Diversity of Cultivable Midgut Microbiota at Different Stages of the Asian Tiger Mosquito, Aedes albopictus from Tezpur, India

Aedes aegypti and Ae. albopictus are among the most important vectors of arboviral diseases, worldwide. Recent studies indicate that diverse midgut microbiota of mosquitoes significantly affect development, digestion, metabolism, and immunity of their hosts. Midgut microbiota has also been suggested to modulate the competency of mosquitoes to transmit arboviruses, malaria parasites etc. Interestingly, the midgut microbial flora is dynamic and the diversity changes with the development of vectors, in addition to other factors such as species, sex, life-stage, feeding behavior and geographical origin. The aim of the present study was to investigate the midgut bacterial diversity among larva, adult male, sugar fed female and blood fed female Ae. albopictus collected from Tezpur, Northeastern India. Based on colony morphological characteristics, we selected 113 cultivable bacterial isolates for 16S rRNA gene sequence based molecular identification. Of the 113 isolates, we could identify 35 bacterial species belonging to 18 distinct genera under four major phyla, namely Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Phyla Proteobacteria and Firmicutes accounted for majority (80%) of the species, while phylum Actinobacteria constituted 17% of the species. Bacteroidetes was the least represented phylum, characterized by a single species- Chryseobacterium rhizoplanae, isolated from blood fed individuals. Dissection of midgut microbiota diversity at different developmental stages of Ae. albopictus will be helpful in better understanding mosquito-borne diseases, and for designing effective strategies to manage mosquito-borne diseases.


Introduction
Among the insects, mosquitoes play a significant role in transmission of various diseases like Dengue, Zika fever, Chikungunya, Yellow fever, Malaria, Japanese encephalitis, lymphatic filariasis etc. Of the medically important mosquito species, Aedes aegypti and Ae. albopictus are the most important vectors of arboviruses, including Zika virus (ZIKV), Dengue virus (DENV), Yellow fever virus (YFV), and Chikungunya virus (CHIKV) [1][2][3][4][5][6]. In the recent the adults were provided with 10% sucrose solution [Sigma Aldrich, St. Louis, USA] as a food source. After 24 hrs, we segregated 30 male and female mosquitoes for dissection and isolation of midgut bacteria. To the remaining mosquitoes, we fed blood through biting of rabbit and after 24 hrs, we took out 30 blood fed mosquitoes for dissection. We identified the mosquitoes based on their macroscopic morphological characteristics prior to dissection [29].
All the dissections were performed under Leica stereomicroscope (Model: Leica EZ4 HD) using sterilized apparatus, according to a protocol described elsewhere [30]. Prior to dissection, all the adult and larvae samples were surface sterilized with 75% ethanol for 5 min, followed by washing with phosphate buffered saline (PBS) twice. All the dissections were carried out in sterile conditions and midgut sections were separately homogenized in 100 μl of PBS [30][31].

Isolation and purification of midgut bacteria
Gut homogenates were serially diluted (10 folds) with PBS and 100 μl of each dilution was pour-plated on nutrient agar media (Himedia, India) and incubated at 37˚C for 24-48 hrs. The last wash (PBS) of larvae and adults was taken as a control, pour plated and incubated as the homogenate of dissected midgut. All the microbiological procedures were carried out in a sterile environment, strictly following aseptic laboratory practices and negative controls (sterile PBS) were included throughout the experiment. We selected morphologically distinct bacterial colonies for subculture on nutrient agar plates and for isolation of pure colonies.

Genomic DNA isolation and PCR amplification of 16S rRNA gene
For amplification of 16S rRNA gene, genomic DNA was isolated, as mentioned previously [30,32]. In brief, genomic DNA was isolated from freshly cultured bacterial cells, and re-suspended in Tris-EDTA buffer (pH-8). For efficient lysis of bacterial cells, a freezing-thawing step (freezing at -80˚C and thawing at 75˚C, for 3 cycles) was incorporated, followed by lysozyme and proteinase-K treatment. Genomic DNA was precipitated in isopropanol, DNA pellets were air dried and re-suspended in TE buffer. An amplicon of approximately 1.5 kb was amplified from the small subunit of 16S rRNA gene using primer set 16S1 (5'-GAGTTTGAT CCTGGCTCA-3') and 16S2 (5'-CGGCTACCTTGTTACGACTT-3') and an automated thermal cycler (BioRad, USA) [33].
