J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 69 Original Article Aerobic Microbial Community of Insectary Population of Phlebotomus papatasi Naseh Maleki-Ravasan 1, *Mohammad Ali Oshaghi 1, Sara Hajikhani 2, Zahra Saeidi 1, Amir Ahmad Akhavan 1, Mohsen Gerami-Shoar 3, Mohammad Hasan Shirazi 2, Bagher Yakhchali 4, Yavar Rassi 1, Davoud Afshar 2 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Department of Parasitology and Mycology, School of Public Health, Tehran University of Medical Sci- ences, Tehran, Iran 4Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran (Received 28 Apr 2013; accepted 1 Oct 2013) Abstract Background: Microbes particularly bacteria presenting in the gut of haematophagous insects may have an important role in the epidemiology of human infectious disease. Methods: The microbial flora of gut and surrounding environmental of a laboratory strain of Phlebotomus papatasi, the main vector of Zoonotic Cutaneous Leishmaniasis (ZCL) in the old world, was investigated. Biochemical reac- tions and 16s rDNA sequencing of the isolated bacteria against 24 sugars and amino acids were used for bacteria species identification. Common mycological media used for fungi identification as well. Results: Most isolates belonged to the Enterobacteriaceae, a large, heterogeneous group of gram-negative rods whose natural habitat is the intestinal tract of humans and animals. Enterobacteriaceae groups included Edwardsiella, Enterobacter, Escherichia, Klebsiella, Kluyvera, Leminorella, Pantoea, Proteus, Providencia, Rahnella, Serratia, Shigella, Tatumella, and Yersinia and non Enterobacteriaceae groups included Bacillus, Staphylococcus and Pseu- domonas. The most prevalent isolates were Proteus mirabilis and P. vulgaris. These saprophytic and swarming mo- tile bacteria were isolated from all immature, pupae, and mature fed or unfed male or female sand flies as well as from larval and adult food sources. Five fungi species were also isolated from sand flies, their food sources and colo- nization materials where Candida sp. was common in all mentioned sources. Conclusion: Midgut microbiota are increasingly seen as an important factor for modulating vector competence in insect vectors so their possible effects of the mirobiota on the biology of P. papatasi and their roles in the sandfly- Leishmania interaction are discussed. Keywords: Symbiont, Microflora, Bacteria, Fungi, Phlebotomus papatasi, Leishmaniasis Introduction Symbiont microorganisms of vector insects are involved in many aspects of the host life including nutrition, reproduction, tolerance to environmental perturbations, maintenance and/or enhancement of host immune system homeostasis, defense, speciation, mucosal bar- rier fortification, xenobiotic metabolism, and pathogen transmission ability (O’Neill et al. 1997, Baumann et al. 2000, Volf 2002, Bourtzis and Miller 2003, Rio et al. 2004, Dillon and Dillon 2004, Weiss and Aksoy 2011). The gut of sand flies is a site where most Leishmania’s entire life cycle takes place, thereby resident gut bacteria could possibly *Corresponding author: Prof Mohammad Ali Oshaghi, E-mail: moshaghi@sina.tums.ac.ir http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 70 have a role in modulating the parasite devel- opment, either enhancing or inhibiting it. In nature, sand flies feed on different kinds of sugars, so having the chance to acquire con- taminating microorganisms, such as bacteria and fungi that can eliminate the leptomonad infection or even kill the sand fly (Schlein et al. 1985, Killick-Kendrick and Killick-Kend- rick 1987, Schlein and Yuval 1987, Cameron et al. 1995). Yeasts and bacteria could reduce the in- fection rate of L. major in Phlebotomus papatasi (Schlein et al. 1985). P. papatasi is of particular interest as a vector of Zoonotic Cutaneous Leishmaniasis (ZCL) to humans in the old world. The conventional vector control measures such as application of in- secticide has got undesirable effects on the environment, human health, and the emer- gence of insecticide resistance in sand flies (Alexander and Maroli 2003). The larvae of sand flies live in contaminated soils of do- mesticated animal shelters, termite mounds and rodent burrows and are exposed to a va- riety of different soil microbes that could be ingested alongside of food (Feliciangeli 2004, Mukhopadhyay et al. 2012). Moreover, gravid P. papatasi attracts to the oviposition sites contaminated by frass and certain soil bac- teria (Radjame et al. 1997, Wasserberg and Rowton 2011). Therefore it is expected that the gut of sand flies will support a range of microorganisms. These microbes could be ge- netically manipulated to express an anti- parasitic molecule and then be reintroduced back into the sand fly gut through larval breeding places. This is a new vector borne disease control method called paratransgenesis that leads to reduce pathogen transmission by an insect vector (Chavshin et al. 2013). This method has been successfully applied for a few insect vectors such as Rhodnius prolixus, vector of Trypanosoma cruzi the causative agent of the Chagas disease in Cen- tral America (Beard et al. 2001), Glossina morsitans, the vector of african sleeping sick- ness (Aksoy et al. 2008, Pontes and Dale 2011), and Anopheles stephensi, a vector of malaria in Asia (Wang et al. 2012). Boulanger et al. (2004) demonstrated that bacteria present in the digestive tract of P. duboscqi induced the secretion of antimicro- bial peptides with a significant antiparasitic activity. Dillon et al. (1996) and Volf et al. (2002) demonstrated that the maximum