Mosquito Population Diversity and Abundance Patterns In Linzhi, Xizang, China

Background: In 2009, China CDC found the distribution of Culex tritaeniorhynchus and Aedes albopictus in Motuo county, Linzhi, Xizang and isolated the epidemic encephalitis V virus from Culex tritaeniorhynchus. However, with the road opend in Motuo County, the trade between people and mosquitoes transmission were more frequent. To illustrate patterns of mosquito population in Linzhi, Xizang, a mosquitoes surveillance was carried. Methods: Adult mosquitoes were collected by light traps and human landing catches, larva by container survey in six counties of Linzhi during August and September 2019. The trapped adult mosquitoes were initially counted and identied according to morphological criteria, and a part of mosquitoes were further identied by PCR. The monitoring data were recorded by Excel 2007, analyzed by SPSS 20.0 software and R. Results: Eight species of mosquitoes belonging to four genera were identied. Culex pipiens pallens (86.80%), Armigeres subalbalus (5.29%) and Aedes vexans (2.99%) were the top three species coolected by 164 light traps on 12 nights with the average mosquito density was 21.20 (mosquitoes per trap per night); Aedes albopictus were only collected in Chayu County Xiachayu Town by human landing catches with high average mosquito density (26.33 mosquitoes per hour per person). The larva were collected from waste tires, cement tanks and drums in Bomi, Milin and Lang Counties. The results of molecular identication are consistent with morphological identication. Aedes albopictus and Culex orientalis were newly recorded species for the rst time. It is preliminarily speculated that there is a high potential risk of dengue fever in Xiachayu Town and a certain epidemic risk of Japanese encephalitis, while the risk of malaria is low in Linzhi. Conclusion: Mosquitoes had heterogeneous distribution throughout Linzhi, Xizang. The dominant mosquito species are Culex pipiens pallens. It is emphasized to carry out mosquito surveillance again during the peak period of mosquito activities so as to better understand the distribution


Background
Mosquito-borne diseases is a major public health problem worldwide [1] .Mosquitoes spread many mosquito-borne viruses and parasites between people and animals, including viral infections such as West Nile disease, malaria, dengue fever and Chikungunya fever [2][3] . Many infections cause high fevers often accompanied with pain or other u-like symptoms [4] . Mosquito-borne diseases are now being reported at high elevations [5][6][7] . Field studies including mosquito surveillance enable effcient mosquito control and yields knowledge of when and where to take appropriate protective measures. In China, Culex pipiens pallens, Culex tritaeniorhynchus, Anopheles sinensis and Aedes albopictus are widely distributed. Culex is the vector of epidemic encephalitis virus, lariasis, West Nile virus and other pathogens. Anopheles sinensis carry Plasmodium, lariasis and other pathogens. The mosquitoes Aedes aegypti and Aedes albopictus are vectors of several globally important arboviruses including dengue virus, yellow fever virus and chikungunya virus (CHIKV). Due to the high risk of transmission caused by many pathogens, mosquito-borne diseases cause considerable incidence rate and mortality [5][6][7][8] , which is a serious public health problem [9] .Changes in the distribution of population and density of mosquitoes are in uenced by climatic conditions, physical geography and human activities [10] , which in turn affect the occurrence and prevalence risk of mosquito-borne diseases [11] .
During the investigation of Japanese encephalitis vector in Motuo County, Linzhi in 2009, China CDC found Culex tritaeniorhynchus and Aedes albopictus. The Japanese Encephalitis Virus Genotype V was isolated from Culex tritaeniorhynchus [12] . Culex tritaeniorhynchus and Aedes albopictus are the vectors of epidemic encephalitis and dengue fever, respectively. Linzhi is located in the southeast of Xizang Autonomous Region of China, in the middle and lower reaches of the Yarlung Zangbo River. It is connected with Lhasa city in the west and shannan city in the southwest, Changduo city in the east and Naqu area in the north, and India and myanmar in the south.Linzhi is a sub-humid tropical climate with su cient sunshine and abundant rainfall, with an annual precipitation of 400 ~ 2200mm. The average altitude of Linzhi is about 3000 m above sea level (Longitude 92°09 −98°47 , Latitude26°52 −30°40 ). Linzhi governs 6 counties and 1 district, which are Gongbujiangda County, Milin County, Motuo County, Bomi County, Chayu County, Lang County and the newly established Bayi District. Motuo County was the only county in China that did not have access to roads in the past. In 2010, it opened the garongla tunnel, and then successfully opened the roads in 2013.
With the opening of the road and the unique geographical location of Motuo County, people in other counties communicate with each other are more and more frequently, which makes the mosquitoes easily transported with the mode of transportation. When the mosquitoes arrive at the new suitable habitat, they propagate and breed by occupying more ecological niches, thus increasing the risk of mosquito distribution and the spread of mosquitoborne diseases. Once exist in high altitude areas, some mosquito species may threaten the health of humans and vertebrates due to their ability to transmit numerous diseases [13][14][15] . In this study, we report the distribution and composition of mosquitoes in Linzhi, Xizang. We investigated the distribution and composition of mosquitoes to provide the rst scienti c assessment of mosquitoes and a foundation for development of measures to control mosquito-borne diseases in Linzhi in the future.

