J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 515 http://jad.tums.ac.ir Published Online: December 30, 2017 Original Article Dynamics of Transgenic Enterobacter cloacae Expressing Green Fluorescent Protein- Defensin (GFP-D) in Anopheles stephensi Under Laboratory Condition Hossein Dehghan 1, *Mohammad Ali Oshaghi 1, *Seyed Hassan Moosa-Kazemi 1, Bagher Yakhchali 2, Hassan Vatandoost 1,3, Naseh Maleki-Ravasan 4, Yavar Rassi 1, Habib Mohammadzadeh 5, Mohammad Reza Abai 1,3, Fatemeh Mohtarami 1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department Industrial and of Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran 3Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 4Malaria and Vector Research Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 5Department of Parasitology and Mycology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran (Received 6 Jan 2017; accepted 23 May 2017) Abstract Background: Enterobacter cloacae bacterium is a known symbiont of the most Anopheles gut microflora and nomi- nated 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 plat- ing 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. Keywords: Bacterial dynamic, Enterobacter cloacae, Anopheles stephensi, Paratransgenesis Introduction Malaria is a mosquito borne disease con- sidered as an important threat to public health in tropical and semi-tropical areas of the world. Although Iran currently is in elimination phase and malaria cases are significantly reduced, the disease is still considered a serious health concern, mostly in the south and southeast corner of the country (1-3). In the southern regions of the country there are six Anopheles mosquito vectors including Anopheles stephensi, An. culicifacies s.l, An. dthali, An. fluviatilis s.l, An. superpictus s.l. and An. pulcherrimus (4-12). However, An. stephensi has been considered the main ma- *Corresponding authors: Dr Mohammad Ali Oshaghi, E-mail: moshaghi@tums.ac.ir, Dr Seyed Hassan Moosa- Kazemi, Email: shm.kazemi@gmail.com http://www.sciencedirect.com/science/article/pii/S0001706X12001817#bib0085 mailto:moshaghi@tums.ac.ir mailto:shm.kazemi@gmail.com J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 516 http://jad.tums.ac.ir Published Online: December 30, 2017 laria vectors in Iran (13-15). This species has shown a wide range of susceptibility/resistant to various insecticides in Iran (16-20). Current- ly, this species is resistant to lambda cyhalo- thrin and cyfluthrin and susceptible to etofenprox, and permethrin and candidate of resistant to deltamethrin in the country (21). Anopheles stephensi is one of the most im- portant malaria vectors in Middle East and Indian subcontinent regions and its resistance to organochlorides, organophosphates, carba- mates and pyrethroids insecticides have been widely reported in these regions (22-24). Emergence of insecticide resistant mos- quitoes plus the emergence of drug-resistant in the parasites highlights the needs for alterna- tive strategies for sustainable malaria con- trol. As an alternative to chemical insecti- cides, paratransgenesis depends on engineered symbiotic microorganisms particularly bacteria of malaria vectors to supply molecules that can kill Plasmodium inside the mosquito gut and or inhibit pathogen transmission (25, 26). Transmission of the Plasmodium parasite is strongly dependent on completion of the par- asite life cycle in the mosquito vector since entering to midgut, across the peritrophic mem- brane (PM), midgut epithelium, salivary glands and transmitted through saliva to new host. The most bottleneck during the development of Plasmodium parasite occurs in ookinete stage. It could be considered as the main target for control of parasite in mosquito vectors (27-31). Some known factors involved in creating parasite bottleneck, including gut digestive enzymes, intestinal microbial flora, and the mosquito's immune response. Microflora per- forms vital role in preventing the develop- ment of pathogens. This effect exerts directly by proliferation of bacteria after blood meal simultaneously with ookinete stage of Plas- modium. Besides, indirect effects on the sur- vival of Plasmodium parasite are applied by inducing the expression of anti-microbial genes against the bacteria (32-38). Accordingly, bac- terial symbionts are genetically modified to express toxic peptides against pathogens, can be considered as an alternative approach for disease control (39). This strategy, commonly named paratransgenesis (40), requires several steps of research on the biology of vectors and vector symbionts and its evaluation in the la- boratory and field conditions (41-43). The midgut bacterial flora of wild-caught mosquitoes is very dynamic and significant fluctuations depending on the stage of life, nutrients and the physiological age (31, 44). Population structure of symbiotic bacteria is considerably changed post blood meal and gram-negative bacteria will be dominant and could survive in harsh condition of midgut with digestive enzymes (31). There are scat- tered studies on insect gut microflora (15, 45, 46 and references herein) and still remained some questions in relation to species compo- sition, stability as well as their acquisition of microbiota (47-49). Punpuni et al. (1996) (33) reported at least nine species of cultivable midgut bacteria with varied composition of An. stephensi, An. gambiae and An. albimanus. In the same study, variety composition of mid- gut bacteria flora was found in An. gambiae and An. funestus and some Anopheles mos- quitoes (49-59). A few of mosquito microflora able to pass from larvae to adult stages because of the dif- ferences between the larval (Aquatic) and adults (Terrestrial) habitats (41, 44). Some bacteria are able to colonize in the malpigh- ian tubules and transstadially pass from lar- vae to adult and presumably remain for long duration in the female gut (15). Therefore, such symbiotic bacteria added to the diet of adult mosquitoes (60). Enterobacter cloacae bacterium is a species of gram-negative, fac- ultative anaerobic, rod-shaped bacteria be- longing to Gammaproteobacteria and Entero- bacteriaceae family. The bacteria species lim- ited the development of Plasmodium berghei and P. falciparum by stimulate the immune system of An. stephensi and increases the J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 517 http://jad.tums.ac.ir Published Online: December 30, 2017 expression of immune responses compounds such as serine protease inhibitors (SRPN6) (31). Enterobacter cloacae bacterium was found as the microflora of An. stephensi (57). Gonzalez-Ceron et al. (51) reported E. cloa- cae restricted the P. vivax development in midgut of An. albimanus. The bacterium also was reported from Culex tarsalis (61), Pso- rophora columbiae (62), Aedes triseriatus (62) and Ae. albopictus and Ae. aegypti (63). Due to the ability of E. cloacae to direct and indi- rect control of Plasmodium parasites, these bacteria could be introduced as a candidate for paratransgenesis approach against the malar- ia parasite. Maleki-Ravasan et al. (46) sug- gested E. cloacae dissolvens as a candidate for paratransgenesis approach to control of Leish- mania transmission in the sand flies vectors. They genetically