prev- alence of bacteria in P. papatasi and P. duboscqi is recorded two days after blood feeding. High diversity in the bacterial gut microbiota, associated with different popu- lations of P. argentipes and Lutzomyia longipalpis, was registered using culture-de- pendent methods (Gouveia et al. 2008, Hillesland et al. 2008). Notwithstanding these studies, there are a few reports available on the micro flora of P. papatasi (Dillon et al. 1996, Guernaoui et al. 2011, Mukhopadhyay et al. 2012). Gut flora is depended upon the environment they live. Male and female sand flies feed on natural sugars, such as nectar, sap, and aphid and coccid secretions (Young et al. 1980, Killick- Kendrick and Killick-Kendrick 1987). These sugars are the main sources of carbohydrates for adults sand flies. Furthermore, females also feed on blood. Sand fly larvae are ter- restrial and feed on soil organic matter (Feliciangeli 2004). It is thus supposed that the diversity in their feeding behavior, as well as conditions that pre-imaginal stages en- counter during their development, affect gut microbiota and could impact their overall ca- pacity to sustain Leishmania development. Sand flies gut bacterial flora has been in- vestigated on the isolated or pooled guts via culture of bacterial gut content and were identified by the use of classical bacteriology, cloning, and ribotyping (16S rDNA sequenc- ing) (Rajendran and Modi 1982, Hillesland et al. 2008, Gouveia et al. 2008, Chavshin et al. 2012). Fungi (yeasts) have been used as larva nutrient (e.g. Wermelinger and Zanuncio 2001) or entomopathogen (Warburg 1991) of http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 71 sand flies. However, there is a little infor- mation about fungi as part of gut flora of sand flies (Schlein et al. 1985). The aim of the present study was to ex- amine different aerobic microbial community (bacteria and fungi) in the gut, food sources and colonization materials of a laboratory- reared colony of P. papatasi (Isfahan strain) in the School of Public Health, Tehran Uni- versity of Medical Sciences (SPH-TUMS). Results of this study may lead to identify ap- propriate candidate/s for paratransgenesis approach. Materials and Methods Sand fly insectary Sand flies of Badrood strain originally collected from Badrood (33° 44´ N, 52° 2 E), Natanz district, Isfahan Province, central Iran, were reared under laboratory condition at 24–27 °C, 80% RH and 14:10 (L: D) pho- toperiod in the sand fly Insectary of SPH- TUMS, following the method of Modi and Tesh for mass rearing of P. papatasi with some modifications (Modi and Tesh 1983). We have examined microbial infestation of a variety of sand fly biological specimens as well as non-biological materials used in the rearing of sand flies in the laboratory. The biological specimens included sand fly eggs, 1–4 instar larvae, pupae, unfed males, honey fed males, unfed females, honey fed females, blood and honey fed females, white mouse auricle, and white mouse muzzles. Tested non-biological materials were larval chow as immature sand fly food sources, saturated sucrose and honey solution 50% as mature sand fly food sources, and sea sand (Merck), plaster (Chalk), and soil as colonization ma- terials (Table 1). Isolation of bacteria Isolation of sand fly guts was conducted in a sterile environment on a sterile glass slide. Before dissection, each fly was surface steri- lized for 1min in 70% ethanol. Bacterial microflora was isolated from each sand fly by one of the two following methods. For adults, the gut from each sand fly was micro- dissected and homogenized in test tubes con- taining 5cc brain heart infusions (BHI) broth. For immature sand flies, the whole body of each individual larva homogenized in BHI broth. Sterile cotton swabs (presenting in a ster- ile test tube) were used to swab the area around the auricle or nose of white mouse where sand flies choose to take blood meal. The infested swabs then were merged in BHI broth. For larval chow, sucrose, honey solu- tion, sea sand, plaster and soil weights equal to 0.7 micro tubes volume were poured in the medium. BHI broth was chosen as a non- selective medium to promote growth a di- verse range of microbes including nutrition- ally fastidious bacteria and even fungi. The complete transparent test tubes were incu- bated aerobically at 37 °C overnight. After 24 hours, opaque test tubes considered as posi- tive and sub cultured in BHI agar medium overnight at the same condition. The colo- nies with different phenotype were sub cul- tured sequentially to obtain single colony of the microbes. Pure cultures for each microbe were used for further identification proce- dure. A test tube containing BHI broth open near the dissection area constituted our ste- rility control during the dissection process. Bacterial identification procedure All of the isolates were differentiated by standard gram staining and morphotypes. MacConkeys Agar, a special selective medium for gram negative bacteria which can differ- entiate those bacteria capable to ferment lac- tose was used for further confirmation. Bio- chemical properties of the gram negative rods belonging to the Entrobacteriaceae were investigated by “DS-DIF-ENTRO-24” kit. This kit is produced for the identification http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 72 and differentiation of 81 species and subspe- cies corresponded to 25 genera of the family Entrobacteriaceae within 24 hours. This ready to use kit has been designed for the perfor- mance of 24 biochemical tests including sim- mons citrate, malonate, esculine, lysine, argi- nine, ornithine, hydrogen