Molecular characterization of midgut microbiota of Aedes albopictus and Aedes aegypti from Arunachal Pradesh, India

Background Microbiota inhabiting midguts of mosquitoes play a key role in the host - parasite interaction and enhance vectorial capacity of viral diseases like dengue and chikungunya fevers. Mosquito midgut is considered to be an important site for host-pathogen interaction and pathogen survival is thought to be an outcome of this interaction. In the present study we examined the bacterial community in the midgut of Aedes mosquitoes in Arunanchal Pradesh, India, a subtropical zone where dengue fever is reported to be emerging. Method Larvae and pupa of Aedes mosquitoes were collected from a biodiversity hotspot, Bhalukpong, Arunachal Pradesh, India. 16S rRNA gene sequences were used for identification of isolated bacterial population from each species of mosquitoes. We used various diversity indices to assess the diversity and richness of the bacterial isolates in both mosquito species. Result On the basis of 16S rRNA gene sequence analysis a total of 24 bacterial species from 13 genera were identified belonging to 10 families of four major phyla. Phylum Proteobacteria was dominant followed by Firmicutes, Bacteroidetes and Actinobacteria. The midgut bacteria belonging to the phylum Proteobacteria and Firmicutes were isolated from both Ae. albopictus and Ae. aegypti, whereas, bacteria belonging to phylum Bacteroidetes and Actinobacteria were isolated only from Ae. albopictus and Ae. aegypti respectively. Enterobacter cloacae was the dominant bacterial species in both Ae. albopictus (33.65 %) and Ae. aegypti (56.45 %). Bacillus aryabhattai (22.78 %) was the second most common bacterial species in Ae. albopictus whereas, in Ae. aegypti the second most common bacterial species was Stenotrophomonas maltophilia (7.44 %). Conclusion The family Enterobacteriaceae of phylum Proteobacteria was dominant in both species of Aedes mosquitoes. To the best of our knowledge, this is the first attempt to study midgut microbiota from a biodiversity hotspot in Northeastern India. Some bacterial genera Enterobacter and Acinetobacter isolated in this study are known to play important roles in parasite-vector interaction. Information on midgut microflora may lead towards the development of novel, safe, and effective strategies to manipulate the vectorial capacity of mosquitoes.


Background
The mosquitoes Aedes aegypti and Aedes albopictus are considered major public health problems. Recent reports have provided evidence of the involvement of Ae. aegypti and Ae. albopictus in outbreaks of arboviral diseases in different parts of the globe [1,2] including dengue and chikungunya fevers. Population growth, rapid urbanization, human travel and failures of preventive public-health measures are the major factors for increasing dengue fever cases [3][4][5][6][7][8]. Of note, dengue cases are increasing not only in urban areas, but also in rural areas [9]. The burden of dengue fever has increased drastically in the last few decades, and about 40 % population living in more than 100 countries is affected. The highest prevalences are documented in South-East Asia, America and regions of the Western Pacific. Currently, about 2.5 billion people are estimated to be at risk of dengue infection with 50-100 million infections occurring annually, worldwide [10].
Dengue fever was first documented in India in the year 1945 [11]. Subsequently, during 1963-64, dengue fever was reported from the Eastern coast of India [9]. According to a survey report in 1963, dengue activity in North East India was recorded in Assam (Darrang district) and Arunachal Pradesh (Lohit district) [12,13]. Currently an increasing number of Dengue cases are being reported from other parts of North Eastern India [13][14][15][16][17].
From the literature available, it has become evident that midgut bacteria of disease vectors directly and/or indirectly affects host-pathogen interactions, and ultimately vector competency, thereby significantly influencing disease transmission potential [18][19][20][21][22]. The mosquito is thought to modulate the composition of its midgut bacterial population [23]. In the highly specialized gut structure of insects, pH, presence of digestive enzymes and food ingested by the host, are factors shown to significantly influence the diversity of microbial communities of insects [24]. The involvement of midgut bacteria in various important functions in relation to host and parasite interaction has been reported, and further studies on midgut microbial composition, its acquisition, and ability to modulate host parasite interaction have become a focus of research for several laboratories, worldwide [25][26][27][28][29].
Considering the global surge in incidences of emerging and re-emerging vector-borne diseases [30], researchers have examined the microbial diversity in different insect vectors, especially mosquitoes, to understand the hostmicrobe-pathogen interactions as well as to investigate the potential application of the host microbes in vector management [19]. Midgut microbiota affects the vectorial capacity of Anopheles mosquitoes by hampering the development of malaria parasites [26]. Inhibition of parasite growth and development has been suggested to be achieved by inducing the production of an effector molecule by genetically modified midgut bacteria [31].
In India, attempts to scrutinize the midgut microflora has remained mainly focused on Culex and Anopheles mosquitoes, which act as vectors for Japanese encephalitis virus, filariasis nematodes and malaria protozoa [25,27,[32][33][34]. Despite being the major vector for dengue, midgut microbial diversity studies in different species of Aedes mosquitoes are rare, especially from India. Although, a recent study reported the midgut microbial diversity in different Ae. aegypti strains (MOYO, MOYO-R, and MOYO-S) with varying vector competency [29], to the best of our knowledge, similar studies on field collected Ae. aegypti and Ae. albopictus have not been reported from India. Therefore, we undertook this comprehensive study to understand and compare the microflora associated with midgut of field collected Ae. aegypti and Ae. albopictus. We collected larvae samples of Ae. aegypti and Ae. albopictus from the same habitats to study the bacterial diversity in the midgut of these two mosquito species. We used 16S rRNA gene sequence based techniques and various diversity indices to explore the species richness, dominance and evenness of bacterial species in the midgut. We report finding differential predominance of bacterial strains in the two species of Aedes, which might have important implications in vector management strategies.

