|Year : 2021 | Volume
| Issue : 1 | Page : 37-46
Genetic differentiation of predominant mosquito species in Hainan province and characterization of mosquito midgut microbiota
Xun Kang1, Biao Liu2, Siping Li1, Qianfeng Xia1
1 Key Laboratory of Tropical Translational Medicine of Ministry of Education, School of Tropical Medicine and Laboratory Medicine, Hainan Medical University, Haikou 571199, China
2 Department of Clinical Laboratorial Examination, Hainan Women and Children’s Medical Center, Haikou 570206, China
|Date of Submission||22-Jul-2021|
|Date of Decision||20-Sep-2021|
|Date of Acceptance||30-Sep-2021|
|Date of Web Publication||03-Nov-2021|
Key Laboratory of Tropical Translational Medicine of Ministry of Education, School of Tropical Medicine and Laboratory Medicine, Hainan Medical University, Haikou 571199
Source of Support: None, Conflict of Interest: None
Objective: To identify the mosquito species collected in the field of 8 cities/counties of Hainan province, China and to analyze the characteristics of genetic differentiation of the predominant mosquito species. The types, contents and composition characteristics of the midgut microbiota of various mosquito species were explored to provide evidence for the control and prevention of mosquito-borne diseases in Hainan province.
Methods: Adult mosquitoes were collected in the field using light traps, human lure or cattle lure methods. Morphology and DNA-barcoding technology (COI gene) were applied to identify the mosquito species. The V3-V4 hypervariable region of microbes 16S rRNA was used for high-throughput sequencing of the midgut microbiota, and SPSS 21.0 and R v3.1.1 software were employed to perform statistical analysis of the sequencing results and the Beta diversity analysis.
Results: Aedes (Ae.) albopictus, Armigeres (Ar.) subalbatus and Culex (Cx.) pipiens pallpens were the three predominant species of the mosquito samples. Ae. albopictus was closely related to MK736660 (India) and JQ235749 (Yunnan), while MT541282 (DF, Dongfang) was a relatively independent population. The composition and structure of bacterial communities significantly varied among most of the samples (P<0.05). At the genus levels, 16 genera overlapped in the midgut in Ae. albopictus, 20 in Ar. subalbatus and 26 in Ar. subalbatus. Besides, Ae. albopictus, Ar. subalbatus and Cx. pipiens pallpens shared 15 out of the 16 overlapping genera.
Conclusions: Ae. albopictus, Ar. subalbatus and Cx. pipiens pallpens were close within the same species in Hainan. The main exchange of mosquito species occurs within Hainan province, but also occurs across provincial or even national boundaries. The gut microbiota of mosquito species collected from the same sites were similar. Most of the core bacteria found in the midgut of Ae. albopictus were also present in Ar. subalbatus and Cx. pipiens pallpens, indicating that the breeding environment of Ae. albopictus may cover the breeding environment of Ar. subalbatus and Cx. pipiens pallpens.
Keywords: Mosquito; Mosquito identification; Phylogenetic tree; 16S rRNA; Midgut microbiota
|How to cite this article:|
Kang X, Liu B, Li S, Xia Q. Genetic differentiation of predominant mosquito species in Hainan province and characterization of mosquito midgut microbiota. One Health Bull 2021;1:37-46
|How to cite this URL:|
Kang X, Liu B, Li S, Xia Q. Genetic differentiation of predominant mosquito species in Hainan province and characterization of mosquito midgut microbiota. One Health Bull [serial online] 2021 [cited 2022 Jan 18];1:37-46. Available from: http://www.johb.info/text.asp?2021/1/1/37/329030
| 1. Introduction|| |
Mosquitoes are closely related to human life, can transmit various pathogens such as Zika virus, plasmodium and dengue virus by biting humans and other vertebrates[1-3]. The rapid developments of science, technology and economy have greatly promoted the economic and trade cooperation and people flow between continents and countries in the world. As a result, the reproduction and transmission of mosquitoes and the spread of mosquito-borne diseases are not limited in the specific regions, but across regions and even across the oceanic plates, triggering the outbreaks of infectious diseases. During the migration, the survival of mosquito species is affected by multiple factors such as environment, climate (temperature and humidity, etc.), food chain, mosquito control measures and local human settlements. According to the rules of species evolution, the structure of mosquito species is bound to have ebb and flow, genetic mutation, changes in the ability to carry pathogens and may even be under the risk of being affected with the new virus, which leads to a huge challenge to the public health around the world.