sulphide, phenyl- alanine, voges-proskauer, indole, glucose, β- galactosidase, lactose, mannitole, sucrose, inositole, sorbitol, arabinose, maltose, adonitol, trehalose, ramnose, dulsitol and urease. The interpretation of the results was based on numerical probabilistic identification using EasyBac software package and the distance method. Gram-positive isolates that were not able to grow on the MacConkeys Agar medium, tested with manual laboratory examinations such as oxidase, catalase, coagulase, novobiocin susceptibility tests and mannitol medium. To test the accuracy of the DS-DIF- ENTRO-24 kit, three bacterial isolates from adult (FU), larvae (34L1) and white mouse muzzle (Mn2Moz) were selected randomly and subjected for molecular ribotyping fol- lowing PCR amplification and PCR-direct sequencing of a subset of about 1500bp of 16S rRNA gene according to Weisburg et al. (1991) protocol. Homologies with the avail- able sequence data in GenBank was checked by using basic local alignment search tool (BLAST) analysis software (www.ncbi.nlm. nih.gov/BLAST). Isolation of fungi For initial isolation of fungi we followed overnight BHI broth test tubes. In the subse- quent subcultures we used Subaru Dextrose Agar with chloramphenicole medium and samples were incubated at 25–27 °C for a week. Positive samples were examined mac- roscopically and then microscopic slides were prepared using the lactophenol cotton blue wet mount. Chrome Agar Candida, a chro- mogenic differential culture medium, used for isolation and identification of the yeast species in incubation conditions of 35 °C for 2 days. Corn Meal Agar medium plus Tween 80 were used for discrimination of Candida albicans from non-albicans species. Results Biochemical identification of bacteria Totally 25 bacterial species were isolated comprising 20 species of Entrobacteriaceae, one species of Pseudomonadaceae, one spe- cies of Bacillaceae, two species of Staphy lococcaceae, and one non-identified species in the Phelebtominae insectary of SPH- TUMS. Details of the bacterial isolates from different stages of sand flies (egg to adults), food sources, and colonization materials are listed in Table 1. Molecular identification of the bacteria Three isolates of 34L1, Mn2Moz and FU, that were biochemically identified as Proteus vulgaris, P. vulgaris and Bacillus sp. respec- tively, were identified accordingly as P. vul- garis, P. penneri and B. flexus based on par- tial sequences of 16S rRNA. Their se- quences with length of 882, 995 and 1279 bp were deposited in GenBank with accession numbers of JQ928898-JQ928900 respectively. Isolation of fungi Five fungal species were identified including three molds species of Cladosporium sp., Penicillium sp., and Aspergillus flavus that were isolated from a honey and blood fed female, Rhizopus sp. from saturated sucrose, and one yeast species, Candida sp., from dif- ferent sources including honey and blood fed females, unfed male, second instar larvae, sea sand, soil, larval chow, saturated su- crose, and honey (Table 1). Although C. albicans formed chlamydospores on Corn Meal Agar medium however mycelia have been observed on the medium. So we en- countered Candida sp. other than C. albicans. http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 73 Table 1. Summary of the isolated bacteria from the sand fly insectary and their sources identified by DS-DIF-ENTRO-24 kit (*based on 16srRNA ribotyping) Sources Species E gg L ar va e 1 L ar va e 2 L ar va e 3, 4 P u p ae U n fe d M al e H on ey f ed M al e U n fe d F em al e H on ey a n d B lo od F ed F em al e S an d L ar va l C h ow S u cr os e H on ey A u ri cl e of W h it e M ou se M u zz le o f W h it e M ou se C h al k S oi l Found in sand flies, insects or other sources [Notes] Bacillus sp (*B. flexus) X X P. papatasi (Mukhopadhyay et al. 2012), Macrotermes carbonarius (Tay et al. 2010) Plants (Sanchez-Gonzalez et al. 2011), Seaweed (Singh et al. 2011) Staphylococcus aureus X X P. argentipes (Hillesland et al. 2008), Cockroaches (Al-bayati et al. 2011), Human skin, somewhen systemic infections (Foster 2005) Staphylococcus saprophyticus X P. papatasi (Mukhopadhyay et al. 2012), P. argentipes (Hillesland et al. 2008), Musca domestica (Butler et al. 2010) Not identified bacterium X ***** Pseudomonas aeruginosa X P. papatasi (Mukhopadhyay et al. 2012), P. argentipes (Hillesland et al. 2008), [Pathogen on human, insects and plants (Silby et al. 2011)] Shigella dysenteriae X X Cockroaches (Salehzadeh et al. 2007, Al-bayati et al. 2011), Musca domestica (Bolaños-Herrera 1959) Edwardsiella ictaluri X [Pathogen on fish (Santander et al. 2010)] Kluyvera ascorbata X Ips typographus (Muratoğlu et al. 2011) Proteus vulgaris X X X X X Cockroaches (Al-bayati et al. 2011), Ips typographus (Muratoğlu et al. 2011) Klebsiella oxytoca X X Ips typographus (Muratoğlu et al. 2011) Bactrocera cacuminata (Thaochan et al. 2010) Klebsiella planticola X Ips typographus (Muratoğlu et al. 2011) Providencia rettgeri X [Pathogens on humans and Drosophila melanogaster (Galac and Lazzaro 2011)] Bactrocera cacuminata (Thaochan et al. 2010) Klebsiella pneumonia ozaenae X X Cockroaches (Al-bayati et al. 2011), Bactrocera cacuminata (Thaochan et al. 2010) Leminorella grimontii X Anopheles stephensi (Rani et al. 2009) Enterobacter amnigenus X X X Anopheles dirus (Khampang et al. 2001) Proteus mirablis X X X X X X X Blow fly maggots (Erdmann 1987, Fleischmann et al. 2004), Lucilia sericata (Ma et al. 2012), Calliphora vicina (Green- berg et al. 1970), Cochliomya hominivorax (Erdmann and Khalil 1986), Lucilia