Sample collection and midgut dissection
The fourth instar larvae and pupa of Aedes mosquitoes were collected from 10 different breeding spots (dump tyres and water storing pots) during the post monsoon season from Bhalukpong, West Kameng district of Arunachal Pradesh, India (Latitude: 27.01°N, Longitude: 92.65°E), a small town located along the southern reaches of the Himalayas. The collected samples were brought to the laboratory and emerged pupae were transferred to the pre-sterilized net cage for adult emergence. The emerged adult mosquitoes were anesthetized using chloroform and the species were identified morpho-taxonomically. Adults emerged from nine samples were either Ae. aegypti or Ae. albopictus, only one sample had mixed population of Ae. aegypti and Ae. albopictus mosquitoes. Adults from mixed population were segregated and analyzed for midgut bacterial diversity. For isolation of midgut bacterial population, a total of 30 adult female mosquitoes were dissected from each species of mosquitoes. Prior to dissection, all the dissecting apparatus, plasticwares, glasswares, buffers and solutions, were sterilized by autoclaving and UV treatment. All the 60 mosquito samples were surface sterilized with 75 % ethanol for 5 min followed by washing with phosphate buffered saline (PBS) twice prior to dissection. Individually dissected midguts were transferred to 1.5 ml micro-centrifuge tube containing 100 μl of PBS and homogenized with a sterilized micropestle [27].

Isolation of midgut bacteria
Gut homogenates were serially diluted (10 folds) in PBS, were directly pour plated on sterile nutrient agar media (Himedia, India), and incubated at 37°C for 24-48 h. All procedures were done in a sterile environment, strictly following aseptic practices and negative controls (PBS) were included throughout the experiment. Bacterial colonies obtained on the plate were differentiated according to their colony morphology like shape, size, colour, margin, opacity, elevation etc. Morphologically distinct colonies were selected for repeated subculture on nutrient agar plates until a presumably pure colony was obtained. Finally, a total of 82 representative colonies were selected for sequencing based on morphological characteristics.

Genomic DNA isolation and PCR amplification of 16S rRNA
Genomic DNA was isolated from bacterial pellets obtained from the centrifugation of fresh over night culture in nutrient broth and re-suspended in Tris-EDTA (TE buffer, pH-8). For the lysis of bacterial cells, a repeated heat shock (freezing and thawing) method was used followed by lysozyme and proteinase-K treatment. Genomic DNA was precipitated in chilled isopropanol and the DNA pellet was air dried and suspended in TE buffer [35]. The small subunit of 16S rRNA gene segment was amplified using primer set 16S1 (5′-GAGT TTGATCCTGGCTCA-3′) and 16S2 (5′-CGGCTACCT TGTTACGACTT-3′) in 50 μl PCR reaction mixture [36]. The program for PCR reaction was set as, initial denaturation at 94°C for 2.5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, followed by a final extension step at 72°C for 7 min. The quality and quantity of amplified PCR product were checked on 1.2 % agarose gel electrophoresis and visualized through staining with Ethidium bromide (final concentration 0.5 μg/ml). PCR products were gel purified, cycle sequencing was done using BigDye ® terminator kit following manufacturer's instructions (Applied Biosystems Inc. ABI, Foster City, CA) and products analyzed on an ABI 3500xL Genetic Analyzer platform (at Chromus Biotech Pvt Ltd, Bangalore, India). Amplicons were sequenced from both directions using forward and reverse primers.