modified the bacterium to produce defensin as a Plasmodium killing ef- fector protein. Defensins are small cysteine- rich cationic proteins and found in plants, ver- tebrates and invertebrates. They are active against fungi, bacteria, and many viruses. To use recombinant bacteria in practice, howev- er, it is required a better understanding of the bacteria dynamics in mosquitoes and its de- livery systems to vectors. In this study, we evaluated the dynamics of E. cloacae dissolvens expressing green flu- orescent and defensin proteins (GFP-D) in midgut of An. stephensi life stages as well as in larval habitats and sugar bait used as two delivery systems for the mosquito in the la- boratory condition. Materials and Methods The mosquitoes Anopheles stephensi, Beech strain origi- nally collected from Pakistan as an addition- al type form ie SDA500 strain originating was provided in 2005 by Professor P.F. Billings- ley, Sanaria, Inc (64). Breeding of the mos- quitoes carried out in 27±1 °C and 60±10% relative humidity with photoperiodic period of 12h. Adult mosquitoes were kept in 30×30×30 cages. Mosquito feeding was carried out using fructose 5% and guinea pigs twice a week. Anopheles gravid, laying eggs in earthenware bowl containing decolorized water. The eggs were slowly transferred to tray having 1500ml of decolorized water before hatching. The fish food and a piece of leaf lettuce used as spe- cific diet for Anopheles larvae. The bacteria Enterobacter cloacae subsp. dissolvens was isolated by sampling microflora of Phleboto- mus papatasi in the field of zoonotic cutane- ous leishmaniasis in Isfahan, central Iran, 2013– 2014 (46). The manipulated strain of E. clo- acae is carrying plasmid expressing difensin and GFP proteins and a gene resistant to tet- racycline (Tet-gene). This strain known as En- terobacter cloacae-GFP-Difensin (E. cloacae- GFP-D) maintained in the School of Public Health, Tehran University of Medical Scienc- es. The bacterium was grown in Brain Heart Infusion (BHI) Broth culture until stationary phase, as determined by spectroscopic opti- cal density (OD) measurements at 600nm. The bacteria were prepared by growing to an OD 600 of = 1 in Tet-BHI broth medium. Sever- al dilutions of the OD 600= 1 were prepared covering a wide region of optical density from 0.1 to 1 and plated onto Tet-BHI agar for viable cell determination. The plates were in- cubated for 13h at 37 °C before counting the number of colony forming units (CFU). The gradients of the calibration curves showed that OD 600nm of 1.0 was corresponding to approximately 1×109 CFU per ml BHI broth medium. Dynamics of Enterobacter cloacae-GFP-D Corncob-bacteria formulation (CCF) and sugar bait-bacteria The method of Arshad et al. (65) was used to prepare corncob formulation (CCF) of E. cloacae-GFP-D and used as the bacterium- floating carrier in larval tray. The corncobs J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 518 http://jad.tums.ac.ir Published Online: December 30, 2017 were autoclaved followed by grinding to small particles less than 0.5mm in diameter. The me- dium containing E. cloacae centrifuged and the precipitated cells washed and suspend in 100µL PBS. The bacteria-PBS buffer was add- ed on corncob and dried at room temperature. Almost 100µL of bacteria-PBS suspension was used for 0.1g grind corncob. Almost 0.1g of the dried CCF containing 5×109 bacterial cells was used for one litter of larval tray water in insectary condition. Sugar bait-bacteria were prepared by using 109 the bacterial cell per 1ml of fructose 5% and 2.5% red food dyes (Fig. 1) according to the method previously described by Wang et al. (66). Introduction Enterobacter cloacae-GFP-D to mosquito larval habitats About 200–300 An. stephensi eggs were transferred to tray containing sterile water and then the hatched larvae were transferred ran- domly to the test and control trays. The lar- vae were fed either on intact corncob and a piece of leaf lettuce in control tray or CCF and a piece of leaf lettuce in test tray. The number of released bacteria in test tray was 109 bacterial cells per liter of sterile water. The CCF was added following emergence of the first (L-I) and fourth (L-IV) instar larvae in test tray. Transstadial and dynamics of the bacteria were investigated by sampling of water and mosquitoes at larvae and adult stages. Dynamics of Enterobacter cloacae-GFP-D in larval habitat To test the course of time on proliferation and stability of E. cloacae-GFP-D in larval habitats of mosquitoes, water sampling was carried out daily from the test trays. For each sample, 10ml of the water was centrifuged at 13000 RPM for 10min followed by remov- ing supernatant; the pellet was mixed with 1mL PBS buffer and vortexed. Finally, serial dilution of the bacterial suspension was pre- pared and 100µL of the proper dilution cul- tured in Tet-BHI agar. The number of colonies (colony forming units: CFUs) was counted un- der a flu- orescent microscope and number of the E. cloacae bacteria in one ml of test tray water was measured. Bacterium transstadial transmission To test the transstadial transmission of E. cloacae-GFP-D bacteria from larvae to adult stage of the mosquitoes we followed the meth- od previously described by Lindh et al. (48). On brief, 5×106 bacteria as CCF per ml sterile water were released in larval breeding trays. Larvae were kept under laboratory condition until pupae stage. Fresh pupae were washed twice in sterile water and transferred to a new tray with 1000mL sterile water until adults emerged. After eclosion, the adult body surface of some specimens was sterilized in 70% al- cohol, then the midgut dissected, homogenized in PBS, and cultivated in Tet-BHI agar/broth for 13h in 37 °C. However, some adults were transferred to 15×15×30cm cage and fed on sterile sugar solution during 24h. The female mosquitoes were blood fed on BALB/c mice and midgut microflora was determined by described method. The number of colonies (CFUs) was counted under a fluorescent mi- croscope for all experiments (Fig. 2). Dynamics of Enterobacter cloacae-GFP-D in An. stephensi larvae In each sample, five larvae were selected and their surface bodies were sterilized by alcohol. Briefly, the larvae were transferred to microtubes containing 500µL sterile water and kept on ice for a few minutes until the larvae were numb, then 500µL of 70% cold ethanol (-5 °C) was added to the microtube after removing water and kept on ice about 5 min. The alcohol was removed and the larvae were washed twice with PBS buffer (4 °C). Finally, the total body of sterilized larvae ho- mogenized in PBS buffer and 100ml of ho- mogenized solution was cultured in Tet-BHI agar plates. Alternatively, the larvae were dis- J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 519 http://jad.tums.ac.ir Published Online: December 30, 2017 sected and their guts were homogenized in PBS buffer, cultured in Tet-BHI agar plates, and CFUs were counted as above. Simultaneously, survival rate of An. stephensi larvae in the test and control (corncob contaminated with/out E. cloacae-GFP-D) trays were investigated. Dynamics of Enterobacter cloacae-GFP-D in Anopheles stephensi adult gut In order to study population dynamics of E. cloacae-GFP-D