Sequencing and phylogenetic analysis PCR products were purified (Chromous Biotech, India) and both the strands were directly sequenced on an ABI 3500xl Genetic Analyzer (Applied Biosystems Inc. Foster City, CA). Sequences were manually checked, edited, analyzed, and aligned using BioEdit software (ver. 7.2) and Chromas Lite (ver. 2.1) and were submitted to the GenBank under the accession numbers (KU550135 to KU550185). The sequences obtained in our study were compared with GenBank database using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST) and the EzTaxon server (http://www.ezbiocloud.net/eztaxon) to search the homologous sequences [34][35]. The homologous sequences were retrieved from the Genbank, and aligned using ClustalW program. Phylogenetic relatedness among 87 sequences (including 36 reference) was determined by tree reconstructed using Neighbor-Joining method (Kimura-2 parameter for distance calculation), incorporated in MEGA 6.0 package [36]. Robustness of the phylogenetic tree was examined through 1000 bootstrap replicates, and the consensus tree was used for analysis.

Statistical analysis
A non-parametric, Friedman test was performed to estimate the differences in prevalence of bacterial species between sugar fed, blood fed, male and larvae samples of Ae. albopictus midguts (p < 0.05, 95% confidence interval). Diversity of isolated bacterial species from the midgut of all samples was analyzed using various indices i.e. Simpson Index [37], Shannon Index, and Evenness [38]. The biodiversity was studied for the determination of diversity richness, evenness and dominance of obtained bacterial species from each sample categories [31]. Good's coverage was calculated by using the formula (1-n/N) Ã 100, where n represents a single bacterial isolate and N denotes total bacterial isolates from one mosquito species [39].

Results
With an aim to examine the diversity of midgut microbiota in various life stages of Ae. albopictus, we collected and isolated cultivable bacteria from the midgut of larvae, male, sugar fed female and blood fed female mosquitoes, collected from Tezpur, Assam. Based on colony morphological characteristics, we selected 113 bacterial isolates for 16S rRNA gene sequence based identification. From all the categories of individuals, we could identify 35 distinct bacterial species from 18 genera, which belonged to four major phyla namely Proteobacteria (40.0%), Firmicutes (40.0%), Actinobacteria (17.14%), and Bacteroidetes (2.86%) (Fig 1 and S1 Table).
The most abundant bacterial species in the sugar fed females and larvae individuals was Acinetobacter pittii with an abundance of 22.22% and 14.71% respectively, while Pseudomonas monteilii (19.35%) was the most abundant species in the blood fed individuals, and Pantoea dispersa (19.04%) in the adult males. All these bacterial species belonged to the phylum Proteobacteria. The phylum Bacteroidetes was represented by only a single bacterial species, Chryseobacterium rhizoplanae, isolated and identified only from blood fed individuals.

