Microbial Ecology

, Volume 78, Issue 1, pp 185–194 | Cite as

Delivery of a Genetically Marked Serratia AS1 to Medically Important Arthropods for Use in RNAi and Paratransgenic Control Strategies

  • Mona Koosha
  • Hassan Vatandoost
  • Fateh Karimian
  • Nayyereh Choubdar
  • Mohammad Ali OshaghiEmail author
Invertebrate Microbiology


Understanding how arthropod vectors acquire their bacteria is essential for implementation of paratransgenic and RNAi strategies using genetically modified bacteria to control vector-borne diseases. In this study, a genetically marked Serratia AS1 strain expressing the mCherry fluorescent protein (mCherry-Serratia) was used to test various acquisition routes in six arthropod vectors including Anopheles stephensi, Culex pipiens, Cx. quinquefaciatus, Cx. theileri, Phlebotomus papatasi, and Hyalomma dromedarii. Depending on the species, the bacteria were delivered to (i) mosquito larval breeding water, (ii) host skin, (iii) sugar bait, and (iv) males (paratransgenic). The arthropods were screened for the bacteria in their guts or other tissues. All the hematophagous arthropods were able to take the bacteria from the skin of their hosts while taking blood meal. The mosquitoes were able to take up the bacteria from the water at larval stages and to transfer them transstadially to adults and finally to transfer them to the water they laid eggs in. The mosquitoes were also able to acquire the bacteria from male sperm. The level of bacterial acquisition was influenced by blood feeding time and strategies (pool or vessel feeding), dipping in water and resting time of newly emerged adult mosquitoes, and the disseminated tissue/organ. Transstadial, vertical, and venereal bacterial acquisition would increase the sustainability of the modified bacteria in vector populations and decrease the need for supplementary release experiments whereas release of paratransgenic males that do not bite has fewer ethical issues. Furthermore, this study is required to determine if the modified bacteria can be introduced to arthropods in the same routes in nature.


Serratia AS1 Delivery methods RNAi Paratransgenesis 



We thank Marcelo Jacobs-Lorena Professor of Molecular Microbiology and Immunology Deputy-Director, at Johns Hopkins Bloomberg School of Public Health, for his immense help with editing and critical reading of the manuscript. Also, we thank Ms. Salimi (Department of Medical Parasitology, SPH, and TUMS) for helping with fluorescent microscopy and Naser Ghasemi (Department of Medical Entomology and Vector Control, SPH, TUMS) for rearing mosquitoes.


This study was funded by the Tehran University of Medical Sciences, Iran, Grant numbers 30353 and 33613.

Compliance with Ethical Standards

All procedures were performed in accordance with the terms of the Iran Animals (Scientific Procedures) Act Project License and were approved by the Tehran University of Medical Sciences Ethical Review Committee.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material