in An. stephensi adult, the 3–5d old female mosquitoes were transferred to 15×15×30cm cage and fed on sugar bait con- taining 109 the bacterial cell /mL fructose 5%, and 2.5% food dyes (red) according to the method previously described by Wang et al. (66) (Fig. 1). To count the bacteria populations (CFUs) in the adult guts, the female mosqui- toes were numbed and immersed in ethanol 70% for 5–7min, placed on glass slides for 3–5min, and then transferred to microtube and homogenized in PBS buffer. Finally, 100µL of homogenized suspension samples was cul- tured in Tet-BHI agar plates. In an alterna- tive method, after body surface sterilization, mosquito midguts were dissected and homog- enized in PBS buffer. To evaluate course of time and sugar/blood meals on the CFUs of the bacterium, two separate experiments were designed. In the first experiment, 3–5d old fe- male mosquitoes were fed on sugar bait, a sub- set of specimens was tested for the number of the bacteria immediately (1 hour) post sugar feeding. Then the sugar bait was re- moved from the cage for 7h and again the bacteria population was tested in a subset of females. Eight hours post sugar feeding, a blood meal was offered to the mosquitoes and then a subset of mosquito guts was tested for the bacteria at 12, 18, 24, 36, 48, 96, and 144h post blood meal. After day-6 (144h), a second blood meal was offered to the flies, and CFUs were counted for the guts 24 and 36h post second blood meal. In the second experiment, 3–4d old female mosquitoes were fed on sugar bait, a subset of specimens was tested for a number of the bacteria immediately (1 hour) post feeding. Then the sugar bait removed from the cage for 7h and again the bacteria populations were tested in a subset of females. Again, eight hours after first sugar meal, second sugar meal was offered to the mosquitoes and the CFUs were measured for the guts 24, 48, and 72h post second sugar meal. Then a blood meal was offered and a subset of mosquito guts was tested for the bacteria CFUs at 24 and 48h post blood meal. In both experiments, 3–4h in advance of offering blood meal, sugar bait containing the bacteria were removed from the cage, and after blood meal, the mosqui- toes were kept on sterile cotton pad soaked with 5% fructose. Stability of Enterobacter cloacae-GFP-D in sugar bait Colony-forming units of the bacteria in sugar bait containing 109 bacteria cell /mL fructose 5% and 2.5% red food dyes was de- termined by daily sampling during eleven d. Sampling was carried out as follow, by press- ing the cotton pad, 10µl of the above suspen- sion was collected and added to 990µl of ster- ile PBS buffer and prepared serial dilutions. About 100µl of the final solution was cultured in Tet-BHI agar plates. After counting the col- onies, number of bacteria per ml of bait was estimated. Statistics and analytical procedure Where it was necessary, the concentrations of CFUs were indicated using logarithmic no- tation, where the value was shown is the base 10 logarithm of the concentration. Bacteria rel- ative abundance was calculated separately for each treatment. The average percentage of lifespan and larval death rate were presented. A significant difference in the bacteria relative abundance between samples was analyzed us- ing the Mann–Whitney test. Multiple-sample comparisons were analyzed using the non- https://en.wikipedia.org/wiki/Common_logarithm https://en.wikipedia.org/wiki/Common_logarithm J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 520 http://jad.tums.ac.ir Published Online: December 30, 2017 parametric Kruskal–Wallis test, and medians were compared using Dunn’s test. GraphPad Prism version 5.00 for Windows (GraphPad Software) was used for all statistics. P< 0.05 was considered statistically significant. Results Corncob formulation (CCF) stability The most attractive features of corncob were considered as lightness and floating on the water surface. When this formulation supplied on mosquito breeding place, Anoph- eles larvae attracted to and fed on corncob par- ticles. Approximately, 90–95% and 50–60% of CCF particles remained floated on the tray water after 24 and 48h of their releasement, respectively. Effect of Enterobacter cloacae-GFP-D on survival of Anopheles stephensi larvae The survival rate of An. stephensi larvae in the test tray containing E. cloacae-GFP-D sig- nificantly was more than the control group at late larval stage. Vis-versa results of this ex- periment showed that death rate of control were significantly lower than the test group at larval stage II (Fig. 3, 4). Peak mortality in test groups occurred about days 8–9, whereas it happened about days 10–14 in control group (Fig. 4). Effect of Enterobacter cloacae-GFP-D on de- velopment rate of Anopheles stephensi larvae Development of An. stephensi larvae took longer time when kept in sterile water or in water containing E. cloacae-GFP-D in com- parison with the ones kept in non-sterile wa- ter. On average adult, mosquitoes appeared 8– 10d from the time of egg hatching when the larvae had kept in non-sterile water whereas it took 14–18d when the larvae bred in sterile water or water containing E. cloacae-GFP-D. Both test and control groups were supplied by a piece of lettuce and corncob. Dynamics of the bacteria in the larval stages and habitat of Anopheles stephensi Population dynamics of E. cloacae-GFP- D had a descendant trend in the larval habitat indicating a weak recycling in water at insec- tary condition. The population of E. cloacae- GFP-D in the water decreased from 2×107 CFUs/ml at day-1 to 2960 CFUs/mL at the end of day-14 (Fig. 5). Trend of the bacteria in the guts of An. stephensi larvae also declined sig- nificantly from about 12000 CFUs in the sec- ond instar larvae (L-II) to less than 100 CFUs in the fourth instar larvae (L-IV) which is cor- responding to the decreasing trend of the bac- teria in the larval habitat water (Fig. 5). Transstadial transmission Enterobacter cloacae-GFP-D can survive and flourish in the guts of An. stephensi larvae but inadequately transmit transstadially from larvae to adult stage. The rate of bacteria pos- itive in larval guts at late instars was more than 75% with a range of 7 to 756 CFUs per gut. However, most of the bacteria were re- moved from midgut in pupal stage due to his- tolysis and histogenesis phenomena. None of the pupae were positive for the bacteria and there were only 2–3 adult specimens (8–12%) were positive for E. cloacae subsp. dissolvens bacteria indicating very low transstadial trans- mission (Table 1). There were only a few (n= 4) bacteria in the newly emerged adult mosqui- to. The number of bacteria increased dramat- ically following a blood meal and reached to 10000 CFUs (Table 1). Course of time and blood meal on Entero- bacter cloacae-GFP-D in adult Anopheles stephensi midguts We tested the effect of blood meal and course of time on loads of the bacteria in adult mosquitoes. Number of the bacteria in the adult mosquito midgut fed on sugar bait containing E. cloacae-GFP-D was on average one million per mosquito gut one-hour post sugar bait feeding. However, populations of the bacteria decreased about 100 folds after seven hours fasting (Fig. 6). Loads of bacte- J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 