cuprina (Mohd Masri et al. 2005), Musca domestica (Greenberg 1959), Parasitoide wasps, Itoplectis (Bucher 1963), Cockroaches (Al-bayati et al. 2011) Escherichia coli X Anopheles gambiae (Dong et al. 2009) Cockroaches (Al-bayati et al. 2011) Enterobacter gergoviae X X Pectinophora gossypiella (Kuzina et al., 2002) Pantoea agglomerans X X X Anopheles gambiae (Dong et al. 2009) Escherichia blattae X X Cockroaches (Burgess et al. 1973) Tatumella ptyseos X X [Pathogen on Human and Pineapple (Marín-Cevada et al. 2010, Hollis et al. 1981)] Serratia plymuthica X Manduca sexta (Toth-Prestia and Hirshfield 1988) Yersinia pseudotuberculosis X [Ubiquitous bacteria affecting humans, animals and fleas (Hinnebusch 2005)] Rahnella aquatilis X X Pacific Coast Wireworm,( Lacey et al. 2007), Southern Pine Beetle (Vasanthakumar et al. 2006), Toxoptera aurantii (Sevim et al. 2012) Providencia alcalifaciens X [Pathogens on humans (Galac and Lazzaro 2011)] http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 74 Discussion We could isolate and identify 25 bacteria and five fungi species from the sand fly strain and its rearing materials. Majority of these microbes including 20 of the bacteria and four of the fungi species, were isolated from sand fly guts. Many species of Candida could be found in the gut of a variety of or- ganisms including C. albicans in mammalian and insect as endosymbionts (Nguyen 2007, Suh 2008). Although bacterial micro flora of some laboratory reared sand flies have been pointed out in a few studies but the source of this flora is the first time that marked and studied in details. In this study we tested mi- crobial infestation of all fields and materials used in sand fly colonization procedure. In this study the correctness of the bio- chemical kit were examined against 16S-rRNA ribotyping. On general there was a great con- cordance between the results of two methods: both methods identified 34L1 and Mn2Moz isolates as Proteus vulgaris. It is noteworthy that P. penneri is formerly known as P. vul- garis biogroup 1. However, the molecular method could identify the FU isolate up to species level (B. flexus) whereas the biochem- ical method identified it up to genus level (Bacillus sp) concluding that the ribotyping is more specified than biochemical one. Although we used a nonselective medium (BHI broth) to promote growth a wide range of bacteria and fungi, however, due to not using various media and culture conditions (e.g. anaerobic condition) the medium gen- erally favored in growth of gram negative Enterobacteriaceae. Similarly, almost all of the studies analyzing the sand fly guts for bacterial communities have also relied on cul- ture dependent techniques in their analyses where Enterobacteriaceae constituted the ma- jority of their findings (Dillon et al. 1996, Hillesland et al. 2008, Mukhopadhyay et al. 2012). Even the studies that have implemented molecular tools used these tools only in the identification and analysis of isolated pure colonies from plate culture, not in the initial isolation of bacteria from the guts (Gouveia et al. 2008, Hillesland et al. 2008). The mo- lecular tools used in both studies were im- plemented in the identification of bacterial colonies obtained by culturing, thereby lim- iting the findings to the small proportion of cultivable microbes. Taking into account the limitations of culture dependent techniques makes these findings incomplete. A correlation between the type of micro- bial gut flora detected and the area inhabited by the sand fly has been addressed by Hillesland et al. 2008, where flies collected from the same region harbored almost the same kinds of bacteria. Therefore, it was sug- gested that gut flora diversity more or less is a reflection of the environment where the sand fly resides (Hillesland et al. 2008). For example, Bacillus megaterium that is present in biofertilizers widely used in the state of Bihar, India, was isolated from the guts of a number of sand flies inhabiting that area. An- other example was that of Brevibacterium lin- ens, the bacterium used in cheese ripening in- dustry that was also isolated from the gut of sand flies collected from regions known to be involved in dairy preparations (Hillesland et al. 2008). Both these bacteria were proposed as candidates for use in a paratransgenesis model, being already employed in biotechno- logical operations without concerns about their safety (Hillesland et al. 2008). Mukhopadhyay et al. 2012 carried out a survey to study the abundance of different natural gut flora of P. papatasi in different habitats (Sheep shed, Rabbit hole, Chick/sheep shed, Human dwell- ings and Lab colony) of Tunisia, Turkey, India and Egypt. They found variation in the species and abundance of gut flora in sand flies collected from different habitats. How- http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 75 ever B. flexus and B. pumilus were common in most of the sites examined. B. flexus is ca- pable to grow under highly alkaline condi- tions that are the case for phlebotomine sand flies (Panizzi and Parra 1991) as alkaline am- ylase producers was found in our study from midgut of unfed female as well. In our study we found Staphylococcus saprophyticus from the saturated sucrose used as adult’s phytophagic meal. This bac- terium which has already isolated from P. argentipes (Hillesland et al. 2008), Musca domestica (Butler 2010) and P. papatasi (Mukhopadhyay 2012) is a very strong oviposition inducer for gravid P. papatasi (Radjame 1997). Mukhopadhyay et al. 2012 could success in introducing of Bacillus