Original Article

Dynamics of Transgenic Enterobacter cloacae Expressing Green Fluorescent Protein-Defensin (GFP-D) in Anopheles stephensi under Laboratory Condition

Abstract

Background: Enterobacter cloacae bacterium is a known symbiont of the most Anopheles gut microflora and nominated as a good candidate for paratransgenic control of malaria. However, the population dynamics of this bacterium with­in An. stephensi and its introduction methods to the mosquitoes have not yet been explored.
Methods:
Enterobacter cloacae subsp. dissolvens expressing green fluorescent protein and defensin (GFP-D) was used to study transstadial transmission and the course of time, larval habitat, sugar, and blood meal on dynamics of the bacterium in the mosquito life stages in the laboratory condition. The bacterial quantities were measured by plating samples and counting GFP expressing colonies on the Tet-BHI agar medium.
Results:
The E. cloacae population remained stable in sugar bait at least for eleven days whereas it was lowered in the insectary larval habitat where the bacteria inadequately recycled. The bacterium was weakly transmitted transstadi­ally from larval to adult stage. The bacterial populations increased smoothly and then dramatically in the guts of An. stephensi following sugar and blood meal respectively followed by a gradual reduction over the time.
Conclusion:
Enterobacter cloacae was highly stable in sugar bait and increased tremendously in the gut of female adult An. stephensi within 24h post blood meal. Sugar bait stations can be used for introduction of the transgenic bacteria in a paratransgenic approach. It is recommended to evaluate the attraction of sugar bait in combination with attractive kairomones as well as its stability and survival rate in the semi-field or field conditions.

Anjomruz M, Oshaghi MA, Pourfatollah AA, Sedaghat MM, Raeisi A, Vatan-doost H, Khamesipour A, Abai MR, Mohtarami F, Akbarzadeh K, Rafie F, Besharati M (2014) Preferential feeding success of laboratory reared Anoph¬eles stephensi mosquitoes ac-cording to ABO blood group status. Acta Trop. 140: 118–123.

Karimian F, Oshaghi MA, Sedaghat MM, Waterhouse RM, Vatandoost H, Hana-fi-Bojd AA, Ravasan NM, Chavshin AR (2014) Phylogenetic analy-sis of the oriental-Palearctic-Afrotropical members of Anopheles (Culicidae: Dip-tera) based on nuclear rDNA and mito-chon¬drial DNA char¬acteristics. Jpn J In-fect Dis. 67(5): 361–367.

Norouzinejad F, Ghaffari F, Raeisi A, No-rouzinejad A (2016) Epidemiologi-cal status of malaria in Iran, 2011–2014. Asian Pac J Trop Med. 9(11): 1055–1061.

Naddaf SR, Oshaghi MA, Vatandoost H, Assmar M (2003) Molecular charac-terization of Anopheles fluviatilis species complex in the Islamic Republic of Iran. East Mediterr Health J. 9(3): 257–265.

Vatandoost H, Emami SN, Oshaghi MA, Abai MR, Raeisi A, Piazak N, Mah-moodi M, Akbarzadeh K, Sartipi M (2011) Ecology of malaria vector Anoph¬eles culicifacies in a malari-ous area of Sistan va Baluchestan Prov-ince, south-east Islamic Republic of Iran. East Mediterr Health J. 17(5): 439–445.

Vatandoost H, Oshaghi MA, Abaie MR, Shahi M, Yaaghoobi F, Baghaii M, Hanafi-Bojd AA, Zamani G, Town-son H (2006) Bi¬onomics of Anophe-les ste¬phensi Liston in the malarious area of Hormozgan Province, south-ern Iran, 2002. Acta Trop. 97(2): 196–203.

Vatandoost H, Shahi H, Abai MR, Hanafi-Bojd AA, Oshagh MA, Zamani G (2004) Larval habitats of main malaria vectors in Hormozgan Province and their susceptibility to different larvicides. South¬east Asian J Trop Med Public Health. 35(2): 22–25.

Emami SN, Vatandoost H, Oshaghi MA, Mohtarami F, Javadian E, Raeisi A (2007) Morphological method for sex¬ing anopheline larvae. J Vector Borne Dis. 44(4): 245–249.