521 http://jad.tums.ac.ir Published Online: December 30, 2017 ria were dramatically increased about 73000 folds on average and maximized up to 155 million (about 73000000 on mean) CFUs 24h post blood meal. The bacteria populations were then declined gradually and reached to about 100 CFUs/gut 144h after (6d) blood meal intake. This trend, i.e. increase, and decrement of the bacteria populations happened again following second blood meal with a slightly lower rate than first blood meal (Fig. 6). To test the effect of only sugar meal (fruc- tose 5%) on the bacteria population, in a sep- arate experiment, the mosquitoes were fed on sugar bait containing E. cloacae-GFP-D and then starved for 8h. Then they were fed on fructose 5% with no bacterai using cotton pad and the load of bacteria was counted daily for three following days. After 24h following nor- mal sugar meal, the bacteria population rais- es up about 10 folds (10000 to 99000 CFUs per gut) and again by course of time, the bac- teria population decreased again (Fig. 7). The number of bacteria dropped about 1000 folds and reached to about 10 CFUs/gut after 72h (3d) (Fig. 7). A blood meal intake following three days causes tremendous (140000 folds) uprise in the number of adult guts bacteria but lower than the previous experiment (14 mil- lion versus 155 million). Dynamics of Enterobacter cloacae-GFP-D in sugar bait In order to determine the course of time on survival of E. cloacae-GFP-D in suspend- ed sugar bait, trend of E. cloacae-GFP-D pop- ulations was investigated in the sugar solution of cotton pad. The number of bacteria was decreased 10 folds (from 1 million to 100000 CFU/ml) throughout 11d in the insectary con- dition (Fig. 8). Table 1. Details of transstadial transmission of Enterobacter cloacae-GFP-D in Anopheles stephensi in insectary condition. Numbers refer to the guts harbor the bacteria out of 25 specimens. Life stages Tet-BHI broth Tet-BHI agar (CFUs/gut) Larvae IV 19 17 (7–756) Pupae 0 0 Adult Blood Fed 2 1 (10,000) Adult Unfed 1 1 (4) Fig. 1. Sugar bait containing 109 bacteria cell/mL (CFUs) fructose 5% and 2.5% food dyes used to introduce the En- terobacter cloacae-GFP-Defensin bacteria to mosquitoes via sugar feeding. The dye made abdomen reddish and visually distinguishable. J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 522 http://jad.tums.ac.ir Published Online: December 30, 2017 Fig. 2. The presence of Enterobacter cloacae-GFP-Defensin in dissected Anopheles stephensi midgut (squares) and BHI agar plates (circles), the bacteria with/out expressing green fluorescent protein in BHI agar plate under non- fluorescent (a and c) and fluorescent (b and d) microscope L -I L -I I L -I II L -I V 0 20 40 60 80 Control E.cloacae-GFP-D Larval stage M e a n o f s u rv iv a l ra te Fig. 3. Mean of survival rate of Anopheles stephensi larvae in test groups (sterile water+corncob-Enterobacter cloa- cae-GFP-Defensin) and control (sterile water+corncob) throughout larval development stages (14d) in insectary con- dition. The bars represent standard error of the mean J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 523 http://jad.tums.ac.ir Published Online: December 30, 2017 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 5 10 15 20 25 E.cloacae-GFP-D Control Time (Day) D e a th R a te Fig. 4. Mortality rate of aquatic stages of Anopheles stephensi in test (sterile water+ CCF: corncob-Enterobacter cloacae-GFP-Defensin) and control (sterile water+corncob) tray throughout 14d in insectary condition. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.0100 1 1.0100 2 1.0100 3 1.0100 4 1.0100 5 1.0100 6 1.0100 7 1.0100 8 1.0100 9 1.0101 0 Water Larvae L-II L-III L-IV Time (Day) C F U s /m l o r C F U s /g u t Fig. 5. Trend of Enterobacter cloacae-GFP-Defensin in the Anopheles stephensi larval habitat water (above) and guts (down) in insectary condition. L-II, L-III, and L-IV represent larval stage II, III, and IV respectively J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 524 http://jad.tums.ac.ir Published Online: December 30, 2017 1 8 12 18 24 36 48 96 144 24 36 1.0100 1.0101 1.0102 1.0103 1.0104 1.0105 1.0106 1.0107 1.0108 1.0109 1st Blood Meal 2 nd Blood Meal Hours after sugarbait & blood meal Sugar bait Meal C F U s /g u t (l o g ) Fig. 6. Dynamics of Enterobacter cloacae-GFP-Defensin populations in the midgut of adult Anopheles stephensi fed on sugar bait containing 109 the bacteria cell/mL fructose 5%. Midgut status 12, 18, 24, and 36h post blood meal is shown underneath. The bars represent standard error of the mean 1 8 24 48 72 24 48 1.0100 1.0101 1.0102 1.0103 1.0104 1.0105 1.0106 1.0107 Sugar Meal Blood Meal Hours after sugar bait, sugar & blood meal Sugar bait Meal C F U s /g u t (l o g ) Fig. 7. Course of time, sugar, and blood meal on the dynamics of Enterobacter cloacae-GFP-Defensin populations in the midgut of adult Anopheles stephensi. Mosquitoes were fed on sugar bait containing 109CFUs/mL fructose 5%, then kept starve for 8h, and then fed on sugar (cotton pad) for three days continuously and on blood meal after 72h. The bars represent standard error of the mean J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 525 http://jad.tums.ac.ir Published Online: December 30, 2017 1 2 3 4 5 6 7 8 9 10 11 1.0100 1.0101 1.0102 1.0103 1.0104 1.0105 1.0106 1.0107 Day C F U s /m l Fig. 8. Trend of Enterobacter cloacae-GFP-Defensin population in the sugar bait solution containing 109CFUs/mL fructose 5% at the beginning of experiment (day zero) in insectary condition throughout11d. Discussion We investigated transstadial transmission and dynamics of E. cloacae-GFP-D within an Anopheles stephensi mosquito colony with the focus on larval habitat and sugar bait as two potential resources or delivery systems for in- troduction of the transgenic bacteria to mos- quitoes. Our observations revealed that the symbiont E. cloacae-GFP-D strain was not able to colonize well in larvae midgut or water hab- itats of An. stephensi in laboratory rearing con- dition. The number of bacteria reduced pro- gressively in both water and larvae midgut. Besides, number of the bacteria transferred transstadially was very low (4%). In this ex- periment, the bacteria were administrated via corncob formulation and drinking water, which is similar to the colonization patterns under a natural condition where bacterial symbionts are acquired throughout feeding or drinking wa- ter. Our larval rearing experiments evidently showed negative effects of E. cloacae-GFP- D on the development speed of An. stephensi larvae. These findings are in agreement with a previous study showed that E. cloacae could not isolate from the midgut of insectary spec- imens of An. albimanus Weidemann from southern Mexico whereas the bacteria was already isolated from field specimens (51). However, a great diversity of microbiota of larvae and adult mosquito gut has been al- ready reported by other researchers (57, 45, and 15). However, it is currently unclear why the E. cloacae symbiont is not recycling in larval habitats at insectary condition. In contrast to the larvae, colonization of the E. cloacae-GFP-D bacteria proceeds rap- idly in adult mosquito midguts following sugar or blood