Distribution of bacterial species in different stages
Bacterial isolates from midgut of sugar fed Ae. Albopictus. A total of 13 different bacterial species from 10 genera were identified from the midgut of sugar fed Ae. albopictus, which belonged to three major phyla namely, Proteobacteria (46.15%), Firmicutes (30.77%) and Actinobacteria (23.08%). Among the phylum Proteobacteria, bacterial species belonging to the class Gamma Proteobacteria was dominant (38.46%), followed by Beta Proteobacteria (7.69%) (Figs 1 and 2). All the bacterial species of sugar fed individuals were classified under nine different families in which Micrococcaceae (23.08%) was the most abundant, followed by Enterobacteriaceae, Staphylococcaceae, Aerococcaceae, Bacillaceae, Comamonadaceae, Moraxellaceae, Pseudomonadaceae, and Xanthomonadaceae (Fig 3). Among all bacterial isolates from the sugar fed mosquitoes, Acinetobacter pittii was the dominant species followed by Enterobacter asburiae, Micrococcus endophyticus, Aerococcus viridians, Staphylococcus cohnii, Pseudomonas Bacterial isolates from midgut of blood fed Ae. Albopictus. A total of ten different bacterial species from six genera were isolated and identified from the midgut of blood fed Ae. albopictus, which belonged to four phyla, Proteobacteria (50.00%), Firmicutes (30.00%), Actinobacteria (10.00%) and Bacteroidetes (10.00%). As in sugar fed individuals, Firmicutes was the second largest phylum with three representative species. Among the phylum Proteobacteria, Gamma Proteobacteria (40.00%) was the dominant class, followed by Beta Proteobacteria (10.00%). Only single species of Micrococcus yunnanensis and Chryseobacterium rhizoplanae were identified from Phyla Actinobacteria and Bacteroidetes, respectively (Figs 1 and 2). All the bacterial species identified in the blood fed individuals belonged to 6 different families, in which Staphylococcaceae (30.00%) constituted most of the species, followed by Pseudomonadaceae, Xanthomonadaceae, Comamonadaceae, Flavobacteriaceae and Micrococcaceae (Fig 3). Two genera Staphylococcus and Pseudomonas were dominant, accounting for 60% species, rest belonged to Micrococcus, Chryseobacterium, Stenotrophomonas and Delftia. Among all the bacterial isolates, Pseudomonas monteilii was the dominant bacterial species followed by Staphylococcus cohnii, Staphylococcus pasteuri, Chryseobacterium rhizoplanae, Stenotrophomonas maltophilia, Delftia lacustris, Staphylococcus saprophyticus, Pseudomonas mosselii, Micrococcus yunnanensis and Pseudomonas geniculata ( Fig 4B).
Bacterial isolates from midgut of male Ae. Albopictus. A total of 12 bacterial species of eight distinct genera were identified from male adult mosquitoes. Similar to the sugar fed mosquitoes, all isolates belonged to three phyla, namely Proteobacteria (41.67%), Firmicutes (41.67%), and Actinobacteria (16.67%). However, unlike sugar fed mosquitoes, in the male individuals, about 83.33% species (10 out of 12 species) belonged to only two major Phyla Proteobacteria and Firmicutes. Only two bacterial species, Micrococcus yunnanensis and Kocuria palustris were represented by phylum Actinobacteria (Figs 1 and 2). Similar to the blood fed individuals, the bacterial species of the adult males were also classifiable within 6 different families, in which the Enterobacteriaceae (25.00%) and Staphylococcaceae (25.00%) were the most abundant, followed by representatives from four different families namely Bacillaceae, Micrococcaceae, Moraxellaceae and Pseudomonadaceae (Fig 3). With three different species, genus Staphylococcus (25.00%) was most abundant, followed by Bacillus (16.67%) and Enterobacter (16.67%). Of the eight genera, these three genera cover about 58.33% species and rest 41.67% species belonging to five other genera. Among all isolated bacterial species, the Pantoea dispersa was dominant followed by Enterobacter xiangfangensis, Staphylococcus warneri, Staphylococcus hominis, Pseudomonas aeruginosa, Acinetobacter pittii, Micrococcus yunnanensis, Kocuria palustris, Bacillus subtilis, Staphylococcus arlettae, Bacillus aerophilus and Enterobacter asburiae (Fig 4C).

Discussion
Aedes aegypti and Ae. albopictus are important vectors, and are responsible for transmission of arboviruses, globally [40]. Specifically, Ae. albopictus mosquitoes have been demonstrated to rapidly expand their territory, worldwide [41]. Due to its sturdy nature and tolerance to a broader temperature range, it is an ideal species for different climatic conditions [42]. The midgut microbiota has been shown to affect the host-pathogen interaction, ultimately influencing the potency of disease transmission of the vectors [43][44][45][46][47]. In this study, we aimed to explore the varying diversity of midgut microbiota of Ae. albopictus at different stages of the life cycle.
In this study, using a combination of culture and 16S rRNA sequence based methods, we could isolate and identify a total of 113 midgut bacterial isolates, belonging to four major phyla of bacteria, namely Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Interestingly, representatives from phyla Proteobacteria, Firmicutes, and Actinobacteria could be isolated from all the life stages of the sampled individuals, but bacterial species belonging to the phylum Bacteroidetes were isolated from the blood fed mosquitoes only.