flexus, B. pumilus, B. licheniformis, B. megaterium and B. subtilis as candidate species for paratransgenesis due to these bacteria ability in induction of sand fly oviposition behavior and real function of those as symbionts and not merely as environmental contaminants. The presence of P. mirabilis and P. vul- garis nearly in all our analyzed samples rais- es questions about the nature of the interac- tion between this microorganism and sand flies. Proteus species are found in the diges- tive tracts of many animals including insects (Guentzel 1991, Rozalski 1997). A field sur- vey of bacteria from the digestive tracts of newly emerged house flies breeding in horse manure showed the predominant flora to consist of P. vulgaris, P. mirabilis, Aerobacter aerogenes, and Citrobacter freundii. A sim- ilar census from flies bred in the laboratory recovered P. vulgaris, P. mirabilis, P. morganii, P. rettgeri, E. coli, and Aerobacter aerogenes (Greenberg 1959). Interestingly, in maggots, the bacterium P. mirabilis se- crete antibacterial toxins (including phenylactic acid and phenylactaldehyde) that kill other microbes but do not harm the maggots (Erdmann 1987, Fleischmann et al. 2004). P. mirabilis is also highly resistant to the action of antimicrobial peptides, such as polymyxin B (PM), protegrin, and the synthetic protegrin analogI B-367 (McCoy 2001). Ma et al. 2012 found that P. mirabilis interkingdom swarm- ing signals attract blow flies. They obtained P. mirabilis from the salivary glands of the blow fly Lucilia sericata, this strain swarmed significantly and produced a strong odor that attracts blow flies. Greenberg et al. (1970) demonstrated that microorganisms ingested by the blowfly Calliphora vicina are rapidly eliminated if P. mirabilis is also present in the ingested material. Several pathogenic mi- croorganisms have been killed by brief expo- sure to 15 days old P. mirabilis culture broth (Erdmann and Khalil 1986). Greenberg (1968) referred to the active anti-bacteria constit- uents as “mirabilicide.” It was also reported that the symbiotic relationship between P. mirabilis and the screwworm could protect the larvae from other harmful bacteria by bac- tericidal agents secreted by P. mirabilis (Erd- mann and Khalil 1986). Interestingly, com- plete sterility of Lucilia cuprina maggots for the purpose of debriding intractable wounds was achieved in all cases, except that P. mi- rabilis was consistently found. However, the presence of this microorganism was consid- ered beneficial (Mohd-Masri et al. 2005). To what extent the bacterium could play a similar role during sand fly larval develop- ment remains unknown and has to be inves- tigated. A few studies have examined the im- pact of the gut microbiota on the establish- ment of human pathogens and parasites in their insect vectors. The possibility that col- onization resistance is involved in suppress- ing medically important parasites such as Plasmodium and Leishmania in their dipteran vectors has been discussed (Dillon et al. 1996, Pumpuni et al. 1996). Although a re- cent study suggesting a dose dependent in- hibitory effect of gut bacteria on Leishmania promastigotes (Muniaraj et al. 2008), howev- er, more investigation need to find the most effective bacteria or bacterium which can be used as bio-agent for combating Leishmania http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 76 parasites. An indigenous biota (not transient one) is present in all individuals of the spe- cies and maintains stable climax communi- ties (Dillon and Dillon 2004) also in some cases an indigenous species may colonize only in the presence of other members of the microbial community (Dillon and Charnley 2002). Indigenous bacteria modulate expres- sion of genes involved in several important intestinal functions including nutrient ab- sorption, mucosal barrier fortification, and xenobiotic metabolism (Dillon and Dillon 2004). Regarding to these criteria, herein we can nominate Proteus spp. as indigenous iso- lates of P. papatasi. However, whether or not, the resident gut microbiota as a micro ecological factor can regulate the prevalence of sand flies with transmissible infections need to be investigated. Koch and Schmid-Hempel (2011) showed that the presence microbiota protects bee hosts against a widespread and highly viru- lent natural parasite, Crithidia bombi. Their results emphasize the importance of consid- ering the host microbiota as an “extended immune phenotype” in addition to the host immune system itself and provide a unique perspective to understanding bees in health and disease. Metacyclic promastigotes are highly adapted for transmission and early survival in the vertebrate host (Kamhawi 2006). Messy con- dition of sandfly midgut and many interac- tions will determine the triumphant. Peritrophic matrix, enzymes, temperature, pH, oxygen pressure, insect immune system, vertebrate host immune system, sugar meals, blood meals, Leishmania, bacteria, fungi and many putative unknown internal/external factors are determinants in these relationships (Young et al. 1980, Killick-Kendrick and Killick- Kendrick 1987, Kamhawi 2006, Bates 2007, Bates 2008, Oshaghi et al. 2009, Weiss and Aksoy 2011). As major contestant, microbiota and Leishmania parasites interactions make it possible to introduce a sand fly as a vector or non-vector rather than other intrinsic and extrinsic criteria. Although midgut bacteria are increasingly seen as an important factor determining vector competence in mosqui- toes and there are some recently examples against or assist this point (Mourya et al. 2002, Xi et al. 