meal. Colonization occurred within a few hours after taking meal and maximized 24h post blood meal. The blood proteins ap- parently caused quick growth of midgut bac- teria and when blood digestion is completed (on day 6 or later) most bacteria were de- feated with blood remains. The peak activity of E. cloacae-GFP-D would be synchronized with ookinete formation of Plasmodium par- asite within the mosquito midguts. By pro- liferation of E. cloacae-GFP-D in the adult J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 526 http://jad.tums.ac.ir Published Online: December 30, 2017 midgut, it could disrupt development of Plas- modium parasite directly by remarkable pro- duction of defensin, a killing parasite mole- cule, and indirectly by induction of Anophe- les immune responses (33, 66, 67, 25). In this experiment, the bacteria were administrated via sugar bait, which is similar to the coloni- zation patterns under a natural condition where bacterial symbionts are acquired throughout sap feeding. Adult mosquitoes may acquire bacteria from at least one of the three following sources: animal host skin while taking blood meal, plant saps through sugar feeding, and transstadially transmitted bacteria from larvae to adult (46, 59,68). Knowing bacterial acquisition routes is essential and play important role for para- transgenic approach against malaria vectors since it commands how to introduce transgenic bacteria to the field condition. We found that E. cloacae-GFP-D is not able to propagate in the larval habitat and midgut and it does not transfer transstadially from larvae to pupae as well as not from pupae to adults, so the mos- quitoes are not able to take up the bacteria from larval habitats. This indicates that in- troduction of E. cloacae-GFP-D to breeding sites of An. stephensi is not recommended in a paratransgenic approach. In contrast, results of introduction of the bacteria via sugar bait containing modified bacteria were promising. Propagation of the bacteria in the adult mid- gut and it’s prolonged stability in sugar bait revealed that sugar bait is an effective mean to introduce engineered bacteria into field mosquito populations. This may be achieved by placing sugar bait stations with attractive material such as fruit juice embedded at at- tractive places such as pit shelters, black box trap, and earthenware crock close to breeding sites of Anopheles mosquitoes (53, 67, 69). Sugar bait station was evaluated in semi-field condition by Mancini et al. (70) and showed that the modified bacteria were effectively capable of spreading at high rate in different An. stephensi and An. gambiae populations, and successfully colonizing in the mosquito midguts. Recently Kotnis and Kuri (71) evalu- ated scenarios to calculate a number of re- quired sugar baits and bait distribution to prevent a malaria outbreak. In our experi- ment, the E. cloacae-GFP-D was stable well in the cotton pads and remained viable at high rate through 11d which support suitability of sugar bait as a worthy mean for bacterial in- troduction to field. However, the stability and survival of E. cloacae-GFP-D in sugar bait should be evaluated in semi-field and field conditions in advance to use it in a real para- transgenic strategy. The E. cloacae strain we used was easily cultivated outside the insect host on normal and cheap microbiological media and also genet- ically manipulated. In this study, we success- fully used a GFP-defensin recombinant strain of the E. cloacae symbiont which allowed trac- ing of cells in plates originated from speci- mens such sugar baits and dissected midgut of larvae and adult mosquitoes. This bacte- rium was orally acquired successfully by adult mosquitoes from sugar bait that can be easily administered under semi-field and field con- ditions. Enterobacter cloacae have already been tested to deliver, express, and spread foreign genes in termite colonies (72) and mulberry pyralid moth, Glyphodes pyloalis (73). The corncob formulation we prepared in this study was floated satisfactory on water surface for two days to supply the modified bacteria in insectary condition. This formula- tion was simple and needs to be developed to other known formulation such granule. Corn- cob has been successfully used as granule to supply Bacillus thuringiensis and B. sphaer- icus to control various larval mosquitoes in semi-field and field conditions (74-76). Fac- tors potentially may influence the efficiency of formulation such as dosage of formulation, precipitation, flooding of the treated sites, and presence of other aquatic animals like fishes should be tested. J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 527 http://jad.tums.ac.ir Published Online: December 30, 2017 Conclusion Sugar bait station is the best method for introduction of E. cloacae-GFP-D into the field condition. The population of the bacteria was increased dramatically within 24h post blood meal. It can interrupt malaria parasite devel- opment in the mosquito midgut. This admin- istration is similar to the colonization patterns under a natural condition where bacterial sym- bionts are acquired throughout sap feeding. On the other hand, lack of proliferation of the bacteria in breeding sites and subsequently in the larval midgut disapproved introduction of the bacteria in Anopheles larval habitat for a paratransgenetic approach. Acknowledgments This study was supported by the Tehran University of Medical Sciences, Iran, Grant number 26231. We thank Ms Talaei (Central Laboratory of SPH, TUMS) and Ms Salimi (Department of Medical Parasitology, SPH, TUMS) for helping in fluorescent microscopy and Fatemeh Rafie (Department of Medical Entomology and Vector Control, SPH, TUMS) for rearing mosquitoes. The authors declare that there is no conflict of interest. References 1. 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 feed- ing success of laboratory reared Anoph- eles stephensi mosquitoes according to ABO blood group status. Acta Trop. 140: 118–123. 2. Karimian F, Oshaghi MA, Sedaghat MM, Waterhouse RM, Vatandoost H, Hana- fi-Bojd AA, Ravasan NM, Chavshin AR (2014) Phylogenetic analysis of the oriental-Palearctic-Afrotropical mem- bers of Anopheles (Culicidae: Diptera) based on nuclear rDNA and mitochon- drial DNA characteristics. Jpn J Infect Dis. 67(5): 361–367. 3. Norouzinejad F, Ghaffari F, Raeisi A, No- rouzinejad A (2016) Epidemiological status of malaria in Iran, 2011–2014. Asian Pac J Trop Med. 9(11): 1055– 1061. 4. Naddaf SR, Oshaghi MA, Vatandoost H, Assmar M (2003) Molecular charac- terization of Anopheles fluviatilis spe- cies complex in the Islamic Republic of Iran. East Mediterr Health J. 9(3): 257–265. 5. Vatandoost H, Emami SN, Oshaghi MA, Abai MR, Raeisi A, Piazak N, Mah- moodi M, Akbarzadeh K, Sartipi M (2011) Ecology of malaria vector Anopheles culicifacies in a malarious area of Sistan va Baluchestan Province, south-east Islamic Republic of Iran. East Mediterr Health J. 17(5): 439–445. 6. Vatandoost H, Oshaghi MA, Abaie MR, Shahi M, Yaaghoobi F, Baghaii M, Hanafi-Bojd AA, Zamani G, Townson H (2006) Bionomics of Anopheles ste- phensi Liston in the malarious area of Hormozgan Province, southern Iran, 2002. Acta Trop. 97(2): 196–203. 