Sequence analyses
Sequence data generated in present study were submitted to the GenBank under the accession numbers (KP717387-KP717416). Homologous sequences were searched in the GenBank database using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) and also using the EzTaxon server (http://www.ezbiocloud.net/ eztaxon) [37]. Bacterial identification was done on the basis of more than 99 % similarity with sequences submitted in the GenBank (Table 1).

Statistical analysis
Mann-Whitney rank sum tests was performed to estimate the differences in prevalence of bacterial species between Ae. albopictus and Ae. aegypti midguts (p < 0.05, 95 % confidence interval). To calculate confidence intervals for comparison of the presence of each bacterial species in the two groups of Aedes mosquito, an Excel spreadsheet tool was used [38]. Various diversity indices i.e. Simpson Index [39], Shannon Index, Sørensen Index, and Evenness [40] of bacterial communities from Ae. albopiictus and Ae. aegypti midgut were calculated [27]. The following formula was used to calculate Good's coverage: percentage of coverage (1-n/N)* 100, where n represents a single bacterial isolate and N denotes total bacterial isolates from one mosquito species [41].

Results
Bacterial isolates obtained from the midguts of Ae. albopictus and Ae. aegypti were screened based on their colony characteristics and identified on the basis of 16S rRNA gene sequences. Based on 16S rRNA gene sequence analysis, a total of 24 species from 13 genera were identified, belonging to 4 major phyla: Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria (Tables 1). Proteobacteria (66.7 %) was the dominant bacterial phylum followed by Firmicutes (25 %). Actinobacteria and Bacteroidetes were the least represented phylum as only one species was identified from each of them.
Bacterial isolates from midgut of Ae. albopictus A total 16 different bacterial species were identified from the midgut of Ae. albopictus out of which phylum Proteobacteria (62.5 %) was the most prominent and 10 of 16 bacterial species belonged to this phylum. The second largest phylum was Firmicutes (31.25 %) containing 5 bacterial species of total 16 identified bacterial species. The least number of bacteria were from the phylum Bacteroidetes (6.25 %). In Proteobacteria phylum, bacteria belonging to the class Gamma Proteobacteria was dominant (56.25 %) followed by Beta Proteobacteria (6.25 %). When the total identified bacterial species were classified according to their family, Enterobacteriaceae (31.25 %) with 5 species was found to be the most abundant, followed by Bacillaceae (25.00 %) with 4 species, Pseudomonadaceae (18.75 %) with 3 species, Staphylococcaceae (6.25 %), Moraxellaceae (6.25 %), Alcaligenaceae (6.25 %) and Flavobacteriaceae (6.25 %) with single number of bacterial species. Enterobacter cloacae was found to be the most dominant bacterial species followed by Bacillus aryabhattai.
Bacterial isolates from midgut of Ae. aegypti A total 14 different bacterial species were identified from the midgut of Ae. aegypti out of which 9 bacterial species from the phylum Proteobacteria (64.29 %) and 4 different bacterial species from the phylum Firmicutes (28.57 %). Proteobacteria was the largest phyla observed in Ae. aegypti and, as in Ae. albopictus, Firmicutes was the second. The least represented phylum overall was Actinobacteria (7.14 %) represented by a single bacterial species. All the bacterial species belonging to the phylum Proteobacteria were found to be of class Gamma Proteobacteria. When classified according to family, the maximum number of bacterial species in Ae. aegypti, belonged to family Enterobacteriaceae (35.71 %) followed by the family Bacillaceae (21.43 %), Pseudomonadaceae (14.29 %), Aeromonadaceae (7.14 %), Xanthomonadaceae (7.14 %), Staphylococcaceae (7.14 %), and Micrococcaceae (7.14 %). Among the bacterial isolates from Ae aegypti, Enterobacter cloacae was the most frequently isolated bacterial species followed by Stenotrophomonas maltophilia.