During the life cycle of mosquitoes, as the first important line of defense against pathogenic organisms, the gut colonizes a large number of microbes, including plankton (Water algae, etc.), bacteria and fungi, etc. At present, bacteria have received more attention than other aspects of research on the gut microbiota of mosquito species. From 2007, with the launch of the Human Microbiome Project, many studies have confirmed that the bacterial communities are closely related to the life activities of the host in many ways,. Mosquitoes mainly live and breed in stagnant water before they mature into adults. Different mosquito species have different requirements for water quality and the environment of adult mosquitoes tends to be more complicated and changeable than that of larval mosquitoes because adult mosquitoes can fly. The source and type of food are different in different mosquito species and stages in development and female mosquitoes require to suck blood of human and vertebrate to fertilize their eggs for reproduction,. The relatively special food chain also provides a necessary way for pathogenic microorganisms to spread among hosts.
Most of the bacteria found in the gut of mosquito species are obtained through the feeding channels, and only a small part is obtained through vertical transmission. Large number of surveys and studies confirmed that habitat was one of the most important factors influencing the structure of the gut of mosquitoes,,,. The gut bacterial communities played a very important role in all the stages of growth and development which was to provide the nutrients to the life activities through itself and secondary metabolites,. Studies have found that the eggs of Aedes (Ae.) aegypti could not develop into adults in sterile environment, and the normal growth and development could be restored after yeast was added,, and increasing the abundance of bacteria in the food for Anopheles could accelerate the growth and development of larvae. It was also found that the preparation of the subspecies of Bacillus thuringiensis Israel has the effect of inhibiting the growth and development of chironomid mosquitoes, and also has an immune regulation on mosquito species,. The gut bacterial communities activated the expression of intestinal immune genes by infecting mosquito species, and the efficacy of Anopheles against Plasmodium infection also changed after being infected by different Serratia. Studies further found that Serratia could also promote the susceptibility of dengue virus, Zika virus, and chikungunya virus to mosquitoes[21-23]. The expression of various antimicrobial peptide genes of the gut epithelial cells of Ae. aegypti could be activated after being infected by Proteus, which can enhance the mosquitoes’ immunity to viruses. Therefore, the study on the midgut bacterial communities was particularly important to the control and prevention of mosquito-borne infectious diseases.
Hainan province is located in southern China and has a tropical marine climate with an annual average temperature of 24.2 °C and an annual average rainfall of 1 684 mm which is suitable for the growth and reproduction of mosquito species. In addition, mosquito species are abundant here with 5 genera and 23 mosquito species reported. Under the context of country’s strong support for the construction of Hainan Free Trade Port, the frequent movement of people, tourism and trade have further facilitated the importation of mosquito-borne infectious diseases, greatly increasing the risk of disease transmission and bringing severe challenges to the control and prevention of infectious diseases.
Currently, researches on local mosquito vectors in Hainan mainly focused on the prevention and control of mosquito species, population surveys and pathogen screening, while there were relatively few studies on the diversity of the midgut bacterial communities based on mosquito species identification. In this study, we used morphology and COI gene to identify the species of field-collected adult mosquitoes in Hainan, in order to detected the species of the midgut microbial communities by V3-V4 of 16S rRNA. Chi-square and other relevant statistical methods were performed to analyze the data, and Mega 7.0 software was applied to compare the genetic differentiation of the obtained gene sequence of mosquito species on Hainan with the sequence of the predominant mosquito species at home and abroad downloaded through the NCBI platform. This study explored the characteristics, structure and composition of the midgut bacterial communities of different mosquito species in different regions of Hainan province, providing a fundamental basis for control and prevention of mosquito-borne infectious diseases.
| 2. Materials and methods|| |
2.1. Sampling materials
Kong Fu Xiao Shuai mosquito lamp trap (LTS-MO2, Ji Xing Medical Technology Co., Ltd., Wuhan), Tissue Grinder (SCIENTZ-48, Scientz Bio Co., Ltd., Ningbo), Thermal cycler (Biometra Tone 96, Jena, Germany), Anatomical microscope (AE200, MOTIC), Mighty Prep reagent for DNA (Cat#9182, TaKaRa Bio Co. Ltd., Dalian), DNeasy Blood & Tissue Kit (QIAGEN# 69506, Nobleyder Technology Co. Ltd., Beijing), MasterMin (BL547A, Labgic Technology Co. Ltd., Beijing), COI forward/reverseprimer (Tian Yi Hui Yuan Biotechnology Co. Ltd., Guangzhou), 50 TAE buffer, 2 KAPA HiFi Mix, Agarose (Carnoss Technology Co., Ltd., Wuhan), Gold View (EP1501, Biosharp), 75% alcohol, absolute alcohol, etc.