7. Vatandoost H, Shahi H, Abai MR, Hanafi- Bojd AA, Oshagh MA, Zamani G (2004) Larval habitats of main malar- ia vectors in Hormozgan Province and their susceptibility to different larvi- cides. Southeast Asian J Trop Med Public Health. 35(2): 22–25. 8. 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. 9. Mehravaran A, Oshaghi MA, Vatandoost H, Abai MR, Ebrahimzadeh A, Roodi AM, https://www.ncbi.nlm.nih.gov/pubmed/25151045 https://www.ncbi.nlm.nih.gov/pubmed/25151045 https://www.ncbi.nlm.nih.gov/pubmed/25151045 https://www.ncbi.nlm.nih.gov/pubmed/25151045 https://www.ncbi.nlm.nih.gov/pubmed/25241686 https://www.ncbi.nlm.nih.gov/pubmed/25241686 https://www.ncbi.nlm.nih.gov/pubmed/25241686 https://www.ncbi.nlm.nih.gov/pubmed/25241686 https://www.ncbi.nlm.nih.gov/pubmed/25241686 https://www.ncbi.nlm.nih.gov/pubmed/15751917 https://www.ncbi.nlm.nih.gov/pubmed/15751917 https://www.ncbi.nlm.nih.gov/pubmed/15751917 https://www.ncbi.nlm.nih.gov/pubmed/15751917 https://www.ncbi.nlm.nih.gov/pubmed/16329986 https://www.ncbi.nlm.nih.gov/pubmed/16329986 https://www.ncbi.nlm.nih.gov/pubmed/16329986 https://www.ncbi.nlm.nih.gov/pubmed/16329986 https://www.ncbi.nlm.nih.gov/pubmed/?term=Vatandoost%20H%5BAuthor%5D&cauthor=true&cauthor_uid=15906629 https://www.ncbi.nlm.nih.gov/pubmed/?term=Shahi%20H%5BAuthor%5D&cauthor=true&cauthor_uid=15906629 https://www.ncbi.nlm.nih.gov/pubmed/?term=Abai%20MR%5BAuthor%5D&cauthor=true&cauthor_uid=15906629 https://www.ncbi.nlm.nih.gov/pubmed/?term=Hanafi-Bojd%20AA%5BAuthor%5D&cauthor=true&cauthor_uid=15906629 https://www.ncbi.nlm.nih.gov/pubmed/?term=Hanafi-Bojd%20AA%5BAuthor%5D&cauthor=true&cauthor_uid=15906629 https://www.ncbi.nlm.nih.gov/pubmed/?term=anopheles+pulcherrimus+oshaghi https://www.ncbi.nlm.nih.gov/pubmed/?term=anopheles+pulcherrimus+oshaghi https://www.ncbi.nlm.nih.gov/pubmed/18092530 https://www.ncbi.nlm.nih.gov/pubmed/18092530 J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 528 http://jad.tums.ac.ir Published Online: December 30, 2017 Grouhi A (2011) First report on Anoph- eles fluviatilis U in southeastern Iran. Acta Trop. 117(2): 76–81. 10. 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 attributes for malaria transmis- sion and implementation of vector con- trol in southern Iran. Acta Trop. 121 (2): 85–92. 11. 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 superpic- tus in Iran using mitochondrial cyto- chrome oxidase (COI and COII) and morphologic markers: a new species complex? Acta Trop. 101(3): 241–248. 12. Oshaghi MA, Yaghobi-Ershadi MR, Shemshad K, Pedram M, Amani H (2008) The Anopheles superpictus complex: introduction of a new ma- laria vector complex in Iran. Bull Soc Pathol Exot. 101(5): 429–434. 13. Oshaghi MA, Yaaghoobi F, Abaie MR (2006) Pattern of mitochondrial DNA variation between and within Anoph- eles stephensi (Diptera: Culicidae) bio- logical forms suggests extensive gene flow. Acta Trop. 99(2–3): 226–233. 14. Mehravaran A, Vatandoost H, Oshaghi MA, Abai MR, Edalat H, Javadian E, Mashayekhi M, Piazak N, Hanafi-Bojd AA (2012) Ecology of Anopheles ste- phensi in a malarious area, southeast of Iran. Acta Med Iran. 50(1): 61–65. 15. Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Terenius O (2014) Isolation and identification of culturable bacteria from wild Anoph- eles culicifacies, a first step in a para- transgenesis approach. Parasit Vectors. 7: 419. 16. Davari B, Vatandoost H, Oshaghi MA, Ladonni H, Enayati AA, Shaeghi M, Basseri HR, Rassi Y, Hanafi-Bojd AA (2007) Selection of Anopheles stephen- si with DDT and dieldrin and cross- resistance spectrum to pyrethroids and fipronil. Pestic Biochem Physiol. 89 (2): 97–103. 17. Abai MR, Mehravaran A, Vatandoost H, Oshaghi MA, Javadian E, Mashayekhi M, Mosleminia A, Piyazak N, Edallat H, Mohtarami F, Jabbari H, Rafi F (2008) Comparative performance of imagicides on Anopheles stephensi, main malaria vector in a malarious area, southern Iran. J Vector Borne Dis. 45(4): 307–312. 18. Soleimani-Ahmadi M, Vatandoost H, Shaeghi M, Raeisi A, Abedi F, Eshraghian MR, Madani A, Safari R, Oshaghi MA, Abtahi M, Hajjaran H. (2012) Field evaluation of permethrin long-lasting insecticide treated nets (Olyset(®)) for malaria control in an endemic area, southeast of Iran. Acta Trop. 123(3): 146–153. 19. Fathian M, Vatandoost H, Moosa-Kazemi SH, Raeisi A, Yaghoobi-Ershadi MR, Oshaghi MA, Sedaghat MM (2014) Susceptibility of Culicidae Mosqui- toes to Some Insecticides Recom- mended by WHO in a Malaria En- demic Area of Southeastern Iran. J Arthropod Borne Dis. 9(1): 22–34. 20. 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. 21. Gorouhi MA, Vatandoost H, Oshaghi MA, Raeisi A, Enayati AA, Mirhendi H, Hanafi-Bojd AA, Abai MR, Sal- im-Abadi Y, Rafi F (2016) Current Susceptibility Status of Anopheles ste- https://www.ncbi.nlm.nih.gov/pubmed/20933492 https://www.ncbi.nlm.nih.gov/pubmed/21570940 https://www.ncbi.nlm.nih.gov/pubmed/21570940 https://www.ncbi.nlm.nih.gov/pubmed/21570940 https://www.ncbi.nlm.nih.gov/pubmed/21570940 https://www.ncbi.nlm.nih.gov/pubmed/17367742 https://www.ncbi.nlm.nih.gov/pubmed/17367742 https://www.ncbi.nlm.nih.gov/pubmed/17367742 https://www.ncbi.nlm.nih.gov/pubmed/17367742 https://www.ncbi.nlm.nih.gov/pubmed/17367742 https://www.ncbi.nlm.nih.gov/pubmed/17367742 https://www.ncbi.nlm.nih.gov/pubmed/19192616 https://www.ncbi.nlm.nih.gov/pubmed/19192616 https://www.ncbi.nlm.nih.gov/pubmed/19192616 https://www.ncbi.nlm.nih.gov/pubmed/16989757 https://www.ncbi.nlm.nih.gov/pubmed/16989757 https://www.ncbi.nlm.nih.gov/pubmed/16989757 https://www.ncbi.nlm.nih.gov/pubmed/16989757 https://www.ncbi.nlm.nih.gov/pubmed/16989757 https://www.ncbi.nlm.nih.gov/pubmed/22267381 https://www.ncbi.nlm.nih.gov/pubmed/22267381 https://www.ncbi.nlm.nih.gov/pubmed/22267381 http://www.ncbi.nlm.nih.gov/pubmed/25189316 http://www.ncbi.nlm.nih.gov/pubmed/25189316 http://www.ncbi.nlm.nih.gov/pubmed/25189316 http://www.ncbi.nlm.nih.gov/pubmed/25189316 https://www.scopus.com/authid/detail.uri?authorId=9743822200&eid=2-s2.0-34548154449 https://www.scopus.com/authid/detail.uri?authorId=9743736500&eid=2-s2.0-34548154449 https://www.scopus.com/authid/detail.uri?authorId=9743736500&eid=2-s2.0-34548154449 https://www.scopus.com/authid/detail.uri?authorId=9742154800&eid=2-s2.0-34548154449 https://www.scopus.com/authid/detail.uri?authorId=6507139052&eid=2-s2.0-34548154449 https://www.scopus.com/authid/detail.uri?authorId=14021995900&eid=2-s2.0-34548154449 https://www.scopus.com/authid/detail.uri?authorId=14021233400&eid=2-s2.0-34548154449 https://www.scopus.com/authid/detail.uri?authorId=11940770000&eid=2-s2.0-34548154449 https://www.scopus.com/authid/detail.uri?authorId=9742808300&eid=2-s2.0-34548154449 https://www.ncbi.nlm.nih.gov/pubmed/22579798 https://www.ncbi.nlm.nih.gov/pubmed/22579798 https://www.ncbi.nlm.nih.gov/pubmed/22579798 https://www.ncbi.nlm.nih.gov/pubmed/22579798 https://www.ncbi.nlm.nih.gov/pubmed/26114141 https://www.ncbi.nlm.nih.gov/pubmed/26114141 https://www.ncbi.nlm.nih.gov/pubmed/26114141 https://www.ncbi.nlm.nih.gov/pubmed/26114141 https://www.ncbi.nlm.nih.gov/pubmed/26114145 https://www.ncbi.nlm.nih.gov/pubmed/26114145 https://www.ncbi.nlm.nih.gov/pubmed/26114145 J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 529 http://jad.tums.ac.ir Published Online: December 30, 2017 phensi (Diptera: Culicidae) to Differ- ent Imagicides in a Malarious Area, Southeastern of Iran. J Arthropod Borne Dis. 10(4): 493–500. 