Statistical analysis
Bacterial prevalence of both the mosquitoes species were compared using Mann Whitney rand sum test. We did not observed statistically significant difference in bacterial species prevalence between Ae. albopictus and Ae. aegypti mosquitoes (p = 0.56). Various diversity indices were calculated for the estimation of diversity among the bacterial communities in the midguts of Ae. albopictus and Ae. aegypti ( Table 2). The Good's coverage was found to be 79.06 % and 82.05 % for Ae. albopictus and Ae. aegypti, respectively. Although not significant, but

Discussion
It has been reported that the midgut bacteria of mosquitoes play a significant role in the vector-parasite interaction [26,42]. The present work was carried out to study the diversity of midgut bacteria of two species of mosquito viz. Ae. albopictus and Ae. aegypti collected from the foothills of Arunanchal Pradesh, North East India. In this study, we only focused on the characterization of culture-dependent aerobic bacteria from the midgut of both species of Aedes mosquitoes, because only culturable bacteria can be used for further applications in management of disease transmission such as paratrangenesis. In our observation, Ae. albopictus was more frequently found as compared to Ae. aegypti among the collection sites. The low abundance of Ae. aegypti may be due to the fact that this species is usually found in urban areas, unlike Ae. albopictus which is commonly found in rural habitats and prefer breeding in natural habitats like bamboo, stumps, tree holes, and bromeliads [43,44]. In present study, we have analyzed the midgut of Ae. aegypti and Ae. albopictus females, sharing the same habitat during their larval development.
A total of 24 different bacterial species identified by a 16S rRNA gene sequence analysis was obtained from both species of Aedes mosquito and most of the bacterial genera had already been reported from the midgut of Aedes as well as other mosquito species. The bacterial genera of Enterobacter, Bacillus, Pseudomonas, Staphylococcus, Klebsiella, Pantoea, Acinetobacter and Aeromonas have been reported from midgut of mosquitoes and the results of the present study corroborate with findings reported by other workers [25,27,34,[45][46][47][48][49][50][51][52][53][54][55][56][57]. Some bacteria species are closely associated with mosquito gut environment and common inhabitants of Aedes as well as other mosquito species [27]. From the results, we observed that in both mosquitoes species, maximum bacterial species belong to families Enterobacteriaceae and Bacillaceae. It has been reported that, in the mosquito's midgut, the bacteria are primarily acquired either through vertical inheritance or through acquisition from the environment [58]. The bacterial species Enterobacter cloacae, Klebsiella michiganensis, Pseudomonas monteilii, Bacillus aryabhattai, Lysinibacillus fusiformis, Staphylococcus hominis have been isolated from both Ae. albopictus and Ae. aegypti. Whereas, there were some other species, which were retrieved either from Ae. albopictus or Ae. aegypti mosquito gut, but their prevalence were very low. For instance, Enterobacter hormaechei, Enterobacter asburiae, Klebsiella oxytoca, Pseudomonas aeruginosa, Pseudomonas geniculata, Acinetobacter pittii, Alcaligenes faecalis, Bacillus cereus, Bacillus tequilensis and Elizabethkingia anophelis were only present in the Ae. albopictus whereas, Enterobacter xiangfangensis, Klebsiella pneumonia, Pantoea dispersa, Pseudomonas mosselii, Aeromonas veronii, Stenotrophomonas maltophilia, Bacillus aerophilus and Micrococcus yunnanensis were exclusively isolated from the Ae. aegypti.
We have observed presence of Enterobacter xiangfangensis from Ae. aegypti midgut for the first time. Earlier, this bacterial species was isolated and identified from Chinese traditional sourdough [59]. Bacillus aryabhattai was also not isolated from Aedes mosquitoes. Earlier, it was reported from Culex quinquefasciatus mosquito and Capsodes infuscatus herbivorous bug [27,60]. Similarly, Aeromonas veronii was previously isolated from the midgut of Cx. quinquefasciatus [27] and larvae of An. gambiae [61] but was not isolated from midgut of Ae. aegypti or Ae. albopictus. Alcaligenes faecalis, was identified from the midgut of the sandfly, Phlebotomus papatasi and hindgut wall of Dermolepida albohirtum larvae [62,63], previously but not recorded from mosquito gut. Similarly, Bacillus tequilensis, Bacillus aerophilus was previously isolated from a herbivorous bug Capsodes infuscatus, but was not reported from the midgut of any mosquitoes [60].