2008, Meister et al. 2005, Cirimotich et al. 2011, Rodrigues et al. 2011). Recent studies have shown the capac- ity of endogenous bacteria to decrease viral and parasitic infections in mosquito and tset- se fly vectors by activating their immune responses or directly inhibiting pathogen de- velopment (Cirimotich et al. 2011). This could be happen in the sand fly gut and this in turn may lead to a reduction in Leishmania infec- tion within the sand fly host. Conclusion Midgut microbiota are increasingly seen as an important factor for modulating vector competence in insect vectors so the possible effects of the mirobiota on the biology of P. papatasi and their roles in the sandfly- Leishmania interaction were discussed. How- ever a more in depth research on the interac- tions between sandfly and their midgut re- siding bacteria and Leishmania is required. Acknowledgements This article is a part of the first author’s dissertation for fulfillment of a PhD degree in Medical Entomology and Vector Control from Department of Medical Entomology and Vec- tor Control, School of Public Health, Tehran University of Medical Sciences. This research was supported financially by the Tehran Uni- versity of Medical Sciences. The authors de- clare that there is no conflict of interest. References Aksoy S, Weiss B, Attardo G (2008) Para- http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 77 transgenesis applied for control of tsetse transmitted sleeping sickness. Adv Exp Med Biol. 627: 35–48. Al-bayati NY, Al-Ubaidi AS, Al-Ubaidi IK (2011) Risks associated with cockroach Periplaneta americana as a transmitter of pathogen agents. Diyala J Med. 1: 91–97. Alexander B, Maroli M (2003) Control of phlebotomine sandflies. Med Vet En- tomol. 17(1): 1–18. Bates PA (2008) Leishmania sandfly inter- action: progress and challenges. Curr Opin Microbiol. 11(4): 340–344. Bates PA (2007) Transmission of Leishmania metacyclic promastigotes by phlebo- tomine sandflies. Int J Parasitol. 37 (10): 1097–1106. Baumann P, Moran NA, Baumann L (2000) Bacteriocyte-associated endosymbionts of insects. In: Dworkin M (ed): The Pro- karyotes. Springer-Verlag, New York. Beard CB, Doston EM, Pennington PM, Eichler S, Cordon-Rosales C, Durvasula RV (2001) Bacterial symbiosis and paratransgenic control of vector-borne Chagas disease. Int J Parasitol. 31: 621–627. Bolaños-Herrera R (1959) Frecuencia de Sal- monella y Shigella em moscas domésticas em la ciudade de San José. Rev Biol Trop. 7(2): 207–210. Boulanger N, Lowenberger C, Volf P, Ursic R, Sigutova L, Sabatier L, Svobodova M, Beverley SM, Spath G, Brun R, Pesson B, Bulet P (2004) Characterization of a defensin from the sand fly Phlebo- tomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major. Infect Immun. 72: 7140–7146. Bourtzis K, Miller TA (2003) Insect Symbi- osis. CRC Press, Boca Raton, FL. Bucher GE (1963) Transmission of bacterial pathogens by the ovipositor of a hymenopterous parasite. J Insect Pathol. 5: 277–283. Burgess NRH, McDermott SN, Whiting J (1973) Aerobic bacteria occurring in the hind-gut of the cockroach, Blatta orientalis. J Hyg (Lond). 71: 1–7. Butler JF, Garcia-Maruniak A, Meek F, Maruniak JE (2010) Wild Florida house flies (Musca domestica) as car- riers of pathogenic bacteria. Fla En- tomol. 93(2): 218–223. Cameron MM, Milligan PJ, Llanos-Cuentas A, Davies CR (1995) An association between phlebotomine sandflies and aphids in the Peruvian Andes. Med Vet Entomol. 9: 127–132. Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Enayati AA, Mardani N, Ghoorchian S (2012) Iden- tification of bacterial microflora in the midgut of the larvae and adult of wild caught Anopheles stephensi: A step to- ward finding suitable paratransgenesis candidates. Acta Trop. 121(2): 129–134. 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: 17–24. Cirimotich CM, Dong Y, Clayton AM, Sandiford SL, Souza-Neto JA, Mulenga M, Dimopoulos G (2011) Natural mi- crobe-mediated refractoriness to Plas- modium infection in Anopheles gambiae. Science. 332: 855–858. Dillon RJ, Dillon VM (2004) The gut bac- teria of insects: nonpathogenic inter- actions. Annu Rev Entomol. 49: 71–92. Dillon RJ, Charnley K (2002) Mutualism be- tween the desert locust Schistocerca gregaria and its gut microbiota. Res Microbiol.153(8): 503–509. Dillon RJ, ElKordy E, Shehata M, Lane RP http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 78 (1996) The prevalence of a microbiota in the digestive tract of Phlebotomus papatasi. Ann Trop Med Parasitol. 90(6): 669–673. Dong Y, Manfredini F, Dimopoulos G (2009) Implication of the Mosquito Midgut Microbiota in the Defense against Ma- laria Parasites. PLoS Pathog. 5(5): e1000423. Erdmann GR (1987) Antibacterial action of myiasis-causing flies. Parasitol Today. 3(7): 214–216. Erdmann GR, Khalil SKW (1986) Isolation and identification of two antibacterial agents produced by a strain of Proteus mirabilis isolated from larvae of the screwworm (Cochliomya hominivorax) (Diptera: Calliphoridae). J Med En- tomol. 23(2): 208–211. Feliciangeli MD (2004) Natural breeding places of phlebotomine sand flies. Med Vet Entomol. 18(1): 71–80. Fleischmann W, Grassberger M, Sherman R (2004) Maggot Therapy: A Handbook of Maggot-Assisted Wound Healing. Stuttgart Georg Thieme. Foster TJ (2005) Immune evasion by staph- ylococci. Nat Rev Microbiol. 3(12): 948–958. Galac MR. Lazzaro BP (2011) Comparative pathology of bacteria in the genus Providencia to a natural host, Dro- sophila melanogaster. Microbes In- fect. 13(7): 673–683. Gouveia C, Asensi MD, Zahner V, Rangel EF, Oliveira SM (2008) Study on the bacterial midgut microbiota associated to different Brazilian populations of Lutzomyia longipalpis (Lutz and Neiva) (Diptera: Psychodidae). Neotrop En- tomol. 