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  1. 1.
    WHO (2017) Fact sheet: vector-borne diseases. Updated October 2017Google Scholar
  2. 2.
    Paoletti MG, Pimentel D (2000) Environmental risks of pesticides versus genetic engineering for agricultural pest control. J Agric Environ Ethics 12:279–303CrossRefGoogle Scholar
  3. 3.
    Hemingway J, Field L, Vontas J (2002) An overview of insecticide resistance. Science 298:96–97CrossRefPubMedGoogle Scholar
  4. 4.
    Nicholson GM (2007) Fighting the global pest problem: preface to the special Toxicon issue on insecticidal toxins and their potential for insect pest control. Toxicon 49:413–422CrossRefPubMedGoogle Scholar
  5. 5.
    Limoee M, Enayati A, Ladonni H, Vatandoost H, Baseri H, Oshaghi M (2007) Various mechanisms responsible for permethrin metabolic resistance in seven field-collected strains of the German cockroach from Iran, Blattella germanica (L.) (Dictyoptera: Blattellidae). Pestic Biochem Physiol 87:138–146CrossRefGoogle Scholar
  6. 6.
    Yu N, Christiaens O, Liu J, Niu J, Cappelle K, Caccia S, Huvenne H, Smagghe G (2013) Delivery of dsRNA for RNAi in insects: an overview and future directions. Insect Sci 20:4–14CrossRefPubMedGoogle Scholar
  7. 7.
    Zamore PD (2001) RNA interference: listening to the sound of silence. Nat Struct Mol Biol 8:746–750CrossRefGoogle Scholar
  8. 8.
    Singh AD, Wong S, Ryan CP, Whyard S, Gordon K (2013) Oral delivery of double-stranded RNA in larvae of the yellow fever mosquito, Aedes aegypti: implications for pest mosquito control. J Insect Sci 13:1–18CrossRefGoogle Scholar
  9. 9.
    Taracena ML, Oliveira PL, Almendares O, Umaña C, Lowenberger C, Dotson EM, Paiva-Silva GO, Pennington PM (2015) Genetically modifying the insect gut microbiota to control Chagas disease vectors through systemic RNAi. PLoS Negl Trop Dis 9:e0003358CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Wang S, Dos-Santos AL, Huang W, Liu KC, Oshaghi MA, Wei G, Agre P, Jacobs-Lorena M (2017) Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria. Science 357:1399–1402CrossRefPubMedGoogle Scholar
  11. 11.
    Wang S, Ghosh AK, Bongio N, Stebbings KA, Lampe DJ, Jacobs-Lorena M (2012) Fighting malaria with engineered symbiotic bacteria from vector mosquitoes. Proc Natl Acad Sci 109:12734–12739CrossRefPubMedGoogle Scholar
  12. 12.
    Durvasula RV, Gumbs A, Panackal A, Kruglov O, Aksoy S, Merrifield RB, Richards FF, Beard CB (1997) Prevention of insect-borne disease: an approach using transgenic symbiotic bacteria. Proc Natl Acad Sci 94:3274–3278CrossRefPubMedGoogle Scholar
  13. 13.
    Ricci I, Damiani C, Rossi P, Capone A, Scuppa P, Cappelli A, Ulissi U, Mosca M, Valzano M, Epis S (2011) Mosquito symbioses: from basic research to the paratransgenic control of mosquito-borne diseases. J Appl Entomol 135:487–493CrossRefGoogle Scholar
  14. 14.
    Hurwitz I, Fieck A, Read A, Hillesland H, Klein N, Kang A, Durvasula R (2011) Paratransgenic control of vector borne diseases. Int J Biol Sci 7:1334–1344CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Coutinho-Abreu IV, Zhu KY, Ramalho-Ortigao M (2010) Transgenesis and paratransgenesis to control insect-borne diseases: current status and future challenges. Parasitol Int 59:1–8CrossRefPubMedGoogle Scholar
  16. 16.
    Maleki-Ravasan N, Oshaghi MA, Afshar D, Arandian MH, Hajikhani S, Akhavan AA, Yakhchali B, Shirazi MH, Rassi Y, Jafari R (2015) Aerobic bacterial flora of biotic and abiotic compartments of a hyperendemic zoonotic cutaneous Leishmaniasis (ZCL) focus. Parasit Vectors 8:63CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Enayati AA, Mardani N, Ghoorchian S (2012) 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 Trop 121:129–134CrossRefPubMedGoogle Scholar