Mehravaran A, Oshaghi MA, Vatandoost H, Abai MR, Ebrahimzadeh A, Roodi AM, Grouhi A (2011) First report on Anopheles fluviatilis U in southeastern Iran. Ac-ta Trop. 117(2): 76–81.

Hanafi-Bojd AA, Vatandoost H, Oshaghi MA, Haghdoost AA, Shahi M, Seda-ghat MM, Abedi F, Yeryan M, Pakari A (2012) Entomological and epidemi-ological attrib¬utes for malaria transmis-sion and implemen¬tation of vector control in southern Iran. Ac¬ta Trop. 121 (2): 85–92.

Oshaghi MA, Shemshad Kh, Yaghobi-Ershadi MR, Pedram M, Vatandoost H, Abaie MR, Akbarzadeh K, Moh-tarami F (2007) Genetic structure of the malaria vector Anopheles superpictus in Iran using mi¬tochondrial cytochrome ox-idase (COI and COII) and morphologic markers: a new spe¬cies complex? Acta Trop. 101(3): 241–248.

Oshaghi MA, Yaghobi-Ershadi MR, Shemshad K, Pedram M, Amani H (2008) The Anopheles superpictus com¬plex: intro¬duction of a new ma-laria vector complex in Iran. Bull Soc Pathol Exot. 101(5): 429–434.

Oshaghi MA, Yaaghoobi F, Abaie MR (2006) Pattern of mitochondrial DNA varia¬tion between and within Anopheles stephensi (Diptera: Culicidae) bio- logical forms sug¬gests extensive gene flow. Acta Trop. 99(2–3): 226–233.

Mehravaran A, Vatandoost H, Oshaghi MA, Abai MR, Edalat H, Javadian E, Mash¬ayekhi M, Piazak N, Hanafi-Bojd AA (2012) Ecology of Anopheles ste-phensi in a malari¬ous area, southeast of Iran. Acta Med Iran. 50(1): 61–65.

Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Tereni-us O (2014) Isolation and identifica-tion of cul¬turable bacteria from wild Anoph¬eles culicifacies, a first step in a paratransgenesis approach. Parasit Vectors. 7: 419.

Davari B, Vatandoost H, Oshaghi MA, Ladonni H, Enayati AA, Shaeghi M, Basseri HR, Rassi Y, Hanafi-Bojd AA (2007) Selection of Anopheles stephensi with DDT and dieldrin and cross-resistance spectrum to pyrethroids and fipronil. Pestic Biochem Phys-iol. 89 (2): 97–103.

Abai MR, Mehravaran A, Vatandoost H, Oshaghi MA, Javadian E, Mashayekhi M, Mosleminia A, Piyazak N, Edallat H, Moh¬tarami F, Jabbari H, Rafi F (2008) Compara¬tive perfor-mance of imagicides on Anophe¬les stephensi, main malaria vector in a ma¬larious area, southern Iran. J Vector Borne Dis. 45(4): 307–312.

Soleimani-Ahmadi M, Vatandoost H, Shaeghi M, Raeisi A, Abedi F, Eshraghi¬an MR, Madani A, Safari R, Oshaghi MA, Ab¬tahi M, Hajjaran H. (2012) Field evaluation of perme-thrin long-lasting insecticide treated nets (Olyset(®)) for malaria control in an endemic area, southeast of Iran. Acta Trop. 123(3): 146–153.

Fathian M, Vatandoost H, Moosa-Kazemi SH, Raeisi A, Yaghoobi-Ershadi MR, Oshaghi MA, Sedaghat MM (2014) Susceptibility of Culicidae Mosquitoes to Some Insecticides Recommended by WHO in a Malaria Endemic Area of Southeast¬ern Iran. J Arthropod Borne Dis. 9(1): 22–34.

Soltani A, Vatandoost H, Oshaghi MA, Ravasan NM, Enayati AA, Asgarian F (2014) Resistance Mechanisms of Anopheles stephensi (Diptera: Culici¬dae) to Temephos. J Arthropod Borne Dis. 9(1): 71–83.