Study sites
Linzhi is located in the southeast of Xizang, in the middle and lower reaches of the Yarlung Zangbo River, with an area of 11,700 km 2 and a population of 231,000. The annual average temperature is 8.7℃, and annual cumulative precipitation is 650 mm.
The present study was undertaken in six sites of Linzhi during August and September 2019 (Fig. 1). Based on this geographic conditions and socio-economic characteristics to the Linzhi, and the actual eld conditions, we chose six counties to fully represent the overall situation of the research. They included Bayi District (formerly Linzhi County), Chayu County, Xiachayu Town, Milin County, Bomi County and Lang County ( Table 1)  Outdoor large and medium-sized water bodies (rivers, ponds, puddles, lakes, canals) and water storage containers in villages, streets and yards were collected for container investigation. Transfer larvae to a mineral water bottle containing half a bottle of raw water with a spoon or a straw and store them in a sampling tube containing 95% ethanol and 75% ethanol after being quickly killed in 80-90℃ hot water.
Each morning, the trapped adult mosquitoes were initially counted and identi ed according to morphological criteria using the key developed by Lu BL [16] . All collected mosquitoes were put into 1.5 mL centrifuge tubes individually and then transported to the laboratory of the Department of Vector Biology and Control in China CDC for further molecular identi cation.
For information on temperature (℃) and relative humidity (%), please visit http://www.weather.com.cn. During the collection process, GPS is used to obtain the longitude and latitude information.

Mosquito species molecular identi cation
Mosquitoes were preserved in 75-95% ethanol and stored dark at 4℃ before molecular analyses. Head and thorax were selected for genomic DNA extraction using Micro Tissue Genomic DNA Extraction Kit (BioTeke, Wuxi, China) and and DNA/RNA Extractor−32 system (BioTeke). A 658 bp fragment of the COI region was PCR-ampli ed using the universal primers LCO1490 and HCO2198 [17] . DNA ampli cation was carried out in 25µl reactions using 12.

Ethics statement
Ethical approval for this study was obtained from the Ethical Committee of China CDC and permission was also obtained from Xizang Autonomous Region CDC and Linzhi CDC.

Mosquito larvae by container survey
The larvae were collected from waste tires, cement tanks and drums in Bomi, Milin and Lang counties. Through the preliminary morphological identi cation of the larvae, it clear that the larva species obtained by container survey methods were consistent with the adult species collected by light traps.