2.2. Sampling location
Adult mosquitoes were collected from July to September in 2018. Hainan’s landforms such as plains, woods, coastal areas, as well as residential areas and construction sites were selected as sampling locations in the four geographic areas of eastern, northern, southern, and central Hainan province. Finally, 2-3 villages located in Lingshui, Dingan, Wenchang, Haikou, Ledong, Sanya, Wuzhishan and Tunchang, respectively were chosen as the sampling sites [Figure 1] and [Supplementary Table 1]. In each sampling sites, we spent 2-3 days to collect the samples.
|Figure 1: Distribution of sampling sites for mosquito species. Ae.=Aedes; Ar.=Armigeres; Cx.=Culex.|
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2.3. Mosquito collection and sample processing
The adult mosquitoes were collected using lamp trap, human lure or cattle lure. Placed the adult mosquitoes on the ice and put them in a petri dish after being faint, identified the mosquito species by morphology. Put single female mosquitoes into 2 mL cryo-storage tubes, marked the sampling time, location, and mosquito species type on the tube wall, then submerged the tubes in liquid nitrogen for storage, and transported it back to the laboratory for further processing.
2.4. Preparation and detection of mosquito DNA
The mosquitoes were removed from the liquid nitrogen, rinsed three times in sterile water, and was surface sterilized with 75% ethanol for 10 min, then rinsed five times in sterile water and once in 0.8% sterile saline, then the mosquitoes were put it in petri dishes and dissected under a dissecting microscope. At least two or more mosquito legs of each mosquito species were kept for further analysis. The legs of each mosquito species were immersed in the wells of a 96-well plate filled with 95% ethanol, marked, and stored at 4 °C to prepare the mosquito species genome. The midgut of the corresponding single mosquito species was dissected, and the midgut of each mosquito species was labeled with a cryotube and stored at –80 °C for later use.
Removed 2-4 legs of each single mosquito from 95% ethanol, immersed them in the EP tube containing 100 μL of Mighty Prep reagent for DNA liquid, then heated at 95 °C for 10 min, centrifuged at 12 000 rpm (13 201 g) for 2 min, finally took the supernatant as a PCR reaction template, and used universal primers designed by multicellular invertebrate COI genes according to references. The expected PCR amplified band fragment size is about 650 bp, the forward primer LOC1490: 5’-GGTCAACAAATCATAAAGATATTGG-3’, and the reverse primer HCO2198: 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’. Reaction system 50 μL: Mix 25 μL, ddH2O 22.5 μL, R 0.75 μL, F 0.75 μL, DNA 1 μL. Program settings: initial denaturation at 94 °C for 5 min, denaturation at 94 °C for 1 min, annealing at 45 °C for 1 min, extension at 72 °C for 1 min, 35 cycles, final extension at 72 °C for 10 min, and storage at 12 °C. The PCR product was separated by electrophoresis on a 1% agarose gel, and the PCR result was confirmed to be positive. The PCR product was sent to Beijing BoyunHuakang Gene Technology Co., Ltd. for sequencing. After obtaining the COI gene sequencing results, the COI gene sequence was used to identify the mosquito species at molecular level by comparing to the BLAST at the National Center for Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). Then the sequence was uploaded to NCBI to obtain the GenBank accession number.
2.5. Phylogenetic tree analysis
Mega 7.0 software was used to build a phylogenetic tree based on the nucleotide sequence of COI gene. After 1 000 Bootstrap method tests, Neighboring-joining tree method was used (CI 70%).
2.6. Midgut DNA preparation and sequencing
According to the results of morphology and molecular identification, 30 qualified midguts of various mosquito species at each sampling site were used for further analysis. The midguts of every 10 mosquito species were combined into one group, and three groups were repeated. The DNA extraction kit (QIAGEN: 69506) was used to extract the total bacterial genome of the midgut of the combined mosquito species which were sent to Beijing BoyunHuakang Gene Technology Co., Ltd. for analyzing the midgut microbiome 16S rRNA gene V3-V4 hypervariable region high-throughput sequencing via HiSeq 4000 system.