Gorouhi MA, Vatandoost H, Oshaghi MA, Raeisi A, Enayati AA, Mirhen-di H, Hanafi-Bojd AA, Abai MR, Salim-Abadi Y, Rafi F (2016) Cur-rent Susceptibility Status of Anopheles ste¬phensi (Diptera: Culicidae) to Dif-ferent Imagicides in a Malarious Ar-ea, Southeastern of Iran. J Arthropod Borne Dis. 10(4): 493–500.

Enayati AA, Vatandoost H, Ladonni H, Townson H, Hemingway J (2003) Molecular evidence for a kdr-like pyrethroid resistance mechanism in the malaria vector mosquito Anophe-les stephensi. Med Vet Entomol. 17(2): 138–44.

Sanil D, Shetty V, Shetty NJ (2014) Dif-ferential expression of glutathione s-transferase enzyme in different life stages of various insecticide-resistant strains of Anopheles stephensi: a malaria vector. J Vector Borne Dis. 51(2): 97–105.

Ahmad M, Buhler C, Pignatelli P, Ran¬son H, Nahzat SM, Naseem M, Sabawoon MF, Siddiqi AM, Vink M (2016) Status of insecticide resistance in high-risk ma-laria provinces in Afghanistan. Ma¬lar J. 15: 98.

Wang S, Jacobs-Lorena M (2013) Ge-net¬ic approaches to interfere with malaria transmission by vector mos-quitoes. Trends Biotechnol. 31(3): 185–193.

WHO Fact Sheet: World Malaria Report (2015) Geneva: World Health Or-ganization, 2015.

http://www.who.int/malaria/media/world-malaria-report-2015/en/

Ghosh A, Edwards M, Jacobs-Lorena M (2000) The journey of the malaria par-asite in the mosquito: hopes for the new century. Parasitol Today. 16(5): 196–201.

Whitten MM, Shiao SH, Levashina EA (2006) Mosquito midguts and malar-ia: cell biology, compartmentalization and immu¬nology. Parasite Immunol. 28: 121–130.

Sinden RE, Dawes EJ, Alavi Y, Waldock J, Finney O, Mendoza J, Butcher GA, An¬drews L, Hill AV, Gilbert SC, Basáñez MG (2007) Progression of Plasmodium berghei through Anopheles stephensi is density-dependent.

PLoS Pathog. 3: e195.

Cirimotich CM, Dong Y, Clayton AM, Sandiford SL, Souza-Neto JA, Mu-lenga M, Dimopoulos G (2011) Nat-ural microbe-mediated refractori-ness to Plasmodium in¬fection in Anopheles gambiae. Science. 332: 855–858.

Eappen AG, Smith RC, Jacobs-Lorena M (2013) Enterobacter-Activated Mos¬quito Immune Responses to Plasmodium Involve Activation of SRPN6 in Anopheles stephen¬si. PLoS ONE. 8(5): e62937.

Pumpuni CB, Beier MS, Nataro JP, Guers LD, Davis JR (1993) Plasmo-dium fal¬ciparum: inhibition of spo-rogonic develop¬ment in Anopheles stephensi by gram-negative bacteria. Exp Parasitol. 77: 195–199.

Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC (1996) Bacterial popu-lation dy¬namics in three anopheline species: the im¬pact on Plasmodium sporogonic develop¬ment. The Am J Trop Med Hyg. 54: 214–218.

Dong Y, Manfredini F, Dimopoulos G (2009) Implication of the mosquito mid-gut microbiota in the defense against malaria parasites. PLoS Pathog. 5: e1000423.

Meister S1, Agianian B, Turlure F, Relógio A, Morlais I, Kafatos FC, Chris¬tophides GK (2009) Anopheles gambiae PGRPLC-mediated defense against bacteria modulates infections with malaria parasites. PLoS Pathog. 5: e1000542.

Cirimotich CM, Dong Y, Garver LS, Sim S, Dimopoulos G (2010) Mos-quito im¬mune defenses against Plasmodium infec¬tion. Dev Comp Immunol. 34: 387–395.