It is thought that the midgut bacteria is generally acquired through vertical inheritance as well as from the surrounding environments [11,17,[48][49]. Recently, Buck and colleagues, based on their studies, proposed that the diversity of mosquito-associated microbiota is a reflection of acquisition through various environments [49]. Additionally, diversity of the midgut microbiota is known to vary according to the life stages of the mosquitoes [11]. A large number of bacterial genera identified in this study, such as Acinetobacter, Microbacterium, Micrococcus, Stenotrophomonas, Klebsiella, Pseudomonas, Enterobacter, Aeromonas, Clostridium and Bacillus have already been reported to be common in environments, where mosquitoes breed and have also been reported to be ingested by the larvae and passed on to the adults [19,50,51]. Moreover, additional bacterial inoculation of the midgut could happen at the adult stages through the horizontal transfer from breeding sites [10,51]. Among the bacterial species isolated from the larvae, a number of species were common to adult mosquitoes, such as A. pittii, B. subtilis subsp. subtilis, K. palustris, P. geniculata, P. monteilii, S. cohnii in the sugar fed adult females, P. geniculata, P. monteilii, S. cohnii, S. pasteuri in blood fed adult females while A. pittii, B. subtilis subsp. subtilis, K. palustris in the adult males. However, more than half of the species such as A. veronii, B. aryabhattai, B. cereus, B. subtilis  subsp. inaquosorum, C. sporogenes, J. hoylei, K. pneumonia, L. kyeonggiensis, M. paraoxydans were specific to larval individuals. This difference in the midgut microbiota diversity at different life stages may be attributable to a complex interplay between the environment and feeding habits, which in turn plays an important role in the metabolism and development of the life stages [11,17,28,49,52].
Of the bacterial isolates identified in the present study, several genera such as the Enterobacter, Klebsiella, Pantoea, Acinetobacter, Pseudomonas, Bacillus, Staphylococcus, Micrococcus, and Aeromonas, have commonly been isolated from the midgut of different mosquito species [17,18,21,27, Apart from the above mentioned bacterial species, during the study we isolated Leucobacter kyeonggiensis, Janibacter hoylei, Chryseobacterium rhizoplanae, Microbacterium paraoxydans, Clostridium sporogenes, which, to the best of our knowledge, have not been previously reported from mosquito's midgut. L. kyeonggiensis is a Gram-positive bacterium and was reported by Kim and Lee in 2011, as a novel species from dye waste water in Korea [63]. Although, till date L. kyeonggiensis has not been reported in insect gut, but a related species, L. holotrichiae was very recently reported from the gut of the scarab beetle larvae [64]. Another isolate, J. hoylei, a Gram-positive bacterium was identified and reported by Shivaji and colleagues, and was isolated from atmospheric samples, collected at very high altitudes (27-41 km) India [65]. C. rhizoplanae, a Gram-negative bacterial species, isolated from present blood fed individuals, was identified by Kämpfer and colleagues from rhizoplane of maize [66]. M. paraoxydans (isolated from larvae in this study) was initially reported in an acute lymphoblastic leukemia patient from Belgium and has subsequently been reported from clinical samples, as well as from fishes [67,68,69]. The bacterial species C. sporogenes (isolated from larvae in the present study) was first isolated from human faeces [70], and was subsequently reported from the gastrointestinal tract of the human and other mammalians [71][72][73].
In addition to other factors, the midgut microbiota diversity has also been shown to be related to the genders of the mosquitoes [17,20]. The results of our study tend to support this observation too. We could identify a number of bacterial species in the female mosquitoes exclusively (A. viridians, D. lacustris, C. rhizoplanae E. asburiae, K. michiganensis, M. endophyticus, P. mosselii, S. haemolyticus and S. maltophilia) and some other bacterial species specifically from males (B. aerophilus, E. cloacae, E. xiangfangensis, P. dispersa, P. aeruginosa, S. arlettae, S. hominis, S. warneri). It is well known that the midgut microbiota plays an important role in the digestion of food [57,[74][75]. Males solely depend upon plant sugars [52]. On the other hand, in addition to plant sugars, female adults require blood meal for development of their ovaries, signifying a shift from carbohydrate rich diet to protein rich diet [23][24][25][26]. This shift consequently results in increased levels of enteric bacteria, while reduction in the overall microbiota diversity [28, [75][76]. In our present study too, we documented the least diversity of midgut microbiota in the blood fed individuals, as compared to all the other category of individuals, supporting the previous findings.