22. Enayati AA, Vatandoost H, Ladonni H, Townson H, Hemingway J (2003) Molecular evidence for a kdr-like py- rethroid resistance mechanism in the malaria vector mosquito Anopheles stephensi. Med Vet Entomol. 17(2): 138–44. 23. 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 ma- laria vector. J Vector Borne Dis. 51 (2): 97–105. 24. Ahmad M, Buhler C, Pignatelli P, Ranson H, Nahzat SM, Naseem M, Sabawoon MF, Siddiqi AM, Vink M (2016) Sta- tus of insecticide resistance in high-risk malaria provinces in Afghanistan. Ma- lar J. 15: 98. 25. Wang S, Jacobs-Lorena M (2013) Genet- ic approaches to interfere with malar- ia transmission by vector mosquitoes. Trends Biotechnol. 31(3): 185–193. 26. WHO Fact Sheet: World Malaria Report (2015) Geneva: World Health Organ- ization, 2015. http://www.who.int/malaria/media/w orld-malaria-report-2015/en/ 27. 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. 28. Whitten MM, Shiao SH, Levashina EA (2006) Mosquito midguts and malar- ia: cell biology, compartmentalization and immunology. Parasite Immunol. 28: 121–130. 29. Sinden RE, Dawes EJ, Alavi Y, Waldock J, Finney O, Mendoza J, Butcher GA, Andrews L, Hill AV, Gilbert SC, Basáñez MG (2007) Progression of Plasmodium berghei through Anoph- eles stephensi is density-dependent. PLoS Pathog. 3: e195. 30. Cirimotich CM, Dong Y, Clayton AM, Sandiford SL, Souza-Neto JA, Mu- lenga M, Dimopoulos G (2011) Nat- ural microbe-mediated refractoriness to Plasmodium infection in Anophe- les gambiae. Science. 332: 855–858. 31. Eappen AG, Smith RC, Jacobs-Lorena M (2013) Enterobacter-Activated Mos- quito Immune Responses to Plasmo- dium Involve Activation of SRPN6 in Anopheles stephensi. PLoS ONE. 8(5): e62937. 32. Pumpuni CB, Beier MS, Nataro JP, Guers LD, Davis JR (1993) Plasmo- dium falciparum: inhibition of sporo- gonic development in Anopheles ste- phensi by gram-negative bacteria. Exp Parasitol. 77: 195–199. 33. Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC (1996) Bacterial popula- tion dynamics in three anopheline species: the impact on Plasmodium sporogonic development. The Am J Trop Med Hyg. 54: 214–218. 34. Dong Y, Manfredini F, Dimopoulos G (2009) Implication of the mosquito mid- gut microbiota in the defense against malaria parasites. PLoS Pathog. 5: e1000423. 35. Meister S1, Agianian B, Turlure F, Relógio A, Morlais I, Kafatos FC, Chris- tophides GK (2009) Anopheles gam- biae PGRPLC-mediated defense against bacteria modulates infections with malaria parasites. PLoS Pathog. 5: e1000542. 36. Cirimotich CM, Dong Y, Garver LS, Sim S, Dimopoulos G (2010) Mos- quito immune defenses against Plas- modium infection. Dev Comp Immu- nol. 34: 387–395. https://www.ncbi.nlm.nih.gov/pubmed/?term=Sanil%20D%5BAuthor%5D&cauthor=true&cauthor_uid=24947216 https://www.ncbi.nlm.nih.gov/pubmed/?term=Shetty%20V%5BAuthor%5D&cauthor=true&cauthor_uid=24947216 https://www.ncbi.nlm.nih.gov/pubmed/?term=Shetty%20NJ%5BAuthor%5D&cauthor=true&cauthor_uid=24947216 https://www.ncbi.nlm.nih.gov/pubmed/24947216 https://www.ncbi.nlm.nih.gov/pubmed/26888409 https://www.ncbi.nlm.nih.gov/pubmed/26888409 https://www.ncbi.nlm.nih.gov/pubmed/26888409 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593784/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593784/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593784/ http://www.who.int/entity/malaria/media/world-malaria-report-2015/en/index.html http://www.who.int/entity/malaria/media/world-malaria-report-2015/en/index.html http://www.who.int/malaria/media/world-malaria-report-2015/en/ http://www.who.int/malaria/media/world-malaria-report-2015/en/ J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 530 http://jad.tums.ac.ir Published Online: December 30, 2017 37. Kumar S, Molina-Cruz A, Gupta L, Ro- drigues J, Barillas-Mury C (2010) A peroxidase/dual oxidase system mod- ulates midgut epithelial immunity in Anopheles gambiae. Science. 327: 1644–1648. 38. 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. 39. Beard CB, Cordon-Rosales C, Durvasula RV (2002) Bacterial symbionts of the Triatominae and their potential use in control of Chagas disease transmission. Annu Rev Entomol. 47: 123–141. 40. Durvasula RV, Gumbs A, Panackal A, Kruglov O, Aksoy S, Merrifield RB, Richards FF, Beard CB (1997) Pre- vention of insect-borne disease: An approach using transgenic symbiotic bacteria. Proc Natl Acad Sci USA. 94: 3274–3278. 41. Riehle MA, Jacobs-Lorena M (2005) Us- ing bacteria to express and display anti-parasite molecules in mosquitoes: current and future strategies. Insect Biochem Mol Biol. 35(7): 699–707. 42. Coutinho-Abreu IV, Zhu KY, Ramalho- Ortigao M (2010) Transgenesis and paratransgenesis to control insect-borne diseases: current status and future chal- lenges. Parasitol Int. 59(1):1–8. 43. 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 molecules involved in vector-pathogen interactions pertain- ing to malaria. Malar J. 12: 216. 44. Gimonneau G, Tchioffo MT, Abate L, Boissière A, Awono-Ambéné PH, Nsango SE, Christen R, Morlais I (2014) Composition of Anopheles coluzzii and Anopheles gambiae mi- crobiota from larval to adult stages. Infect Genet Evol. 28: 715–24. 45. 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 para- transgenesis candidates. Acta Trop. 121: 129–134. 46. 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 biotic and abiotic compartments of a hyperen- demic Zoonotic Cutaneous Leishman- iasis (ZCL) focus. Parasit Vectors. 8: 63. 47. Merritt RW, Dadd RH, Walker ED (1992) Feeding behavior, natural food, and nutritional relationships of larval mos- quitoes. Annu Rev Entomol. 37: 349– 376. 48. Lindh JM, Borg-Karlson AK, Faye I (2008) Transstadial and horizontal transfer of bacteria within a colony of Anoph- eles gambiae (Diptera: Culicidae) and oviposition response to bacteria-con- taining water. Acta Trop. 107(3): 242– 250. 49. 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. 50. Straif SC, Mbogo CN, Toure AM, Walk- er ED, Kaufman M, Toure YT, Beier JC (1998) Midgut bacteria in Anoph- eles gambiae and An. Funestus (Dip- tera: Culicidae) from Kenya and Ma- li. J Med Entomol. 35: 222–226. 51. Gonzalez-Ceron L, Santillan F, Rodriguez http://www.ncbi.nlm.nih.gov/pubmed?term=Riehle%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=15894187 http://www.ncbi.nlm.nih.gov/pubmed?term=Jacobs-Lorena%20M%5BAuthor%5D&cauthor=true&cauthor_uid=15894187 http://www.ncbi.nlm.nih.gov/pubmed/?term=using+bacteria+to+express+and+display+anti+parasite+molecule http://www.ncbi.nlm.nih.gov/pubmed/?term=using+bacteria+to+express+and+display+anti+parasite+molecule http://www.ncbi.nlm.nih.gov/pubmed/19819346 http://www.ncbi.nlm.nih.gov/pubmed/19819346 http://www.ncbi.nlm.nih.gov/pubmed/19819346 http://www.ncbi.nlm.nih.gov/pubmed/19819346 http://www.ncbi.nlm.nih.gov/pubmed?term=Sreenivasamurthy%20SK%5BAuthor%5D&cauthor=true&cauthor_uid=23802619 http://www.ncbi.nlm.nih.gov/pubmed?term=Dey%20G%5BAuthor%5D&cauthor=true&cauthor_uid=23802619 http://www.ncbi.nlm.nih.gov/pubmed?term=Ramu%20M%5BAuthor%5D&cauthor=true&cauthor_uid=23802619 http://www.ncbi.nlm.nih.gov/pubmed?term=Kumar%20M%5BAuthor%5D&cauthor=true&cauthor_uid=23802619 http://www.ncbi.nlm.nih.gov/pubmed?term=Gupta%20MK%5BAuthor%5D&cauthor=true&cauthor_uid=23802619 http://www.ncbi.nlm.nih.gov/pubmed/23802619 http://www.ncbi.nlm.nih.gov/pubmed/25283802 http://www.ncbi.nlm.nih.gov/pubmed/25283802 http://www.ncbi.nlm.nih.gov/pubmed/25283802 https://www.ncbi.nlm.nih.gov/pubmed/25630498 https://www.ncbi.nlm.nih.gov/pubmed/25630498 https://www.ncbi.nlm.nih.gov/pubmed/25630498 https://www.ncbi.nlm.nih.gov/pubmed/25630498 J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 531 http://jad.tums.ac.ir Published Online: December 30, 2017 MH, Mendez D, Hernandez-Avila JE (2003) Bacteria in midguts of field- collected Anopheles albimanus block Plasmodium vivax sporogonic devel- opment. J Med Entomol. 