2.7. 16S rRNA biological information and statistical analysis
Green gene database was used for comparison and species annotation, Excel 2019 software for sorting out the data, R v3.1.1 for analyzing the beta diversity of the bacterial communities, SPSS 21.0 software for χ2 test for comparisons of frequencies and rates. P-value less than 0.05 was considered as statistically significant.
| 3. Results|| |
3.1. COI gene amplification electrophoreses
The size of the vitro amplified band of the COI gene of the mosquito species is about 650 bp. The gel electrophoreses results of representative samples from each region are shown in Supplementary [Figure 1]. According to the COI gene sequencing results, the NCBI public database sharing platform was used to perform BLAST molecular comparison and identification of nucleic acid sequences, combing with the morphological identification results. The results showed that the mosquito species samples were Ae. albopictus, Ar. subalbatus and Cx. pipiens pallpens, respectively. Uploaded to the NCBI database and got the GenBank accession number of the representative sequence of each region assigned by the platform. Ae. albopictus: MT890464 (HK), MT906353 (TC), MT906351 (SY), MT906350 (WC), MT890489 (DA), MT906156 (LS), MT890465 (LD), MT906352 (WZS); Ar. subalbatus: MW446161 (HK), MW446163 (WC), MW446158 (SY), MW446159 (WZS); Cx. pipiens pallpens: MW446164 (HK), MW446160 (WZS). According to the results of mosquito species identification, 10 mosquito species were collected in each group from each sampling site, combined with midgut, and three biological repetitions were performed.
The representative sequences of mosquito species were searched and downloaded in Genbank, which were Ae. albopictus: JN406730 (Madagascar), MK736660 (India), MG198604 (Russia), JQ235749 (Yunnan, China), KX886325 (Henan, China); Ar. subalbatus: MK644935 (American), KC970289 (India), KJ768190 (Pakistan), JQ728219 (Yunnan, China), MF179278 (Guangdong, China); Cx. pipiens pallpens: MK033269 (Madagascar), MK402922 (Spain), KF406803 (Pakistan), MF278809 (Chongqing, China), MF179182 (Guangdong, China). The representative sequences of other regions in Hainan province, which were Ae. albopictus: MT541249 (Lingao, LG), MT541261 (Danzhou, DZ), MT541282 (Dongfang, DF), MT541303 (Qionghai, QH), MT541318 (Wanning, WN); Ar. subalbatus: MT541045 (DZ), MT541078 (LS); Cx. pipiens pallpens: MT566522 (SY), MT566586 (LD), MT566640 (DZ), MT566752 (DF), MT566778 (QH). Mega 7.0 software was used to build phylogenetic trees with Neighbor-joining, Anopheles vagus MH425409 was used as the out-group, and phylogenetic trees were constructed based on the nucleotide sequence of COI gene for each species. After 1 000 Bootstrap method tests, we selected the Neighboring-joining tree method (CI 70%).
3.2. Phylogenetic analysis of Ae. albopictus
The phylogenetic tree of COI gene sequence was established by using neighbor-joining method. The phylogenetic tree showed that JN406730 (Madagascar), KX886325 (Henan), MG198604 (Russia), MT541282 (DF), and mosquito species from other regions were assigned to 4 main groups, respectively. Among them, relationship between MT906352 (WZS) and MT906351 (SY) as well as between MT890489 (DA) and MT906350 (WC) were the closest, while MT541261 (DZ) was relatively closed to MK736660 (India), JQ235749 (Yunnan), MT890456 (LD), and MT890464 (HK). However, only MT541282 (DF) was an independent group. What is more interesting is that MK736660 (India) and JQ235749 (Yunnan) were also closely related to Ae. albopictus from Hainan province [Figure 2]A.
|Figure 2: (A) Ae. albopictus neighbor-joining (NJ) genetic phylogenetic tree constructed on the basis of COI. Sampled mosquito species, shown in Latin (region, mosquito species, GenBank registration number). (B) Ar. Subalbatus NJ genetic phylogenetic tree constructed on the basis of COI. Sampled mosquito species, shown in Latin (region, mosquito species, GenBank registration number). (C) Cx.pipiens pallpens NJ genetic phylogenetic tree constructed on the basis of COI. Sampled mosquito species, shown in Latin (region, mosquito species, GenBank registration number).|
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3.3. Phylogenetic analysis of Ar. subalbatus
The representative sequences of Ar. subalbatus extracted in this study, MW446161 (HK), MW446163 (WC), MW446159 (WZS), MW446158 (SY), were compared with mosquito species in other regions. The relationship between MW446163 (WC) and MT541045 (DZ) was found to be the closest with stable reconstruction. MF179278 (Guangdong), MK644935 (American), MW446159 (WZS), and MW446161 (HK) were closely related to the above two mosquito species, while MW446158 (SY) and MT541078 (LS) were an independent group, respectively. The genetic relationships among KJ768190 (Pakistan), KC970289 (India), and JQ728219 (Yunnan) were relatively distant, which was consistent with the physical distances among each sampling site [Figure 2]B.