The biodiversity index is used to quantify the number of different individuals and their distribution in any community. Simpson index is used to measure the probability of randomly drawn species of any two individuals from infinitely large community of different species [31,37]. The Simpson index is directly proportional to diversity. In the present study, the value of Simpson's diversity index ranged from 0.87 to 0.92 (maximum in larvae and minimum in blood fed individuals). Another widely used index for comparing the diversity between various habitats is the Shannon and the values ranged from 1.5 to 3.5. Values greater than 3, indicate rich and stable diversity of habitat, whereas values less than 1.5, indicate unstable diversity, due to the pollution and degradation of habitat structure [77]. In the present study, the value of Shannon diversity index ranged from 2.17 to 2.66 (maximum in larvae and minimum in blood fed individuals). In the previous study, lowest bacterial diversity was calculated from the blood-fed mosquitoes [28] and the value of Simpson and Shannon index in our study indicate the minimum bacterial diversity from the blood fed individuals and maximum from larvae individuals. Evenness index is used to estimate the closeness of the species and determines how well they are evenly distributed among any habitat. The values of evenness in the present study ranged from 0.82 (Sugar fed) to 0.89 (larvae), which indicated that bacterial species were evenly distributed among the larvae individuals as compared to other categories of individuals. The maximum value of Good's coverage was recorded from blood fed Ae. albopictus (93.55) and lowest from male individuals (71.43), indicating that an additional 6 and 29 operational taxonomic units (OTUs) would be found if 100 additional colonies were sequenced in these two categories [78].
It is now well understood that midgut microbiota of any species is involved in various important function including digestion of food, development, providing immunity etc [11,[61][62][74][75][79][80]. In vectors, specifically, midgut bacterial populations have also been shown to be involved in host-parasites interaction, resulting changes in the vectorial capacity [11,31,52,79]. Recent evidences also suggest a prominent role of the midgut microbiota in modulating the sporogonic development of parasites, augmentation of immunity against invading parasites, resulting in altered vectorial capacity [27,46,[59][60][79][80]. In case of Aedes mosquitoes, susceptibility to Dengue viruses has been demonstrated to increase significantly in the presence Aeromonas culicicola and Escherichia coli in the midgut [48,81], while Serratia odorifera has been shown to enhance the susceptibility of Aedes mosquitoes to Dengue and Chikungunya viruses [48,82]. Interestingly, a recent study showed three bacteria isolated from Ae. albopictus i.e, Enterobacter ludwigii, Pseudomonas rhodesiae, and Vagococcus salmoninarium inhibit La Crosse virus in vitro, suggesting an anti-viral effect of these bacterial species [83].
In the recent years, the interest in midgut-associated bacteria has increased manifolds. Although many studies have been done on different mosquito species, diversity, functions and genetic potential of bacteria associated with Aedes mosquitoes is poorly understood. In recent years the population density and geographic expansion of Aedes mosquitoes has increased worldwide, posing significant threat of transmitting viruses such as Dengue, Chikugunya, Zika etc. Therefore, the understanding of diversity of symbiotic bacteria is essential for better understanding the adaptation and vectorial capacity, as well as to develop effective vector management strategies. The midgut microbial flora of the mosquitoes is a dynamic niche changing rapidly with the development of the vectors. Thus, understanding of bacterial communities at different life stages of vectors is important to control the vectors at different developmental stages. The findings of our present work gives a glimpse of the midgut associated microbiota of the Asian Tiger mosquito Ae. albopictus, and this information could be helpful in investigating disease transmission, and control of disease outbreaks.
Supporting Information S1