37: 597–601. Greenberg B (1959) Persistence of bacteria in the developmental stages of the house fly .II. Quantitative study of the host- contaminant relationship in flies breed- ing under natural conditions. Am J Trop Med Hyg. 8: 412–416. Greenberg B (1968) Model for destruction of bacteria in the midgut of blow fly maggots. J Med Entomol. 5(1): 31–38. Greenberg B, Kowalski JA, Klowden MJ (1970) Factors affecting the transmis- sion of Salmonella by flies. Natural re- sistance to colonization and bacterial in- terference. Infect Immun. 2(6): 800–809. Guentzel MN (1991) Escherichia, Klebsiella, Enterobacter, Serratia, Citrobacter and Proteus. In: Baron S (Ed): Medical Mi- crobiology. New York, Churchill Living- stone, pp. 377–387. Guernaoui S, Garcia D, Gazanion E, Ouhdouch Y, Boumezzough A, Pesson B, Fontenille D, Sereno D (2011) Bac- terial flora as indicated by PCR-tem- perature gradient gel electrophoresis (TGGE) of 16S rDNA gene fragments from isolated guts of phlebotomine sand flies (Diptera: Psychodidae). J Vector Ecol. 36: 144–147. Hillesland H, Read A, Subhadra B, Hurwitz I, McKelvey R, Ghosh K, Das P, Durvasula R (2008) Identification of aerobic gut bacteria from the kala azar vector, Phlebotomus argentipes: A platform for potential paratransgenic manipulation of sand flies. Am J Trop Med Hyg. 79(6): 881–886. Hinnebusch BJ (2005) The Evolution of Flea- borne Transmission in Yersinia pestis. Curr Issues Mol Biol. 7(2): 197–212. Hollis DG, Hickman FW, Fanning GR, Farmer JJ, Weaver RE, Brenner DJ (1981) Tatumella ptyseos gen. nov., sp. nov., a member of the family Enterobacteriaceae found in clinical specimens. J Clin Microbiol. 14: 79e88. Kamhawi S (2006) Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends Parasitol. 22(9): 439–445. Khampang P, Luxananil P, Tanapongpipat S, http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 79 Chungjatupornchai W, Panyim S (2001) Recombinant Enterobacter amnigenus highly toxic to Anopheles dirus mos- quito larvae. Curr Microbiol. 43(6): 448–451. Killick-Kendrick R, Killick-Kendrick M (1987) Honeydew of aphids as a source of sugar for Phlebotomus ariasi. Med Vet Entomol. 1(3): 297–302. Koch H, Schmid-Hempel P (2011) Socially transmitted gut microbiota protect bum- ble bees against an intestinal parasite. PNAS. 108(48): 19288–19292. Kuzina LV, Miller ED, Ge BX, Miller TA (2002) Transformation of Enterobacter gergoviae isolated from pink bollworm (Lepidoptera: Gelechiidae) gut with Ba- cillus thuringiensis toxin. Curr Mic- robiol. 44: 1–4. Lacey LA, Unruh TR, Simkins H, Thomsen- Archer K (2007) Gut bacteria associ- ated with the Pacific Coast wireworm, Limonius canus, inferred from16S rDNA sequences and the triplications forcontrol. Phytoparasit 35: 479–489. Ma Q, Fonseca A, Liu W, Fields AT, Pimsler ML, Spindola AF, Tarone AM, Crippen TL, Tomberlin JK, Wood TK (2012) Proteus mirabilis interkingdom swarm- ing signals attract blow flies. ISME J. 6(7): 1356–66. Marín-Cevada V, Caballero-Mellado J, Bustillos-Cristales R, Muñoz-Rojas J, Mascarúa-Esparza MA, Castañeda-Lucio M, López-Reyes L, Martínez-Aguilar L, Fuentes-Ramírez LE (2010) Tatumella ptyseos, an unrevealed causative agent of pink disease in pineapple. J Phy- topathol. 158(2): 93–99. McCoy AJ, Liu H, Falla TJ, Gunn JS (2001) Identification of Proteus mirabilis mu- tants with increased sensitivity to an- timicrobial peptides. Antimicrob Agents Chemother. 45(7): 2030–2037. Meister S, Kanzok SM, Zheng XL, Luna C, Li TR, Hoa NT, Clayton JR, White KP, Kafatos FC, Christophides GK, Zheng L (2005) Immune signaling path- ways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae. Proc Natl Acad Sci USA. 102: 11420–11425. Modi GB, Tesh RB (1983) A simple tech- nique for mass rearing Lutzomyia longipalpis and Phlebotomus papatasi (Diptera: Psychodidae) in the labora- tory. J Med Entomol. 20: 568–569. Mohd Masri S, Nazni WA, Lee HL, Tengku Rogayah TAR, Subramaniam S (2005) Sterilization of Lucilia cuprina (Wiedemann) maggots used in therapy of intractable wounds. Trop Biomed. 22(2): 185–189. Mukhopadhyay J, Braig HR, Rowton ED, Ghosh K (2012) Naturally occurring culturable aerobic gut flora of adult Phlebotomus papatasi, Vector of Leish- mania major in the old world. PLoS ONE. 7(5): e35748. Muniaraj M, Dinesh D, Sinha P, Das P, Bhattacharya S (2008) Dual culture method to determine the relationship of gut bacteria of sand fly (Phlebotomus argentipes) with promastigotes of Leish- mania donovani. J Commun Dis. 40 (2): 133–138. Muratoğlu H, Sezen K, Demirbağ Z (2011) Determination and pathogenicity of the bacterial flora associated with the spruce bark beetle, Ips typographus (L.) (Cole- optera: Curculionidae: Scolytinae) Turk J Biol. 35: 9–20. Nguyen NH, Suh SO, Blackwell M (2007) "Five novel Candida species in insect- associated yeast clades isolated from Neuroptera and other insects". My- cologia. 99(6): 842–858. O’Neill SL, Hoffmann AA, Werren JH (1997) Influential Passengers. Oxford Univer- sity Press, Oxford. Oshaghi MA, Maleki Ravasan N, Javadian E, Rassi Y, Sadraei J, Enayati AA, Vatan- http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 80 doost H, Zare Z, Emami SN (2009) Ap- plication of predictive degree day mod- el for field development of sand fly vec- tors of visceral leishmaniasis in north- west of Iran. J Vector Borne Dis. 46: 1–8. Panizzi AR, Parra JRP (1991) Ecologia nut- ricional de insetos e suas implicações no manejo de pragas. São Paulo, Manole/ Brasilia: CNPq. Pontes MH, Dale C (2011) Lambda Red- mediated genetic modification of the insect endosymbiont Sodalis glossini- dius. Appl Environ Microbiol. 