Kumar S, Molina-Cruz A, Gupta L, Ro-drigues J, Barillas-Mury C (2010) A peroxi¬dase/dual oxidase system mod¬ulates midgut epithelial immun-ity in Anopheles gambiae. Science. 327: 1644–1648.

Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C (2010) Hemocyte differentiation mediates innate im-mune memory in Anopheles gambiae mosquitoes. Science. 329: 1353–1355.

Beard CB, Cordon-Rosales C, Durvasula RV (2002) Bacterial symbionts of the Tria¬tominae and their potential use in control of Chagas disease trans-mission. Annu Rev En¬tomol. 47: 123–141.

Durvasula RV, Gumbs A, Panackal A, Kruglov O, Aksoy S, Merrifield RB, Rich¬ards FF, Beard CB (1997) Pre-vention of in¬sect-borne disease: An approach using trans¬genic symbiotic bacteria. Proc Natl Acad Sci USA. 94: 3274–3278.

Riehle MA, Jacobs-Lorena M (2005) Us¬ing bacteria to express and dis-play anti-parasite molecules in mosqui-toes: current and future strategies. In-sect Biochem Mol Biol. 35(7): 699–707.

Coutinho-Abreu IV, Zhu KY, Ramalho-Ortigao M (2010) Transgenesis and para¬transgenesis to control insect-borne diseases: current status and future chal¬lenges. Parasi¬tol Int. 59(1): 1–8.

Sreenivasamurthy SK, Dey G, Ramu M, Kumar M, Gupta MK, Mohanty AK, Harsha HC, Sharma P, Kumar N, Pandey A, Kumar A, Prasad TS (2013) A compendium of mol¬ecules in¬volved in vector-pathogen interac¬tions pertain¬ing to malaria. Malar J. 12: 216.

Gimonneau G, Tchioffo MT, Abate L, Boissière A, Awono-Ambéné PH, Nsango SE, Christen R, Morlais I (2014) Composi¬tion of Anopheles coluzzii and Anopheles gambiae mi-crobiota from larval to adult stages. Infect Genet Evol. 28: 715–24.

Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Enayati AA, Mardani N, Ghoorchian S (2012) Iden¬tification of bacterial microflora in the mid¬gut of the larvae and adult of wild caught Anopheles stephensi: A step toward finding suitable para-transgenesis candidates. Acta Trop. 121: 129–134.

Maleki-Ravasan N, Oshaghi MA, Afshar D, Arandian MH, Hajikhani S, Akha-van AA, Yakhchali B, Shirazi MH, Rassi Y, Jafari R, Aminian K, Fazeli-Varzaneh RA, Durvasula R (2015) Aerobic bacterial flora of bi-otic and abiotic compartments of a hyperendemic Zoonotic Cutaneous Leishman¬iasis (ZCL) focus. Parasit Vectors. 8: 63.

Merritt RW, Dadd RH, Walker ED (1992) Feeding behavior, natural food, and nutritional relationships of larval mos¬qui¬toes. Annu Rev Entomol. 37: 349–376.

Lindh JM, Borg-Karlson AK, Faye I (2008) Transstadial and horizontal transfer of bacteria within a colony of Anopheles gambiae (Diptera: Cu-licidae) and oviposi¬tion response to bacteria-con¬taining water. Acta Trop. 107(3): 242–250.

Wang Y, Gilbreath TM, Kukutla P, Yan G, Xu J (2011) Dynamic gut micro-biome across life history of the ma-laria mosquito Anopheles gambiae in Kenya. PLoS One. 6: e24767.

Straif SC, Mbogo CN, Toure AM, Walk-er ED, Kaufman M, Toure YT, Beier JC (1998) Midgut bacteria in Anoph-eles gambi¬ae and An. Funestus (Dip-tera: Culicidae) from Kenya and Ma-li. J Med Entomol. 35: 222–226.

Gonzalez-Ceron L, Santillan F, Rodri¬guez MH, Mendez D, Hernandez-Avila JE (2003) Bacteria in midguts of field-collected Anopheles albimanus block Plasmodium vi¬vax sporogonic devel-opment. J Med Ento¬mol. 40: 371–374.