In our study, we found that Enterobacter cloacae was the dominant species in both Ae. albopictus (33.65 %) and Ae. aegypti (56.45 %). This finding is important since a number of studies have been done and this species of bacteria has been found to block the development of Plasmodium falciparum in Anopheles gambiae and sporogonic development of Plasmodium vivax in An. albimanus [64,65], induce the expression of mosquito immune components in midgut of An. stephensi  [66]. In addition, E. cloacae has also been found to inhabit the midgut of the sand fly Phlebotomus papatasi and its potential application in paratransgenic approach to reduce the transmission of Leishmania has been suggested recently [67]. Apart from these potential applications, E. cloacae have also been successfully used to deliver, express, and spread foreign genes in termite colonies [68]. E. cloacae transformed with ice nucleation (IN) gene have also been shown to be useful for reduction of mulberry pyralid moth, Glyphodes pyloalis [69]. Considering these findings, direct application of E. cloacae for pathogen reduction, or through paratransgenic approach, appears to be a potential strategy towards effective management of vector-borne diseases. The bacterial genera Serratia and Enterobacter produce hemolytic enzymes that might take part in the digestion of blood in hematophagous Diptera [52,70,71]. Other important bacterial genera Acinetobacter obtained from Ae. albopictus in our study are known to be involved in blood digestion. Minard et al., [28] reported that the bacterial species Acinetobacter baumannii and A. johnsonii isolated from Ae. albopictus may have a role in assimilation of nectar and blood digestion.
In the recent years, it is reported that some midgut inhabiting bacteria play an important role in disease transmission, host-parasites interaction and also affects the vectorial capacity of mosquitoes. The midgut serves as the first contact point between parasites and the epithelial surfaces, where significant parasite numbers are reduced [22]. The microbiota involved in the blocking of the Plasmodium development may be used in the modulation of vectorial capacity of mosquitoes [26]. Midgut microbiota is known to augment the immune response of the mosquito [26,53,64,[72][73][74]. Since immunocompetent mosquitoes are thought to be less likely to transmit other parasites such as malaria [75], similar strategies might also be helpful in dengue control through use of bacterial species that augment the mosquito immune system.
The midgut microbiota composition has an important role on susceptibility of chikungunya and dengue viruses. It has been demonstrated that the susceptibility of Ae. aegypti to chikungunya and dengue virus increases in the presence of midgut bacteria Serratia odorifera due to the suppression of immune response of Ae. aegypti [42,76]. It has been showed that susceptibility to DENV-2 enhanced when Ae. aegypti were fed with the Aeromonas sp. and Escherichia coli [42,77].
From the above discussion, it is clear that the midgut bacteria can be significantly involved in host-parasite interaction and may decrease or increase the vectorial capacity through various mechanisms including enhancement of immune response or precluding the development of parasites. Midgut microbiota may be genetically manipulated to express molecules against the parasites, which could be used as a novel strategy for vector management. The understanding of midgut microbiota and the mosquitoes could be used for the development of novel, eco-friendly and highly effective defense mechanism to reduce the vectorial capacity of mosquitoes and hence disease transmission control.

Conclusion
To the best of our knowledge, this is the first time attempt towards a comprehensive study of the midgut microbiota of Ae. albopictus and Ae. aegypti in Arunanchal Pradesh, North East India. The involvement of midgut bacteria in the defense mechanism of the vector has been reported, but this information is still very limited.
Enterobacter was found to be the predominant culturable gut bacteria genera in both Ae. albopictus and Ae. aegypti and previously reported data supports its involvement in P. falciparum development blockage and blood digestion. Other important bacterial genera such as Acinetobacter were also identified from Ae. albopictus which is known to take part in blood digestion of mosquitoes. Comprehensive knowledge about midgut bacteria may leads towards better understanding the direct or indirect involvement of microbiota in the immune response, nutrition and reproduction of mosquitoes.