3.4. Analysis of Cx. pipiens pallpens phylogenetic trees
Culex species from Hainan had a relatively close genetic relationship. More interestingly, KF406803 (Pakistan) and MW446164 (HK) had a closer genetic relationship than the species in other regions. MT566522 (SY), MT566586 (LD), KF406803 (Pakistan), MF179182 (Guangdong), MT566752 (DF) belonged to the same group, while MW446160 (WZS) was distantly related to MW446164 (HK). According to the physical distance among regions, the genetic relationship among MF278809 (Chongqing), MK402922 (Spain), and MK033269 (Madagascar) was more distant [Figure 2]C.
| 4. Diversity analysis of midgut|| |
4.1. Beta diversity analysis of midgut bactertia flora of Ae. albopictus
Beta analysis was used to compare the differences in species composition among samples. Heatmap of the Bray-Curtis matrix in [Figure 3]A showed that CB of WZS, LM and XHX of LD, and CW of HK were quite different from other regions. The midgut bacterial flora of XHX of LD and WC of WC also showed significantly different structures from flora of different regions, while the structure of the midgut bacterial flora of GLZ of LS, FJ of DA, NB and CC of SY showed little difference.
|Figure 3: (A) Heatmap of the Bray-Curtis array of Ae. albopictus midgut microbiota in each of the eight sampling areas. (B) Heatmap of the Bray-Curtis array of Ar. subalbatus midgut microbiota in each of the four sampling areas. (C) Heatmap of the Bray-Curtis array of Cx. pipiens pallpens midgut microbiota in each of the two sampling areas. The label on the right side of [Figure 3] indicates the value is closer to 1, the greater the difference between the samples, values closer to 0 indicate the most similar samples.|
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Through clustering analysis of the composition of the sample bacterial flora, the Bray-Curtis matrix at the top of [Figure 3]A showed that all the tested samples were clustered into two main groups, with JB of WC, CB of WZS, LM and XHX of LD as a large group, and the other samples as the other group. Flora from different sampling sites in the same sampling area had different bacterial structures, but within the same sampling sites, the bacterial structures of repeated groups were similar.
4.2. Beta diversity analysis of midgut bactertia flora of Ar. subalbatus
As shown in [Figure 3]B, the bacterial structure of Ar. subalbatus midgut samples collected at each sampling site was quite different, especially for NY of SY, SW of HK, and WZS of WZS. Secondly, HS of WC and WZSO of WZS were quite different, and the bacterial structure was also very different between HS and TR of WC sampling site. It also showed that all the tested samples were clustered into three large groups: WZSO of WZS and WC, TR and HS of WC; CHO and WZS of WZS; SW of HK and NY of SY. The structure of the midgut bacterial flora of Ar. subalbatus was also different at different sampling sites in the same sampling area. Different sampling sites in the same sampling area had different bacterial structures, but within the same sampling sites, the bacterial structures of repeated groups were similar.
4.3. Beta diversity analysis of midgut bacterial flora of Cx.pipiens pallpens
The midgut samples collected in 3 sampling sites of 2 sampling areas were detected and analyzed. As shown in [Figure 3]C, the structures of midgut bacterial flora in 3 sampling sites were significantly different, and the differences of each detecting group in XH and XY sampling sites of HK were also significant, while the bacterial structure of each group of WZSCB was similar.
As shown in [Figure 3]C, all the tested samples were clustered into two main groups. The three repeatedly tested groups of WZSCB were clustered into a group, the XH and XY sampling sites of HK were clustered into a group. The tested samples at the WZSCB sampling site were highly similar, and there were certain differences among the tested samples of HK.