Lindh JM, Terenius O, Faye I (2005) 16S rRNA gene-based identification of midgut bacteria from fieldcaught Anopheles gambiae sensu lato and An. funestus mosquitoes reveals new species re-lated to known insect symbionts. Appl Environ Microbiol. 71: 7217–7223.

Favia G, Ricci I, Damiani C, Raddadi N, Crotti E, Marzorati M, Rizzi A, Urso R, Bru¬setti L, Borin S, Mora D, Scuppa P, Pasqual¬ini L, Clementi E, Genchi M, Corona S, Negri I, Grandi G, Al-ma A, Kramer L, Es¬posito F, Bandi C, Sacchi L, Daffonchio D (2007) Bac-teria of the genus Asaia stably asso-ciates with Anopheles stephensi, an Asian malarial mosquito vec-tor. Proc Natl AcadSci USA. 104: 9047–9051.

Damiani C, Ricci I, Crotti E, Rossi P, Rizzi A, Scuppa P, Esposito F, Bandi C, Daffonchio D, Favia G (2008) Pa-ternal transmission of symbiotic bac-teria in malaria vectors. Curr Bi-ol. 18(23): R1087–1088.

Damiani C, Ricci I, Crotti E, Rossi P, Rizzi A, Scuppa P, Capone A, Ulissi U, Epis S, Genchi M, Sagnon N, Faye I, Kang A, Chouaia B, White-horn C, Moussa GW, Mandrioli M, Esposito F, Sacchi L, Bandi C, Daf-fonchio D, Favia G (2010) Mosqui-to-bacteria symbiosis: the case of Anoph-eles gambiae Asaia. Microb Ecol. 60: 644–654.

Terenius O, De Oliveira CD, Pinheiro WD, Tadei WP, James AA, Marinotti O (2008) 16S rRNA gene sequences from bac¬teria associated with adult Anoph-eles darling (Diptera: Culicidae) mosqui-toes. J Med Entomol. 45: 172–175.

Rani A, Sharma A, Rajagopal R, Adak T, Bhatnagar R (2009) Bacterial di-versity analysis of larvae and adult midgut microflora using culture-de-pendent and culture-independent meth¬ods in lab-reared and field-collected Anopheles stephensi an Asian malarial vector. BMC Microbiol. 9: 96.

Ricci I, Damiani C, Rossi P, Capone A, Scuppa P, Cappelli A, Ulissi U, Mosca M, Valzano M, Epis S, Crotti E, Daffonchio D, Alma A, Sacchi L, Mandrioli M, Bandi C, Favia G (2011) Mosquito symbioses: from basic re-search to the paratransgenic control of mosquito-borne diseases. J Appl En-tomol. 135: 487–493.

Chavshin AR, Oshaghi MA, Vatandoost H, Yakhchali B, Zarenejad F, Tereni-us O (2015) Malpighian tubules are im-portant de¬terminants of Pseudomonas transstadial transmission and longtime persistence in Anopheles stephensi. Par-asit Vectors. 8: 36.

Moll RM, Romoser WS, Modrakowski MC, Moncayo AC, Lerdthusnee K (2001) Meconial peritrophic mem-branes and the fate of midgut bacte-ria during mosquito (Diptera: Cu-licidae) metamorphosis. J Med En-tomol. 38: 29–32.

Chao J, Wistreich G (1959) Microbial isolation from the midgut of Culex tarsalis Coquillett. J Insect Pathol. 1: 311–318.

Demaio J, Pumpuni CB, Kent M, Beier JC (1996) The midgut bacterial flora of wild Äedes triseriatus, Culex pipiens and Psoro¬phora columbiae mosqui-toes. Am J Trop Med Hyg. 54: 219–223.

Yadav KK, Bora A, Datta S, Chandel K, Gogoi HK, Prasad GB, Veer V (2015) Mo¬lecular characterization of midgut mi-crobiota of Aedes albopictus and Aedes aegypti from Arunachal Pradesh, India. Parasit Vectors. 8: 641.

