The results obtained from the current research demonstrated that the species composition of mosquitoes in Yongcheng city includes Cx. tritaeniorhynchus, Cx. pipiens pallens, An. sinensis, Ae. albopictus, and Ar. subalbatus(Coquillett). This was the first time that the species composition of mosquitoes was reported in Yongcheng city. Though Cx. tritaeniorhynchus, Cx. pipiens pallens, Ae. albopictus, and Ar. subalbatus(Coquillett) are not vectors of P. vivax malaria, these species could be considered as the major nuisance on the basis of their high proportions. The influence of culicine species, especially Cx. tritaeniorhynchus and Cx. pipiens pallens should not be ignored, since Cx. tritaeniorhynchus is the primary vector of JE virus, and Cx. pipiens pallens is the primary vector of JE virus and filariasis in China . The ribosomal DNA PCR assay revealed that An. sinensis was the sole species of Hyrcanus Complex in identified female Anopheline mosquitoes in the study sites. The results of this study were consistent with those reported earlier in China [5, 25, 27]. Based on previous researches and results of the current PCR assay, it was probable that there weren't any other species of Anopheline mosquitoes of Hyrcanus Complex except for An. sinensis present in Yongcheng city.
Mosquito host-seeking behavior can be studied by different methods [30, 33, 74–77]. In the northeastern USA, host-seeking activities and avian host preferences of mosquitoes associated with West Nile virus (WNV) transmission were studied by a custom-designed trap baited with dry ice . To study potential WNV vectors, horse- and bird-baited traps and HLC methods were carried out weekly from May to October 2004 at two Camargue sites . In the Toledo District, Belize, Central America, host-feeding preferences of Anopheline species were collected by manual aspiration, mechanical aspiration, and a vehicle-mounted trap . In the present study, baited bednet traps [34, 79] were adopted to study the host-seeking behavior of culicine species and An. sinensis. The current study was similar to a study in Bernalillo County, New Mexico. Hosts used in their study were calves, chickens, dogs, and horses . The rationale for using baited bednet traps in the current study was that they would separate the zoophilic and the anthropophilic species while other methods of collection, such as HLC and IRS may not be suitable to use for this purpose.
Latin square design (LSD) was employed in the present study because the experimental set-up consisted of several baits tested on 5-6 experimental nights. Previous studies had established that the LSD would greatly reduce potential bias related to the location and alternated position of the baited-bednet samplers, allowing comparison of the attractiveness of different hosts to the mosquitoes in the field [34, 46].
The results of variance analysis demonstrated that there were significant differences among the abundance of Cx. tritaeniorhynchus for each bait, the abundance of An. sinensis for each bait, but no differences of female Cx. pipiens pallens for each bait. There were some major characteristics, which might influence the abundance of female Cx. tritaeniorhynchus and An. sinensis in these villages. First, during the study period, the locations of the bednet traps of Renhu village were near to a wide canal and many ponds (harboring endogenous lotus). High density of culicine and Anopheline mosquito larvae were observed from these breeding sites. In another experiment concerning breeding sites of Anopheline mosquitoes in 2010, Renhu village had more breeding sites of Anopheline mosquitoes than the other two villages. Second, the number of animal baits from high to low was consistent with the abundance of collected female culicine species and An. sinensis in the study sites. Differences in the availability of blood meals for female culicine species and female An. sinensis might exist within the study villages. Third, the locations selected for our study were sampled at different periods. This could possible explain the observed differences in the number of both Culicine and Anopheline mosquitoes caught between the villages. Fourth, the local selective pressures within which mosquitoes thrive might be added to the variability of the results.
By far, field studies have revealed that each mosquito species had its own host preference [81, 82]. Pair wise comparison showed that the rank of abundance of host-seeking female Cx. tritaeniorhynchus and An. sinensis for each bait from high to low was pigs, goats, calves, dogs, humans, and chickens, respectively. There was a consistent trend of the rank of abundance of host-seeking activity between female Cx. tritaeniorhynchus and An. sinensis. In Thailand, An. sinensis was almost entirely zoophilic in comparative biting tests involving man and cow; almost none of them were attracted to man . This phenomenon could be explained by an opportunistic feeding behavior present in these species. The anti-vector behavior at the community level, namely practices often used by local people for protection from mosquitoes, also had some influence on the host-seeking behavior of these species. Olfaction may be the major sensory modality involved in the resource searching behavior of insects . In mosquitoes, it is mainly exploited in host-seeking and finding a suitable place for oviposition. Differences in host-preference in the current study were likely to be reflected in their response to different host odors offered [85–88]. However, the focus on the current research was only one aspect of behavior in mosquitoes and cannot fully explain the dramatic re-emergence of malaria in the Huang-Huai River region of central China. Zhou et al  suggested that the spatial distribution between malaria cases and water-body, the changing of meteorological factors, and the increasing vectorial capacity and basic reproductive rate of An. sinensis were the possible determinants of malaria re-emergence in these areas.