Discussion
Mosquito-borne diseases are spreading all over the world at an amazing speed. In recent years, there are more and more reports about mosquito-borne diseases [18][19][20] . Based on morphological and molecular identi cation, this study revealed the composition and density of mosquito vectors and other relevant information in Linzhi, Xizang. Using light traps, it is found that the dominant mosquito species in Linzhi were Culex pipiens pallens, followed by Armigeres subalbalus, Aedes vexans, Culex orientalis and Anopheles. Culex pipiens pallens distributed in areas north of the Yangtse River, which is mainly distributed in the area below 3000 m above sea level [21] , The highest elevation at which Culex pipiens pallens has been observed in China is 2,900 metres, in Mainling County, Nyingchi area, Tibet [12] , which is basically consistent with the ndings of this study. The female mosquito of Armigeres subalbalus can stab and suck human blood ercely. It is the mosquito family with greater harassment [16] . Previous studies in Europe have demonstrated that Aedes vexans is not an effective vector of chikungunya virus but may play a secondary role in temperate regions where the mosquito is abundant [22] . It is di cult to distinguish between Culex orientalis and Culex mimulus in morphology. The middle white ring of the beak is obvious which mainly distributed in Heilongjiang, Jilin and Liaoning in China, South Korea, Japan and other places abroad [16] . In South Korea, it has been found that Culex orientalis can carry epidemic encephalitis V virus, which can be viewed as the second vector to transmit Japanese encephalitis except Culex tritaeniorhynchus [23] . It was found that Bayi District had the highest mosquito density in the survey (42.96 mosquitoes per trap per night), Bayi District was located in the North Bank of the Yarlung Zangbo River in the south of Linzhi, Xizang, with su cient water resources and wide wetland coverage area. Although the altitude was 2985 m, the vegetation coverage was good, and the population density of Bayi District is high. The distribution and density of mosquito species are closely related to the natural environment and human factors, and more mosquito breeding places will be formed due to frequent human activities. The distribution of Culex orientalis was found for the rst time in Chayu and Bomi Counties. The density value of Chayu (3.77 mosquitoes per trap per night) is much higher than Bomi County (0.25 mosquitoes per trap per night). It is closely related to the climatic conditions of Chayu County. It has a low altitude area with high temperature, high precipitation and short sunshine time, which is suitable for mosquito breeding. Anopheles were found little in the survey, which were distributed in Chayu County, Xiachayu Town, Milin County, Bomi County and Lang County, it might be related to missing the local maximum temperature in the investigation season, and the other possible reason was that the cowshed preferred by Anopheles was in the vacant period. Compared with the collection of adult mosquitoes in 2009, the species of mosquito vectors are basically the same, but the diversity is lower than that in Motuo County. Due to the investigation of Japanese encephalitis vector in Linzhi, the selection of mosquito catching sites focused on the pig pen and outdoor environment suitable for the survival of Culex tritaeniorhynchus, resulting in the dominant species were Culex pipiens pallens in Linzhi different with Motuo (Culex tritaeniorhynchus).
Using human landing catches, Aedes albopictus was rst found in Chayu County xiachayu town and only caught in this area with the high density (26.33 mosquitoes per hour per person). Xiachayu town has low altitude, mild climate and high rainfall, which is suitable for the breeding of Aedes albopictus [24] . Combined with the discovery of Aedes albopictus in Motuo in 2009 [12] , it is preliminarily speculated that there is a high potential epidemic risk of dengue fever in Chayu and Motuo County. Some research results show that seasonal weather change, vegetation height, population and land coverage will affect the population and distribution of Aedes albopictus, which are indicators to assess the risk of dengue transmission [25,26] .
The container survey only collected larva in Bomi, Milin and Lang Counties. The results show that the preferred breeding place for mosquitoes is waste tires, cement cans and barrels, which are closely related to human beings.
Old tires are often found along the main roads or waste factories, where they are usually unattended and exposed to rain and sunshine for a long time. In communities with intermittent or inadequate water supply, cement tanks and buckets are the most common water storage containers. Although Aedes albopictus preferred water, no Aedes albopictus was found in this kind of habitat. It can be preliminarily inferred that Aedes albopictus did not occur population diffusion in Linzhi.
Aedes albopictus and Culex orientalis were found for the rst time and were new records. In South Korea, Japan and other places, Culex orientalis is considered as an important vector for the transmission of Japanese encephalitis except Culex tritaeniorhynchus [23] . It can not be considered that there is no epidemic risk of Japanese encephalitis in this area, so further virological testing is needed for certi cation. It has been reported to explore the correlation between malaria and meteorological factors in Motuo County, Xizang. The results show that temperature, relative humidity, rainfall and other meteorological variables are important environmental factors for malaria transmission [27] . It can be preliminarily inferred that with the progress of malaria elimination, the local malaria epidemic risk has been at a very low risk level. Climate change, population ow and environmental change play an important role in mosquito distribution in Linzhi [26] . In 2009, Aedes albopictus was rst found in Motuo County, while in this study, Aedes albopictus was only found in Xiachayu. It is preliminarily speculated that there is mosquito diffusion barrier in Linzhi. Therefore, in order to better understand the local ecological characteristics of Aedes albopictus, it is urgent to further explore the possibility of population diffusion of Aedes albopictus in Linzhi.
In the future, further warming is expected, and further economic development in Linzhi will lead to even greater movement of freight and people. These conditions raise the risk of outbreak of mosquito-borne diseases in a population with no prior exposure to such infections [29,30] . Therefore, it is urgent to strengthen the detection and monitoring of mosquito-borne diseases.
This study has limitations due to time and tra c restrictions, the research sites did not cover the whole area of Linzhi, but the six counties selected can fully represent the mosquito surveillance results of Linzhi. However, due to the maintenance of Motuo road tra c, the epidemic risk of mosquito diseases in Motuo County can only be preliminarily speculated. Secondly, the survey was carried out in August and September 2019 when the temperature is between 10℃-26℃ slightly lower than the peak of mosquito activity. However, the results of this study can be used to preliminarily explore the species and distribution of mosquitoes in Linzhi, so as to speculate the epidemic risk of mosquito-borne diseases. The agricultural area along the border between China and Myanmar is rich in mosquitoes with obvious seasonality. The composition and density of mosquito vectors are greatly affected by the natural environment, and the risk of mosquito-borne disease transmission is relatively high [31] . As a neighboring country of China, India has a wide range of mosquito species, and the prevalence of dengue fever, chikungunya fever, malaria and other mosquito borne diseases is serious [32,33] . Therefore, it is speculated that the mosquito density will increase in high temperature season, the diversity of mosquito species will increase, and the potential epidemic risk of various diseases will increase slightly. It is suggested that mosquito surveillance should be carried out again in Linzhi during the peak period of mosquito activity and the opening of Motuo road to obtain more accurate mosquito distribution and epidemic risk assessment of mosquito diseases. Mosquito monitoring is of great signi cance for the assessment of mosquito-borne diseases, which is consistent with the results of most studies [34] .
Mosquitoes had heterogeneous distribution throughout Linzhi, Xizang. With the increasingly developed tra c and global warming, it is necessary to further determine the composition and density of mosquitoes in this area, so as to assess the risk of disease transmission and establish an effective mosquito surveillance system. With the continuous progress and development of Xizang's economy, people pay more attention to the pursuit of health. Mosquitoes can breed and breed in the high-altitude urban environment of Xizang, so that people have the risk of disease. Strengthened community health education and engagement should be conducted to better guarantee the health and life safety of local citizens [35] . Mosquito-borne viruses pose a serious health threat to people. Mosquito surveillance is an essential part to control mosquito-borne diseases, and sometimes it is the only effective way to block or reduce the spread of these diseases (such as dengue fever) [36,37] . Using effective monitoring tools to characterize the species composition and density of mosquitoes is the most important step to control and assess the risk of mosquito-borne diseases outbreak [28,38] . It is suggested that Aedes vector monitoring should be carried out again in the high-risk counties in order to better grasp the species, distribution and growth and decline of mosquito vectors in the area, and increase the number and frequency of monitoring appropriately through monitoring data, so as to provide theoretical basis for scienti c mosquito control and control.