4.4. Abundance analysis of midgut bacteria of different mosquito species at geneus level
All midgut samples were detected by Illumina’s Hiseq platform. According to the abundance ratio of bacteria genera obtained from each sample, 41 bacterial genera were detected [Figure 4]. Of which, the largest genera was found in Ae. albopictus (39) from LD, followed by HK’s Cx. pipiens pallpens (37), Ae. albopictus (36), Cx. pipiens pallpens (35) of HK, Ae. albopictus (36) of SY, Ae. albopictus (36) of WC, Ar. subalbatus (31) and Ar. subalbatus (31) of WZS. Ae. albopictus, Ar. subalbatus and Cx. pipiens pallpens of HK had a similar number of bacterial genera, with 36, 35, and 37, respectively. Ar. subalbatus of SY (23) had fewer genera, followed by Ae.albopictus of TC (24) and Ae. albopictus of WZS (24). According to [Figure 4], Wolbachia was the dominant bacterium with a higher abundance. Its distribution in the midgut of Ae. albopictus was as follows: DA (84.69%), HK (56.89%), LS (68.77%), SY (84.34%), TC (38.28%), WC (31.57%), 23.16% in Ar. subalbatus of HK, and 93.81% in SY. Among the three mosquito species tested for WZS, Wolbachia was found in 0.36% of Ar. subalbatus, and less than 0.01% of Ae. albopictus and Cx. pipiens pallpens. However, it was interesting that 60.30% and 23.81% of bacterial genera in Ae. albopictus and Ar. subalbatus of WZS, respectively could not be classified. More particularly, 99.14% of bacterial genera in Cx. pipiens pallpens failed to be identified. In the midgut sample of Ae. albopictus of WZS, Enterococcus accounted for 38.41%, In Ar. subalbatus Enterococcus and Enterobacter accounted for 16.03% and 7.30%, respectively. Besides, Elizabethkingia accounted for 19.84% in Ar. subalbatus, which was different from other mosquito species. In the Ar. subalbatus of WZS, Staphylococcus accounted for 5.52%. The unrecognized bacterial genera with a large proportion were found in Cx. pipiens pallpens of HK (27.57%), Ae. albopictus (48.31%) of TC, and Ae. albopictus (27.50%) of WC.
|Figure 4: Taxonomic abundance of midgut microbiota of mosquito species. The right column shows a color (letter) to indicate a genus of bacteria, and the value in the figure is the percentage of the abundance of the bacterial group (marked with a value of 5% and above).|
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Among the tested samples, Actinomyces accounted for 10.99% in Ae. albopictus of HK, Acinetobacter accounted for 8.30%, Thorsellia accounted for 39.50% in Ar. subalbatus of HK, Escherichia accounted for 5.82%. Swaminathania accounted for 13.36% and Serratia accounted for 7.05% in Ae. albopictus of LS, Enterobacter accounted for 7.09% in Ae. albopictus of SY, Swaminathania accounted for 10.13% in Ae. albopictus of TC and Enterococcus accounted for 14.59% in Ae. abopictus of WC. Especially, Enterococcus accounted for 47.55% in Ar. subalbatus of WC, Dysgonomonas accounted for 45.52%. Providencia (22.78%), Enterococcus (16.03%), Enterobacter (7.30%), Elizabethkingia (19.84%), and Staphylococcus (5.52%) accounted for a relatively high proportion in Ar. subalbatus of WZS.
On the whole, the distribution proportion of a single genus in Ae. albopictus of DA, HK and SY and Ar. subalbatus of SY was relatively higher, with abundance of a single genus higher than 5%. Ae. albopictus (3), Ar. subalbatus (3) and Cx. pipiens pallpens (4) of HK contained three or more bacterial genera Ae. albopictus (5) of LD and Ar. subalbatus (5) of WZS had a relatively uniform distribution of genera in the midgut.
The abundance of the midgut bacterial flora in each region was analyzed by the χ2 test, and it was found that the differences in the composition of the bacterial floras in most samples were statistically significant (P<0.05) [Table 1]. There was no significant difference between Ae. albopictus of DA and that of HK, LS and SY (P>0.05), Ae. albopictus of DA and Ar. subalbatus of SY (P>0.05), Ae. albopictus of LS and Ae. albopictus, Ar. subalbatus of SY (P>0.05), Ae. albopictus and Ar. subalbatus of SY (P>0.05), Ae. albopictus of SY and Ae. albopictus of TC (P>0.05).