  18. 18.
    Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Terenius O (2014) Isolation and identification of culturable bacteria from wild Anopheles culicifacies, a first step in a paratransgenesis approach. Parasit Vectors 7:419CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Dehghan H, Oshaghi MA, Moosa-Kazemi SH, Yakhchali B, Vatandoost H, Maleki-Ravasan N, Rassi Y, Mohammadzadeh H, Abai MR, Mohtarami F (2017) Dynamics of transgenic Enterobacter cloacae expressing green fluorescent protein-defensin (GFP-D) in Anopheles stephensi under laboratory condition. J Arthropod Borne Dis 11:515–532PubMedPubMedCentralGoogle Scholar
  20. 20.
    Lindh JM, Terenius O, Faye I (2005) 16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts. Appl Environ Microbiol 71:7217–7223CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Riehle MA, Jacobs-Lorena M (2005) Using bacteria to express and display anti-parasite molecules in mosquitoes: current and future strategies. Insect Biochem Mol Biol 35:699–707CrossRefPubMedGoogle Scholar
  22. 22.
    Mysore K, Sun L, Tomchaney M, Sullivan G, Adams H, Piscoya AS, Severson DW, Syed Z, Duman-Scheel M (2015) siRNA-mediated silencing of doublesex during female development of the dengue vector mosquito Aedes aegypti. PLoS Negl Trop Dis 9:e0004213CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Tian H, Peng H, Yao Q, Chen H, Xie Q, Tang B, Zhang W (2009) Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a non-midgut gene. PLoS One 4:e6225CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Araujo R, Santos A, Pinto F, Gontijo N, Lehane M, Pereira M (2006) RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection. Insect Biochem Mol Biol 36:683–693CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Walshe D, Lehane S, Lehane M, Haines L (2009) Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA. Insect Mol Biol 18:11–19CrossRefPubMedGoogle Scholar
  26. 26.
    Yoshida S, Ioka D, Matsuoka H, Endo H, Ishii A (2001) Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes. Mol Biochem Parasitol 113:89–96CrossRefPubMedGoogle Scholar
  27. 27.
    Aksoy S, Weiss B, Attardo G (2008) Paratransgenesis applied for control of tsetse transmitted sleeping sickness. Transgenesis and the management of vector-borne disease: 35–48Google Scholar
  28. 28.
    Vallet-Gely I, Lemaitre B, Boccard F (2008) Bacterial strategies to overcome insect defences. Nat Rev Microbiol 6:302–313CrossRefPubMedGoogle Scholar
  29. 29.
    Damiani C, Ricci I, Crotti E, Rossi P, Rizzi A, Scuppa P, Esposito F, Bandi C, Daffonchio D, Favia G (2008) Paternal transmission of symbiotic bacteria in malaria vectors. Curr Biol 18:R1087–R1088CrossRefPubMedGoogle Scholar
  30. 30.
    Favia G, Ricci I, Damiani C, Raddadi N, Crotti E, Marzorati M, Rizzi A, Urso R, Brusetti L, Borin S (2007) Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector. Proc Natl Acad Sci 104:9047–9051CrossRefPubMedGoogle Scholar
  31. 31.
    Watanabe K, Yukuhiro F, Matsuura Y, Fukatsu T, Noda H (2014) Intrasperm vertical symbiont transmission. Proc Natl Acad Sci 111:7433–7437CrossRefPubMedGoogle Scholar
  32. 32.
    Lindh JM, Terenius O, Eriksson-Gonzales K, Knols BG, Faye I (2006) Re-introducing bacteria in mosquitoes—a method for determination of mosquito feeding preferences based on coloured sugar solutions. Acta Trop 99:173–183CrossRefPubMedGoogle Scholar
  33. 33.
    Lindh J, Borg-Karlson A-K, Faye I (2008) Transstadial and horizontal transfer of bacteria within a colony of Anopheles gambiae (Diptera: Culicidae) and oviposition response to bacteria-containing water. Acta Trop 107:242–250CrossRefPubMedGoogle Scholar
  34. 34.