77: 1918–1920. Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC (1996) Bacterial population dynamics in three Anopheline species: the impact on Plasmodium sporogonic development. Am J Trop Med Hyg. 54 (2): 214–218. Radjame K, Srinivasan R, Dhanda V (1997) Oviposition response of phlebotomid sandfly Phlebotomus papatasi to soil bacteria isolated from natural breeding habitats. Indian J Exp Biol. 35: 59–61. Rajendran P, Modi GB (1982) Bacterial flora of Sandfly gut (Diptera: Psychodidae) Indian J Public Health. 26(1): 49–52. Rani A, Sharma A, Rajagopal R, Adak T, Bhatnagar RK (2009) Bacterial diver- sity 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: 96. Rio RV, Hu Y, Aksoy S (2004) Strategies of the home-team: symbioses exploited for vector-borne disease control. Trends Microbiol. 12(7): 325–336. Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C (2011) Hemocyte dif- ferentiation mediates innate immune memory in Anopheles gambiae mos- quitoes. Science. 329(5997): 1353–1355. Rozalski A, Sidorczyk Z, Kotelko K (1997) Potential virulence factors of Proteus bacilli. Microbiol molec Biol Rev. 61 (1): 65–89. Salehzadeh A, Tavacol P, Mahjub H (2007) Bacterial, fungal and parasitic contam- ination of cockroaches in public hospi- tals of Hamadan, Iran. J Vect Borne Dis. 44: 105–110. Sanchez-Gonzalez M, Blanco-Gamez A, Es- calante A, Valladares AG, Olvera C, Roberto P (2011) Isolation and charac- terization of new facultative alkaphilic Bacillus flexus strains from maize pro- cessing waste water (nejayote) Lett Appl Microbiol. 52(4): 413–419. Santander J, Xin W, Yang Z, Curtiss R (2010) The Aspartate-Semialdehyde Dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology. PloS ONE. 5(12): e15944. Schlein Y, Polacheck I, Yuva B (1985) My- coses, bacterial infections and anti- bacterial activity in sandflies (Psy- chodidae) and their possible role in the transmission of leishmaniasis. Parasitol. 90: 57–66. Schlein Y, Yuval B (1987) Leishmaniasis in the Jordan Valley. IV. Attraction of Phlebotomus papatasi (Diptera: Psy- chodidae) to plants in the field. J Med Entomol. 24(1): 87–90. Sevim E, Celebi O, Sevim A (2012) Deter- mination of the bacterial flora as a mi- crobial control agent of Toxoptera aurantii (Homoptera: Aphididae). Bio- logia. 67(2): 397–404. Silby MW, Winstanley C, Godfrey SAC, Levy SB, Jackson RW (2011) Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev. 35(4): 652–680. Singh RP, Mantri VA, Reddy CRK, Jha B (2011) Isolation of seaweed associated bacteria and their morphogenesis-in- ducing capability in axenic cultures of http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 69–81 N Maleki-Ravasan et al.: Aerobic Microbial … 81 the green alga Ulva fasciata. Aquat Biol. 12: 13–21. Suh SO, Nguyen NH, Blackwell M (2008) Yeasts isolated from plant-associated beetles and other insects: seven novel Candida species near Candida albicans". FEMS. Yeast Res. 8(1): 88–102. Tay BY, Lokesh BE, Lee CY, Sudesh K (2010) Polyhydroxyalkanoate (PHA) accumulating bacteria from the gut of higher termite Macrotermes carbonarius (Blattodea: Termitidae). World J Mic- robiol Biotechnol. 26(6): 1015–1024. Thaochan N, Drew RAI, Hughes JM, Vijaysegaran S, Chinajariyawong A (2010) Alimentary tract bacteria iso- lated and identified with API-20E and molecular cloning techniques from Aus- tralian tropical fruit flies, Bactrocera cacuminata and B. tryoni. J Insect Sci. 10:131. Toth-Prestia C, Hirshfield IN (1988) Iso-lation of plasmid-harboring Serratia plymuthica from facultative gut microflora of the tobacco hornworm, Manduca sexta. Appl Environ Microbiol. 54(7): 1855– 1857. Vasanthakumar A, Delalibera I, Handelsman J, Klepzig KD, Schloss PD, Raffa KF (2006) Characterization of gut-associ- ated microorganisms in larvae and adults of the southern pine beetle, Dendroctonus frontalis Zimmerman. En- viron Entomol. 35: 1710–1717. Volf P, Kiewegova A, Nemec A (2002) Bac- terial colonisation in the gut of Phlebo- tomus duboscqi (Diptera Psychodidae): transtadial passage and the role of fe- male diet. Folia Parasitol. 49: 73–77. Wang S, Ghosh AK, Bongio N, Stebbings KA, Lampe DJ, Jacobs-Lorena M (2012) Fighting malaria with engineered sym- biotic bacteria from vector mosquitoes. Proc Natl Acad Sci USA. 109(31): 12734–12739. Warburg A (1991) Entomopathogens of phlebotomine sand flies: laboratory experiments and natural infections. J Invertebr Pathol. 58(2): 189–202. Wasserberg G, Rowton ED (2011) Sub-ad- ditive effect of conspecific eggs and frass on oviposition rate of Lutzomyia longipalpis and Phlebotomus papatasi. J Vector Ecol. 36(1): S138–S143. Weisberg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplfication for phylogenetic study. J Bacteriol. 173(2): 697–703. Weiss B, Aksoy S (2011) Microbiome influ- ences on insect host vector competence. Trends Parasitol. 27: 514–522. Wermelinger ED, Zanuncio JC (2001) Dev- elopment of Lutzomyia intermedia and Lutzomyia longipalpis (Diptera: Psy- chodidae: Phlebotominae) larvae in dif- ferent diets. Braz J Biol. 61(3): 405– 408. Xi Z, Ramirez JL, Dimopoulos G (2008) The Aedes aegypti toll pathway controls dengue virus infection. PLoS Pathog. 4(7): e1000098. Young CJ, Turner DP, Killick-Kendrick R, Rioux JA, Leaney AJ (1980) Fructose in wild-caught Phlebotomus ariasi and the possible relevance of sugars taken by sand flies to the transmission of leishmaniasis. Trans R Soc Trop Med Hyg. 74(3): 363–366. http://jad.tums.ac.ir Published Online: December 18, 2013