In the current study, successful amplification was obtained by template DNA from the whole blood and female blood-fed An. sinensis in the research villages. All bednet traps sampled approximately the same proportions of female blood-fed An. sinensis. The results of multiplex PCR assay showed that only 3 blood meals were human in origin, while most of blood meals originated from domestic animals, such as pigs, calves, and goats. The overall HBI calculated from this study including mixed blood meals was 2.94% and 3.70% when compared with only the single blood meal. Similar studies were reported in Japan in 1951 ; in north Kyonggi-do in 1962 (1.7%), in 1999 (0.7%), and in 2000 (0.8%) . Though the HBI of these tests was very low, they readily fed on humans in high numbers where domestic animals were not nearby for feeding . The HBI of the current study was higher than that of these findings mentioned above. Gonotrophic discordance, or taking multiple blood meals during the gonotrophic cycle, has been reported in many different mosquito taxa. In these studies, approximately 10% to 40% of blood-fed field specimens contained multiple blood meals [92–99]. Amplification of template derived from mosquito abdomens containing mixed blood meals confirmed that multiple blood meals from different mammals could be detected in a single mosquito. Our studies demonstrated that high proportions of mixed blood meals were encountered (21/102), and cryptic blood meals were likely to be more numerous. Two to three sources of blood meal were detected from the abdomen of female blood-fed An. sinensis, and this phenomenon could be explained by a modification of the traditional view of the gonotrophic cycle . This host-feeding behavior can influence pathogen transmission through increased frequency of vector-human contact, or possibly reduce vector-human contact if some blood meals are taken from alternative mammalian hosts . In the current study, we failed to identify the origin of the blood meal in 11 out 102 field specimens examined. Insufficiency in host DNA concentrations due to the low blood meal volume in some mosquito specimens is presumed to be the cause of this failure. In addition, the process of blood digestion may have denatured host DNA, or the mosquito may have fed on an animal not included in the diagnostic assay .
The current study demonstrated that the host-seeking activity of female An. sinensis showed two peaks. The primary host-seeking activity occurred between 19:00 and 21:00 and a smaller peak between 4:00 and 5:00. It reported that An. sinensis bites man and animals soon after dark and throughout the night, with a peak generally in the first quarter of the night . In east China, its peak was from 19:00 to 21:00 . Chow (1949) found that the peak of An. sinensis biting buffalo in Chongqing was from 20:00 to 22:00. Zhou et al  reported that the biting time of An. sinensis was from 19:30 to 24:00 locally, with a peak time from 21:00 to 24:00 in Yongcheng city. The primary peak time in the present study was earlier than that of Zhou' s report  and consistent with a previous study in east China . This pattern suggested that vector abatement directed at adult An. sinensis would be most effective if initiated from 19:00 to 21:00, namely before human host go to bed at night.
Previous studies have shown that meteorological factors, such as the average temperature, rainfall, and relative humidity have a certain correlation to mosquito density in the corresponding period [101–105]. Temperature was found to be the most important environmental factor, followed by rainfall and relative humidity in the Delphi evaluation. However, relative humidity was found to be more important than rainfall and temperature in the ranking list according to the three single environmental factor regression models . Paaijmans et al  believed that the incubation period for malaria parasites within the mosquito is exquisitely temperature-sensitive, and temperature is a major determinant of malaria risk. Yang et al  found that the distribution of the mosquito vector was mainly related to relative humidity, which determined the extent of malaria transmission. In the current study, there were no correlations between the density of female An. sinensis and the average temperature in the study sites, while significant correlations between the density of female An. sinensis and the average relative humidity (r = 0.859, P = 0.029) were observed in Wangshanzhuang village. It could be explained due to the average relative humidity affecting the distribution and breeding of An. sinensis.
Care needs to be taken in interpreting the results of this study. Several factors may have affected the variability in the abundance and host-seeking behaviour of the culicine mosquitoes and An. sinensis in our study. First, biases inherent in the trapping method may have affected the observed number and species composition of mosquitoes within baited traps. Some mosquitoes may fly out from the gaps under the baited bednet traps more easily than others, resulting in some species being underrepresented. Second, the physiological status and infected proportions of mosquitoes were not recorded in the field; therefore, the importance of gravid female An. sinensis is not emphasized in the current report. Third, the abundance of Ae. albopictus, Ar. subalbatus (Coquillett), and other mosquito species in each site were not calculated because they were poorly represented in our samples. Further research should be undertaken using this multiplex PCR protocol for the identification of the origin of blood meals of female blood-fed An. sinensis, in addition, further studies are needed to better characterize the malaria vector and its respective role in malaria transmission. An olfactometry assay could be conducted in the future to clarify if behavior of An. sinensis is not directional and possibly provide an indication of how this species will behave in different circumstances. The origin of blood meals of Cx. tritaeniorhynchus could also be studied in the future because of its high proportions and epidemiologic implications .