    Chavshin AR, Oshaghi MA, Vatandoost H, Yakhchali B, Raeisi A, Zarenejad F (2013) Escherichia coli expressing a green fluorescent protein (GFP) in Anopheles stephensi: a preliminary model for paratransgenesis. Symbiosis 60:17–24CrossRefGoogle Scholar
  35. 35.
    Chavshin AR, Oshaghi MA, Vatandoost H, Yakhchali B, Zarenejad F, Terenius O (2015) Malpighian tubules are important determinants of Pseudomonas transstadial transmission and longtime persistence in Anopheles stephensi. Parasit Vectors 8:36CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Modi GB, Tesh RB (1983) A simple technique for mass rearing Lutzomyia longipalpis and Phlebotomus papatasi (Diptera: Psychodidae) in the laboratory. J Med Entomol 20:568–569CrossRefPubMedGoogle Scholar
  37. 37.
    Troughton DR, Levin ML (2007) Life cycles of seven ixodid tick species (Acari: Ixodidae) under standardized laboratory conditions. J Med Entomol 44:732–740CrossRefPubMedGoogle Scholar
  38. 38.
    Martínez-García E, Calles B, Arévalo-Rodríguez M, de Lorenzo V (2011) pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiol 11:38CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Gonzalez-Ceron L, Santillan F, Rodriguez MH, Mendez D, Hernandez-Avila JE (2003) Bacteria in midguts of field-collected Anopheles albimanus block Plasmodium vivax sporogonic development. J Med Entomol 40:371–374CrossRefPubMedGoogle Scholar
  40. 40.
    Boissière A, Tchioffo MT, Bachar D, Abate L, Marie A, Nsango SE, Shahbazkia HR, Awono-Ambene PH, Levashina EA, Christen R (2012) Midgut microbiota of the malaria mosquito vector Anopheles gambiae and interactions with Plasmodium falciparum infection. PLoS Pathog 8:e1002742CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Pumpuni C, Beier M, Nataro J, Guers LD, Davis J (1993) Plasmodium falciparum: inhibition of sporogonic development in Anopheles stephensi by gram-negative bacteria. Exp Parasitol 77:195–199CrossRefPubMedGoogle Scholar
  42. 42.
    Rani A, Sharma A, Rajagopal R, Adak T, Bhatnagar RK (2009) Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi—an Asian malarial vector. BMC Microbiol 9:96CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Sharma P, Sharma S, Maurya RK, De TD, Thomas T, Lata S, Singh N, Pandey KC, Valecha N, Dixit R (2014) Salivary glands harbor more diverse microbial communities than gut in Anopheles culicifacies. Parasit Vectors 7:235CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Gusmão DS, Santos AV, Marini DC, Russo ÉS, Peixoto AMD, Bacci Júnior M, Berbert-Molina MA, Lemos FJA (2007) First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association. Mem Inst Oswaldo Cruz 102:919–924CrossRefPubMedGoogle Scholar
  45. 45.
    Maleki-Ravasan N, Oshaghi MA, Hajikhani S, Saeidi Z, Akhavan AA, Gerami-Shoar M, Shirazi MH, Yakhchali B, Rassi Y, Afshar D (2014) Aerobic microbial community of insectary population of Phlebotomus papatasi. J Arthropod Borne Dis 8:69PubMedGoogle Scholar
  46. 46.
    C-h LI, Jie C, Y-z ZHOU, H-s ZHANG, H-y GONG, J-l ZHOU (2014) The midgut bacterial flora of laboratory-reared hard ticks, Haemaphysalis longicornis, Hyalomma asiaticum, and Rhipicephalus haemaphysaloides. J Integr Agric 13:1766–1771CrossRefGoogle Scholar
  47. 47.
    Patton WS, Cragg F (1913) On certain haematophagous species of the genus Musca, with descriptions of two new species. Indian J Med Res 1Google Scholar
  48. 48.
    Lavoipierre M (1965) Feeding mechanism of blood-sucking arthropods. Nature 208:302–303CrossRefPubMedGoogle Scholar
  49. 49.
    Brown SJ, Rosalsky JH (1984) Blood leukocyte response in hosts parasitized by the hematophagous arthropods Triatoma protracta and Lutzomyia longipalpis. The American journal of tropical medicine and hygiene 33:499–505CrossRefPubMedGoogle Scholar
  50. 50.