|Table 1: Abundance of midgut bacteria in different regions of Hainan province.|
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4.5. Venn analysis of midgut bacterial genera in the same mosquito species from different regions
We analyzed the midgut bacterial genera in the same species from different regions using Venn diagram. There were 16 overlapping species in the midgut of Ae. albopictus collected in 8 regions [Figure 5]A, 20 overlapping species in the midgut of Ar. subalbatus in 4 regions, 26 overlapping species in the midgut of Ar. subalbatus in 2 regions [Figure 5]B. Cx. pipiens pallpens had the most overlapping bacteria genera among the three mosquito species [Figure 5]C. Of the 16 overlapping bacteria genera that appeared in Ae. albopictus, 15 of them also appeared in Ar. subalbatus and Cx. pipiens pallpens, respectively: Acinetobacter, Asticcacaulis, Bacillus, Brevibacillus, Carnobacterium, Elizabethkingia, Enterobacter, Enterococcus, Escherichia, Paenibacillus, Serratia, Sphingomonas, Staphylococcus, Thermus, Wolbachia.
|Figure 5: (A) Venn diagram of the taxonomy of the midgut microbiota of Ae. albopictus in different regions. The numerical value in the figure on the left is the number of bacterial species and the one on the right is the name of the main bacterial genus. 16 overlapping species in the midgut: Acinetobacter, Asticcacaulis, Bacillus, Brevibacillus, Carnobacterium, Elizabethkingia, Enterobacter, Enterococcus, Escherichia, Pacnibacillus, Pseudomonas, Serratia, Sphingomonas, Staphylococcus, Thermus, Wolbachia. (B) Venn diagram of the taxonomy of the midgut microbiota of Ar. subalbatus in different regions. The numerical value in the figure on the left is the number of bacterial species and the one on the right is the name of the main bacterial genus. 20 overlapping species in the midgut: Acinetobacter, Asticcacaulis, Bacillus, Brevibacillus, Carnobacterium, Chryseobacterium, Comamonas, Elizabethkingia, Enterobacter, Enterococcus, Escherichia, Morganella, Paenibacillus, Providencia, Pseudomonas, Serratia, Sphingomonas, Staphylococcus, Thermus, Wolbachia. (C) Venn diagram of the taxonomy of the midgut microbiota of Cx. pipiens pallpens in different regions. The numerical value in the figure on the left is the number of bacterial species and the one on the right is the name of the main bacterial genus. 26 overlapping species in the midgut: Acidovorax, Acinetobacter, Actinomycetospora, Asticcacaulis, Bacillus, Brevibacillus, Carnobacterium, Chryseobacterium, Comamonas, Elizabethkingia, Enterobacter, Enterococcus, Erwinia.|
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| 5. Discussion|| |
In this study, Ae. albopictus collected in 8 cities and counties, Ar. subalbatus collected in 4 cities and counties and Cx. pipiens pallpens collected in 2 cities and counties in Hainan province were used for molecular identification of COI gene, and a phylogenetic tree was constructed using Mega 7.0 software to analyze and trace the nucleotide sequence of COI gene of mosquito species. Ae. albopictus, also known as the “Asian tiger mosquito”, originates from Asia. The result shows that most of the Ae. albopictus in Hainan province cluster together except in Dongfang. It indicates that the population communication of Ae. albopictus in Hainan mainly occurs on the island, but Ae. albopictus in India also has a relatively close relationship with that in Hainan province. The genetic differentiation distance of the population communication in the island was consistent with the physical distance of the geographical distribution, and the genetic relationship of mosquito species in neighboring areas was relatively close. Dongfang is different from other regions. Since it is a coastal city and main port, mosquito species from overseas could invade the local mosquito species due to logistics in Dongfang. Besides, coastal ports have been regarded as the main source of invasion of extraterritorial mosquito species and pathogens.
Ar. subalbatus belongs to the genus Armigera and could carry and transmit filarial worm and encephalitis virus [30, 31]. The phylogenetic analysis of Ar. subalbatus in this study indicates that the genetic relationship of Ar. subalbatus mainly cycles in the province, but it is still possible to communicate with the mosquito species outside the province, and it is also closely related to the mosquito species of Guangdong and American. Ar. subalbatus is known as the “odoriferous water mosquito” due to its relatively concentrated breeding environment and special living environment where is dirty and humid. This study does not reflect the significant relationship between the genetic relationship of Ar. subalbatus in the province and the physical distance between regions.