40: 371–374. 52. Lindh JM, Terenius O, Faye I (2005) 16S rRNA gene-based identification of mid- gut bacteria from fieldcaught Anophe- les gambiae sensu lato and An. funestus mosquitoes reveals new species relat- ed to known insect symbionts. Appl Environ Microbiol. 71: 7217–7223. 53. Favia G, Ricci I, Damiani C, Raddadi N, Crotti E, Marzorati M, Rizzi A, Urso R, Brusetti L, Borin S, Mora D, Scuppa P, Pasqualini L, Clementi E, Genchi M, Corona S, Negri I, Grandi G, Al- ma A, Kramer L, Esposito F, Bandi C, Sacchi L, Daffonchio D (2007) Bac- teria of the genus Asaia stably asso- ciates with Anopheles stephensi, an Asian malarial mosquito vector. Proc Natl AcadSci USA. 104: 9047–9051. 54. 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. 55.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) Mosquito- bacteria symbiosis: the case of Anoph- eles gambiae Asaia. Microb Ecol. 60: 644–654. 56. Terenius O, De Oliveira CD, Pinheiro WD, Tadei WP, James AA, Marinotti O (2008) 16S rRNA gene sequences from bacteria associated with adult Anoph- eles darling (Diptera: Culicidae) mos- quitoes. J Med Entomol. 45: 172–175. 57. 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. 58. 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. 59. Chavshin AR, Oshaghi MA, Vatandoost H, Yakhchali B, Zarenejad F, Tereni- us O (2015) Malpighian tubules are im- portant determinants of Pseudomonas transstadial transmission and longtime persistence in Anopheles stephensi. Par- asit Vectors. 8: 36. 60. Moll RM, Romoser WS, Modrakowski MC, Moncayo AC, Lerdthusnee K (2001) Meconial peritrophic mem- branes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. J Med Entomol. 38: 29–32. 61. Chao J, Wistreich G (1959) Microbial isolation from the midgut of Culex tarsalis Coquillett. J Insect Pathol. 1: 311–318. 62. Demaio J, Pumpuni CB, Kent M, Beier JC (1996) The midgut bacterial flora of wild Äedes triseriatus, Culex pipiens and Psorophora columbiae mosqui- toes. Am J Trop Med Hyg. 54: 219– 223. 63. Yadav KK, Bora A, Datta S, Chandel K, Gogoi HK, Prasad GB, Veer V (2015) Molecular characterization of midgut microbiota of Aedes albopictus and Ae- https://www.ncbi.nlm.nih.gov/pubmed/25604581 https://www.ncbi.nlm.nih.gov/pubmed/25604581 https://www.ncbi.nlm.nih.gov/pubmed/25604581 https://www.ncbi.nlm.nih.gov/pubmed/25604581 http://www.ncbi.nlm.nih.gov/pubmed/26684012 http://www.ncbi.nlm.nih.gov/pubmed/26684012 J Arthropod-Borne Dis, December 2017, 11(4): 515–532 H Dehghan et al.: Dynamics of … 532 http://jad.tums.ac.ir Published Online: December 30, 2017 des aegypti from Arunachal Pradesh, India. Parasit Vectors. 8: 641. 64. Basseri HR, Mohamadzadeh Hajipirloo H, Mohammadi Bavani M, Whitten MM (2013) Comparative susceptibility of different biological forms of Anoph- eles stephensi to Plasmodium berghei ANKA strain. PLoS One. 8(9): e75413. 65. Arshad A, Rui-De X, Rechard L, Naph- tali C (1994) Evaluation of granular corncob formulations of Bacillus thu- ringiensis serovar israelensis against mosquito larvae using a semi-field bi- oassay method. J Am Mosq Control Assoc. 10: 492–495. 66. 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. 67. Riehle MA, Moreira CK, Lampe D, Lau- zon C, Jacobs-Lorena M (2007) Using bacteria to express and display anti- Plasmodium molecules in the mosquito midgut. Int J Parasitol. 37: 595–603. 68. Coon KL, Vogel KJ, Brown MR, Strand MR (2014) Mosquitoes rely on their gut microbiota for development. Mol Ecol. 23(11): 2727–2739. 69. Müller GC, Beier JC, Traore SF, Toure MB, Traore MM, Bah S, Doumbia S, Schlein Y (2010) Successful field tri- al of attractive toxic sugar bait (ATSB) plant-spraying methods against ma- laria vectors in the Anopheles gambi- ae complex in Mali, West Africa. Malar J. 9: 210. 70. 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, Fa- via G (2016) Paratransgenesis to con- trol malaria vectors: a semi-field pilot study. Parasit Vectors. 9: 140. 71. Kotnis B, Kuri J (2016) Evaluating the use- fulness of paratransgenesis for malaria control. Math Biosci. 277: 117–125. 72. Husseneder C, Grace JK (2005) Genet- ically engineered termite gut bacteria (Enterobacter cloacae) deliver and spread foreign genes in termite colo- nies. Appl Microbiol Biotechnol. 68: 360–367. 73. 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 Microbiol. 88: 90–97. 74. Ali A, Xue RD, Lobinske R, Carandang N (1994) Evaluation of granular corncob formulations of Bacillus thuringiensis serovar israelensis against mosquito lar- vae using a semi-field bioassay meth- od. J Am Mosq Control Assoc. 10(4): 492–495. 75. Mulla MS, Rodcharoen J, Ngamsuk W, Tawatsin A, Pan-Urai P, Thavara U (1997) Field trials with Bacillus sphaer- icus formulations against polluted water mosquitoes in a suburban area of Bang- kok, Thailand . J Am Mosq Control Assoc. 13(4): 297–304. 76. Vilarinhos PT, Monnerat R (2004) Lar- vicidal persistence of formulations of Bacillus thuringiensis var. israelensis to control larval Aedes aegypti. J Am Mosq Control Assoc. 20(3): 311–314. http://www.ncbi.nlm.nih.gov/pubmed/26684012 http://www.ncbi.nlm.nih.gov/pubmed/26684012 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412027/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412027/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412027/ https://www.ncbi.nlm.nih.gov/pubmed/?term=Coon%20KL%5BAuthor%5D&cauthor=true&cauthor_uid=24766707 https://www.ncbi.nlm.nih.gov/pubmed/?term=Vogel%20KJ%5BAuthor%5D&cauthor=true&cauthor_uid=24766707 https://www.ncbi.nlm.nih.gov/pubmed/?term=Brown%20MR%5BAuthor%5D&cauthor=true&cauthor_uid=24766707 https://www.ncbi.nlm.nih.gov/pubmed/?term=Strand%20MR%5BAuthor%5D&cauthor=true&cauthor_uid=24766707 https://www.ncbi.nlm.nih.gov/pubmed/?term=Strand%20MR%5BAuthor%5D&cauthor=true&cauthor_uid=24766707 https://www.ncbi.nlm.nih.gov/pubmed/24766707 https://www.ncbi.nlm.nih.gov/pubmed/24766707 https://www.ncbi.nlm.nih.gov/pubmed/?term=M%C3%BCller%20GC%5BAuthor%5D&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=Beier%20JC%5BAuthor%5D&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=Traore%20SF%5BAuthor%5D&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=Toure%20MB%5BAuthor%5D&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=Toure%20MB%5BAuthor%5D&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=Traore%20MM%5BAuthor%5D&cauthor=true&cauthor_uid=20663142 http://www.ncbi.nlm.nih.gov/pubmed/26965746 http://www.ncbi.nlm.nih.gov/pubmed/26965746 http://www.ncbi.nlm.nih.gov/pubmed/26965746