITS2 (Internal Transcribed Spacer 2) for specific molecular characterization of malaria vectors Anopheles species. Med Arch 2021;75:418–23. https://doi.org/10.5455/medarh.2021.75.418-423.

Hasanah LN, Masruroh D, Wahyuni I, et al. Internal transcribed spacer 2 (ITS2) based molecular identification of malaria vectors from Bangsring Banyuwangi-Indonesia. As Pac J Mol Biol Biotechnol 2022;30:57-68. https://doi.org/10.35118/apjmbb.2022.030.3.06.

Budiyanto A, Ambarita LP, Salim M. Konfirmasi Anopheles sinensis dan Anopheles vagus sebagai vektor malaria di Kabupaten Muara Enim Provinsi Sumatera Selatan. [Confirmation of Anopheles sinensis and Anopheles vagus as malaria vectors in Muara Enim District, South Sumatra province], Aspirator 2017;9:51-60. Indonesian. https://dx.doi.org/10.22435/aspirator.v9i2%20Des.5998.51-60.

Scolari F, Casiraghi M, Bonizzoni M. Aedes spp. and their microbiota: a review. Front Microbiol 2019;10:2036. https://doi.org/10.3389/fmicb.2019.02036.

Dickson LB, Ghozlane A, Volant S, et al. Diverse laboratory colonies of Aedes aegypti harbor the same adult midgut bacterial microbiome. Parasites Vectors 2018;11:207. https://doi.org/10.1186/s13071-018-2780-1.

Young KI, Medwid JT, Azar SR, Identification of mosquito bloodmeals collected in diverse habitats in Malaysian Borneo using COI barcoding. Trop Med Infect Dis 2020;5:51. doi: 10.3390/tropicalmed5020051.

Senjarini K, Oktarianti R, Abdullah MK, Sholichah RN, Tosin A, Wathon S. Morphological characteristic difference between mosquitoes vector for malaria and dengue fever. Bioedukasi 2020;18:53-8. https://doi.org/10.19184/bioedu.v18i2.18890.

Yadav KK, Bora A, Datta S, et al. Molecular characterization of midgut microbiota of Aedes albopictus and Aedes aegypti from Arunachal Pradesh, India. Parasites Vectors 2015;8:641. https://doi.org/10.1186/s13071-015-1252-0.

Leboffe MJ, Pierce BE. Microbiology: laboratory theory and application. Morton Publishing Company; 2019.

Susanti E, Suharti, Ramadhan HR, Fatma F. Isolasi dan seleksi bakteri proteolitik potensial dari tauco Surabaya. [Isolation and selection of potential proteolytic bacteria from fermented soy bean paste]. Prosiding Seminar Nasoinal Kimia dan Pembelajarannya: Universitas Negeri Malang; 2017. Indonesian.

Green MR, Sambrook J. Molecular cloning: a laboratory manual. 4th ed. New York: Cold Spring Harbor Laboratory Press;2012.

Bando H, Okado K, Guelbeogo WM, et al. Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity. Sci Rep 2013;3:1641. doi: 10.1038/srep01641.

Azambuja P, Garcia ES, Ratcliffe NA. Gut microbiota and parasite transmission by insect vectors. Trends Parasitol 2005;21:568–72. https://doi.org/10.1016/j.pt.2005.09.011.

Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Terenius O. Isolation and identification of culturable bacteria from wild Anopheles culicifacies, a first step in a paratransgenesis approach. Parasit Vectors 2014;7:419. https://doi.org/10.1186/1756-3305-7-419.

Lazaro JEH, Nitcheu J, Predicala RZ, et al. Heptyl prodigiosin, a bacterial metabolite, is antimalarial in vivo and non-mutagenic in vitro. J Nat Toxins 2002;11:367–77.

Bakhshi H, Mousson L, Moutailler S, et al. Detection of arboviruses in mosquitoes: Evidence of circulation of chikungunya virus in Iran. PLoS Negl Trop Dis 2020;14:e0008135. https://doi.org/10.1371%2Fjournal.pntd.0008135.

Elkington PTG, O’Kane CM, Friedland JS. The paradox of matrix metalloproteinases in infectious disease. Clin Exp Immunol 2005;142:12–20. https://doi.org/10.1111/j.1365-2249.2005.02840.x.

Walterson AM, Stavrinides J. Pantoea: insights into a highly versatile and diverse genus within the Enterobacteriaceae. FEMS Microbiol Rev 2015;39:968–84. https://doi.org/10.1093/femsre/fuv027.

Yang CH, Qiao FJ, Lu Z, et al. Interspecific competitions between Frankliniella intonsa and Frankliniella occidentalis on fresh lentil bean pods and pepper plants. Insects 2022;14:1. https://doi.org/10.3390/insects14010001.

Jayakrishnan L, Sudhikumar A, Aneesh EM. Role of gut inhabitants on vectorial capacity of mosquitoes. J Vector Borne Dis 2018;55:69-78. https://doi.org/10.4103/0972-9062.242567.

Rajasekhar K, Nizamuddin N, Surur AS, Mekonnen YT. Synthesis, characterization, antitubercular and antibacterial activity, and molecular docking of 2,3-disubstituted quinazolinone derivatives. Res Rep Med Chem 2016;6:15–26. https://doi.org/10.2147/RRMC.S91474.

Arruda A, Ferreira GEM, Santos Júnior A, et al. Diversity of culturable bacteria isolated from the feces of wild Anopheles darlingi (Diptera: Culicidae) mosquitoes from the Brazilian Amazon. J Med Entomol 2021;58:1900–7. https://doi.org/10.1093/jme/tjab028.

Silva BE, Zingoni ZM, Koekemoer LL, Dahan-Moss YL. Microbiota identified from preserved Anopheles. Malaria J 2021;20:230. https://doi.org/10.1186/s12936-021-03754-7.

Eappen AG, Smith RC, Jacobs-Lorena M. Enterobacter-activated mosquito immune responses to Plasmodium involve activation of SRPN6 in Anopheles stephensi. PLoS ONE 2013;8:e62937. https://doi.org/10.1371/journal.pone.0062937.

Gonzalez-Ceron L, Santillan F, Rodriguez MH, Mendez D, Hernandez-Avila JE. Bacteria in midguts of field-collected Anopheles albimanus block Plasmodium vivax sporogonic development. J Med Entomol 2003;40:371–4. https://doi.org/10.1603/0022-2585-40.3.371.

Ezemuoka 26 LC, Akorli EA, Aboagye-Antwi F, Akorli J. Mosquito midgut Enterobacter cloacae and Serratia marcescens affect the fitness of adult female Anopheles gambiae s.l. PLoS ONE 2020;15:e0238931. https://doi.org/10.1371/journal.pone.0238931.