The phylogenetic analysis of Cx. pipiens pallpens in Hainan province showed that the traceability result of Cx. pipien spallpens was similar to that of Ar. subalbatus, mainly cycled in the province. The genetic distance of Cx. pipiens pallpens collected in HK and WZS was much farther than that of the other parts of Hainan province, which is consistent with the physical distance between the two sampling sites. Besides, climates are also different and multiple factors would affect the communication between mosquito species. One thing to point out is that Cx. pipiens pallpens in all regions of Hainan province are also relatively close to those in Pakistan, Guangdong, and Spain.
Hainan province is an independent island, physically separated from other regions. The tracing of the COI gene of mosquito species showed that mosquito species in most areas were closely related in the island, especially the results of the phylogenetic tree of Ae. albopictus were more obvious, which was consistent with its strong environmental adaptability. Compared with Ae.albopictus, Ar. subalbatus and Cx. pipiens pallpens had relatively little communication on the island because the living environment of these two mosquito species is relatively stable, and they are less disturbed by external factors. However, it was found that the genetic distance was also relatively close between these two mosquito species and the species outside the region, which could be due to logistics. In conclusion, Ae. albopictus, Ar. subalbatus and Cx. pipiens pallpens in Hainan province are relatively closely related within the same species, mainly communicate within the province, and also communicate with the species outside the region. It is suggested that there is a greater risk of imported mosquito species from outside the territory, and the monitoring and early warning of mosquito species and related pathogens should be strengthened.
The Bray-Curtis matrix diagram showed that in Ae.albopictus, the CB of WZS, LM and XHX of LD, and CW of HK were significantly different from those of other regions. The composition of midgut bacterial flora of the XHX of LD and the WC of WC was quite different from other regions, it is because that WZS is located in the central mountainous area of Hainan province and has relatively little economic and trade exchanges with the outside world; LD is located in the southwest and has a longer coastline; HK and WC are located in the north, with denser population and frequent foreign economic and trade exchanges with outside. Ar. subalbatus and Cx. pipiens pallpens collected from different regions were all significantly different, because of the geographical location, climate and local cultural and economic structure. All of the above factors have certain effects on the survival of mosquito species, thus affecting the structure of midgut bacterial flora of mosquito species, and the differences are more obvious in different regions, which is similar to the results of other researches.
In this study, a total of 41 genera were detected through the high-throughput detection of the V3-V4 hypervariable region of the 16S rRNA of the mosquito species gut bacterial flora. Compared with about 200 species that had been identified worldwide, the number is slightly lower, but it is still considerable compared with the gut bacterial flora of a single mosquito species. The Venn diagram showed visually the common and unique bacterial genera among multiple samples so that to find the microbiota of the different samples. In this study, Venn analysis at the genus level found that there were 16 overlapping midgut species of Ae.albopictus collected from 8 regions, and 20 overlapping midgut species of Ar. subalbatus collected from 4 regions, 26 overlapping midgut species of Ar. subalbatus collected in two regions. It was interesting that out of 16 overlapping genera that appeared in Ae. albopictus, 15 appeared in Ar. subalbatus and Cx. pipiens pallpens. The overlapping bacterial genera of different mosquito species in the same area were significantly higher than that in different areas, and the unique bacterial genera of the midgut of different mosquito species in the same area were significantly less than that of the same mosquito species in different areas. The analysis results of Venn also further emphasizes that the mosquito breeding environment in Hainan province is very important for the construction of gut bacteria, which is consistent with the findings of other research groups. With the development of economy and trade and the rapid circulation of ship logistics, especially the establishment and construction of free trade island, it will inevitably promote international communication of various mosquito species, and also greatly increase the invasive risk of the mosquito species from outside and related pathogens in Hainan province.
Conflict of interest statement
We declare that we have no conflict of interest.
This work was supported by the Major Science and Technology Program of Hainan Province (ZDKJ202003), National Key Plan for Scientific Research and Development of China (2019YFC1200504), and the National Natural Science Foundation of China (81560002, 81960002).
We thank Manage station staff, Li Jiwen and Liu Chengli who made possible and contributed to sampling in various cities and counties in Hainan. We are grateful to research staff from Institute of Zoology, Chinese Academy of Sciences for their help during sampling in Hainan. We thank Qi Yuan and Liu Jinjin who provided insightful comments and English revision of the manuscript. We thank the anonymous referees who contributed to the manuscript revision.
Kang X was responsible for literature collection and writing the draft of this article. Liu B analyzed synthesize study data and the application of statistics. Li SP prepared study materials, materials, laboratory samples, or other analysis tools. As the corresponding author, Xia QF controlled the content of the whole review. All authors contributed to the article and approved the submitted version.
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