Basseri HR, Mohamadzadeh Hajipirloo H, Mohammadi Bavani M, Whitten MM (2013) Comparative susceptibility of differ¬ent biological forms of Anoph¬eles stephensi to Plasmodium berghei ANKA strain. PLoS One. 8(9): e75413.

Arshad A, Rui-De X, Rechard L, Naph-tali C (1994) Evaluation of granular corn¬cob formulations of Bacillus thu¬rin¬giensis serovar israelensis against mos¬quito larvae using a semi-field bi¬oassay method. J Am Mosq Con-trol Assoc. 10: 492–495.

Wang S, Ghosh AK, Bongio N Stebbings KA, Lampe DJ, Jacobs-Lorena M (2012) Fighting malaria with engi-neered symbiotic bacteria from vec-tor mosquitoes. Proc Natl Acad Sci U S A. 109(31): 12734–12739.

Riehle MA, Moreira CK, Lampe D, Lau-zon C, Jacobs-Lorena M (2007) Using bacte¬ria to express and display anti-Plasmodium molecules in the mosquito mid¬gut. Int J Parasitol. 37: 595–603.

Coon KL, Vogel KJ, Brown MR, Strand MR (2014) Mosquitoes rely on their gut mi¬crobiota for development. Mol Ecol. 23(11): 2727–2739.

Müller GC, Beier JC, Traore SF, Toure MB, Traore MM, Bah S, Doumbia S, Schlein Y (2010) Successful field trial of attractive toxic sugar bait (ATSB) plant-spraying methods against ma-laria vectors in the Anopheles gam-biae complex in Mali, West Africa. Malar J. 9: 210.

Mancini MV, Spaccapelo R, Damiani C, Accoti A, Tallarita M, Petraglia E1, Rossi P, Cappelli A, Capone A, Pe-ruzzi G, Valzano M, Picciolini M, Diabaté A, Facchinelli L, Ricci I, Favia G (2016) Paratransgenesis to con-trol malaria vectors: a semi-field pilot study. Parasit Vectors. 9: 140.

Kotnis B, Kuri J (2016) Evaluating the use-fulness of paratransgenesis for ma¬laria control. Math Biosci. 277: 117–125.

Husseneder C, Grace JK (2005) Genet-ically engineered termite gut bacte-ria (En¬terobacter cloacae) deliver and spread for¬eign genes in termite colonies. Appl Micro¬biol Biotech-nol. 68: 360–367.

Watanabe K, Abe K, Sato M (2000) Bio-logical control of an insect pest by gut-colonizing Enterobacter cloacae trans-formed with ice nucleation gene. J Appl Microbi¬ol. 88: 90–97.

Ali A, Xue RD, Lobinske R, Carandang N (1994) Evaluation of granular corncob formulations of Bacillus thurin¬giensis serovar israelensis against mosquito lar-vae using a semi-field bioassay meth¬od. J Am Mosq Control Assoc. 10(4): 492–495.

Mulla MS, Rodcharoen J, Ngamsuk W, Tawatsin A, Pan-Urai P, Thavara U (1997) Field trials with Bacillus sphaer-icus formu¬lations against polluted wa¬ter mosquitoes in a suburban area of Bang-kok, Thailand .J Am Mosq Control Assoc. 13(4): 297–304.

Vilarinhos PT, Monnerat R (2004) Lar-vicidal persistence of formulations of Bacil¬lus thuringiensis var. is-raelensis to control larval Aedes ae-gypti. J Am Mosq Control Assoc. 20(3): 311–314.

Files
IssueVol 11 No 4 (2017) QRcode
SectionOriginal Article
Keywords
Bacterial dynamic Enterobacter cloacae Anopheles stephensi Paratransgenesis

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
1.
Dehghan H, Oshaghi MA, Moosa-Kazemi SH, Yakhchali B, Vatandoost H, Maleki-Ravasan N, Rassi Y, Mohammadzadeh H, Abai MR, Mohtarami F. Dynamics of Transgenic Enterobacter cloacae Expressing Green Fluorescent Protein-Defensin (GFP-D) in Anopheles stephensi under Laboratory Condition. J Arthropod Borne Dis. 2017;11(4):515-532.