Conclusion
In summary, our results revealed the mosquito density and population composition in Linzhi, which made up for the blank of mosquito surveillance in this area. The results showed that the dominant mosquito species in Linzhi were Culex pipiens pallens, and mosquitoes had heterogeneous distribution throughout Linzhi, Xizang. The mosquito density and species composition changed with the change of longitude, latitude and altitude. Among them, Aedes albopictus and Culex orientalis were found for the rst time and were new records. It is speculated that it has a high potential epidemic risk of dengue fever in Chayu County Xiachayu Town, while the risk of malaria ow in Linzhi is low, and the epidemic risk of Japanese encephalitis can not be excluded. Due to the fact that there is no epidemic history of mosquito-borne diseases in the local, people's awareness of mosquito-borne diseases will be relatively weak. In view of the above situation, it is suggested that local citizens use physical barriers such as screens, nets and insecticides when the peak of mosquitoes. In the process of raising livestock, the distance between livestock and houses should be expanded as much as possible, and the awareness of mosquito prevention should be enhanced through the publicity of local communities or CDC to prevent the occurrence of mosquito-borne diseases. At the same time, local staff should timely clean up the waste tires, garbage stations and other mosquito breeding places to prevent the occurrence of mosquito breeding places, so as to minimize the risk of mosquito-borne diseases by controlling vector mosquitoes.