    Buczek A, Bartosik K, Zając Z, Stanko M (2015) Host-feeding behaviour of Dermacentor reticulatus and Dermacentor marginatus in mono-specific and inter-specific infestations. Parasit Vectors 8:470CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Hussein MA, Roby NH, Doha SA, Ghany SASA (2015) Effect of different blood sources on the feeding time of sand fly, Phlebotomus Papatasi. J Egypt Soc Parasitol 45:555–558CrossRefPubMedGoogle Scholar
  52. 52.
    Ribeiro J (2000) Blood-feeding in mosquitoes: probing time and salivary gland anti-haemostatic activities in representatives of three genera (Aedes, Anopheles, Culex). Med Vet Entomol 14:142–148CrossRefPubMedGoogle Scholar
  53. 53.
    Nuttall P, Labuda M (2003) Dynamics of infection in tick vectors and at the tick-host interface. Adv Virus Res 60:233–272CrossRefPubMedGoogle Scholar
  54. 54.
    Killick-Kendrick R, Leaney A, Ready P, Molyneux D (1977) Leishmania in phlebotomid sandflies-IV. The transmission of Leishmania mexicana amazonensis to hamsters by the bite of experimentally infected Lutzomyia longipalpis. Proc R Soc Lond B 196:105–115CrossRefPubMedGoogle Scholar
  55. 55.
    Beach R, Kiilu G, Hendricks L, Oster C, Leeuwenburg J (1984) Cutaneous leishmaniasis in Kenya: transmission of Leishmania major to man by the bite of a naturally infected Phlebotomus duboscqi. Trans R Soc Trop Med Hyg 78:747–751CrossRefPubMedGoogle Scholar
  56. 56.
    Kamhawi S (2000) The biological and immunomodulatory properties of sand fly saliva and its role in the establishment of Leishmania infections. Microbes Infect 2:1765–1773CrossRefPubMedGoogle Scholar
  57. 57.
    Rohousová I, Volf P (2006) Sand fly saliva: effects on host immune response and Leishmania transmission. Folia Parasitol 53:161–171CrossRefPubMedGoogle Scholar
  58. 58.
    Jadin J, Vincke I, Dunjic A, Delville J, Wery M, Bafort J, Scheepers-Biva M (1966) Role of Pseudomonas in the sporogenesis of the hematozoon of malaria in the mosquito. Bull Soc Pathol Exot Filiales 59:514PubMedGoogle Scholar
  59. 59.
    Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC (1996) Bacterial population dynamics in three anopheline species: the impact on Plasmodium sporogonic development. The American journal of tropical medicine and hygiene 54:214–218CrossRefPubMedGoogle Scholar
  60. 60.
    Briones AM, Shililu J, Githure J, Novak R, Raskin L (2008) Thorsellia anophelis is the dominant bacterium in a Kenyan population of adult Anopheles gambiae mosquitoes. The ISME journal 2:74–82CrossRefPubMedGoogle Scholar
  61. 61.
    Hosokawa T, Kikuchi Y, Fukatsu T (2007) How many symbionts are provided by mothers, acquired by offspring, and needed for successful vertical transmission in an obligate insect–bacterium mutualism? Mol Ecol 16:5316–5325CrossRefPubMedGoogle Scholar
  62. 62.
    Kremer N, Huigens ME (2011) Vertical and horizontal transmission drive bacterial invasion. Mol Ecol 20:3496–3498CrossRefPubMedGoogle Scholar
  63. 63.
    Zug R, Hammerstein P (2012) Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PLoS One 7:e38544CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Knell RJ, Webberley KM (2004) Sexually transmitted diseases of insects: distribution, evolution, ecology and host behaviour. Biol Rev 79:557–581CrossRefPubMedGoogle Scholar
  65. 65.
    Moran NA, Dunbar HE (2006) Sexual acquisition of beneficial symbionts in aphids. Proc Natl Acad Sci 103:12803–12806CrossRefPubMedGoogle Scholar
  66. 66.
    Moll RM, Romoser WS, Modrakowski MC, Moncayo AC, Lerdthusnee K (2001) Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. J Med Entomol 38:29–32CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Medical Entomology and Vector Control, School of Public HealthTehran University of Medical SciencesTehranIran

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