4.1 Distribution of Cx. quinquefasciatus
Each of mosquito species has specific biological and ecological requirements for their occurrence and survival (Moise et al., 2018). Identification and transmission status of mosquito borne disease in endemic areas are not only depending on vector abundance and vectorial capacity, but also on vector distribution (Sutherst, 2004; Mackey et al., 2014; Achee et al., 2019). Kruskal Wallis test was performed to test the significance of differences in several independent samples on abnormal data (Subagyo, 2006), and there was significant differences (ρ<0.05) of the number and mosquitoes activities in geographically from the four sites of collection. The result of this study indicated that different geographical location would contribute to a number and type of factors that influence the mosquito distribution. These factors were likely to contribute the number and diversity of mosquitos collected.
The sampling areas, point 1-4 were all urban areas with abundance shrubbery, and point 5 and 6 were rural areas (Fig. 1). The occurrence of breeding sites and resting places (shrubs) would made the areas suitable for mosquito. Moise et al. (2018) suggested that the abundance of inter-annual mosquito was vary over time. Moreover, studies established that the amount of suitable breeding sites were related significantly to the risk of filariasis in the area by 8.707 times, meanwhile the presence of shrubbery will increase the risk by 4,194 times (Mulyono, 2008; Windiastuti et al., 2013).
4.2. RAPD-PCR
The five primers indicate as suitable primers for studying mosquito polymorphism, because of it revealed the consistency of the RAPD band. There was variation number of fragment from the PCR results of mosquitoes using different primer. The number copies of the DNA fragment (amplicon) and target produced at the end of PCR cycle in this study was good enough. As Handoyo and Rudiretna (2001)and Sasmito et al. (2014), stated that by PCR it is possible to obtain DNA fragments exponential in short period of time.
The same primer used for mosquito samples from different site of collection showed different number of fragments, as monomorphic or polymorphic fragments. By using the different primers this study revealed that the band size ranges from 100-3010 bp, and each of primers can produce band from 1-15 The highest band was produced by OPA12 primer. The variation number of mosquitos captured in this study showed that the primers can produce the genetic characters of sampling areas and their show up the genetic variation intra and inter population. This study was in line with Preet and Gupta (2017), they said that from 140 primers screened only 4 primers revealed with a good band quality, which were OPA02, OPM02, OPM04, and OPR08. Their research showed that each primer can produced band from 2 to 12 band from sample with the band size range from 346-2741 bp.
The variation of genetic characters inter and intra population in sampling areas could be accounted to several factors such as environmental conditions (temperature, humidity, rainfall), the availability of breeding sites and resting sites (de-Morais et al., 2010; Astuti et al., 2016; Koval and Vazques-Prokopec, 2018). Pekalongan City is a very dense settlement and mosquito breeding site can be observed in the environment that close to housing. Compare to other location, in Semarang Regency the houses are far apart interspersed with salacca or rubber plantation, so there were few of breeding sites, however, it showed high (11.2) of ratio genetic character. The four sites of mosquito collection has the environmental conditions that suitable for breeding and resting sites of Cx. quinquefasciatus.
4.3 Genetic variation and dendogram tree
The number of character variations found in sampling areas is not always related to number of mosquitos captured, for example Semarang City has 29 captured with 271 characters detected, however it has lower character variation per individual mosquito (9.3) compare to Pekalongan City (14.4), Pekalongan Regency (12.5) and Semarang Regency (11.2). This data shows that in some areas, Cx. quinquefasciatus population have more vary characters. Some samples show distinctive characters that made them located in different cluster of a dendogram tree. These unique sample were i.06.R and i.20.B from Pekalongan City (Fig. 2A), o.20.R and i.02.B from Pekalongan Regency (Fig. 2B), i.22.R and i.18.B from Semarang City (Fig. 3A), and one of i.22.R from Semarang Regency (Fig. 3B).
The interesting thing is the 2 samples (o.01.B, Fig. 3A) in the same period of collection definitely showed the genetic character differences and separated into different main group. This mosquitoes may come from the different breeding sites around housing that each sites may have genetic pool in a high variation. Moreover, It was explained that in a big population, naturally, random mating happens. The parental genetic combination will support to produce high genetic variation (Frankham, 2002). In other study, found that genetic larvae diversity of Cx. quinquefasciatus indicated moderate variation among the larvae based on the value of GST (genetic differentiation). Moreover, the high genetic differentiation may be resulted from migration rates, that may be indicated from the high gene flow in such areas (Preet and Gupta, 2017).
In Semarang Regency, the environment is very different from the three other sites of mosquito collection. There were a lot of canopy and bush, and also a lot of domestic animals. The site of collection showed dense vegetation, as an example salacca plantation and other fruit trees. This condition may have impact in the number of Cx quinquefasciatus cathched. The compilation of the two sites of collection in Semarang showed that the samples from Semarang Regency (C) were close to the main group A of mosquito from Semarang City (Fig 4.). Herein, genetically there was moderate to high character differences among the mosquites samples from the sites of study.
As earlier study, the Culex mosquito has an abundance and different temporal pattern for their biting and resting activities (Astuti et al, 2016; Moise, 2018). This activities pattern maight be influenced by the environmental changing as the temperature, humidity, and wind velocity. In the same areas, increasing the air temperature had positif effect in the indoor and outdoor resting activities, but it moderate influenced the outdoor activities. However, the increasing of wind velocity had strongly negatife effect for their biting activities (Astuti et al., 2019).
Price et al. (2011) reported that phylogenetic study facilitated for further better understanding in insect biodiversity due to the environmental changing. As Moise et al. (2018) had been suggested that identifying the local environment and socioeconomic factors were needed to understand the mosquito distribution and their abundance. From this point, in future the phylogenetic study will be useful for undertanding the establishment of parasites in vectors, transmission of disease and implementation of mosquito control strategy (Takken and Koenraadt, 2013; Vythilingam, 2016).
Herein, the each site of mosquitoes collection was different geographycally and topografically. This condition may produce the differences of the life cycle period and their abundant, furthermore it will develop the genetic variation in the population on each site of collection. Besides that, the differences of mosquitoes breeding sites of the settlements may play important role as genetic pool source that have high variation.
As Frankham et al. (2002) said that individually, mosquito can well adapt to their environment. The diversity of genetic character will increase in line with how fast mosquito spreads and expands to other areas, the intervention of new comer with the local mosquitoes, mating behaviour in the population (Frankham et al., 2002; Indrawan et al., 2007). As in Fig. 5, almost all of the mosquitoes from SKT (Semarang City) and SKB (Semarang Regency) were get together on the same coordinates, it showed that the breeding place of population may from same areas. However, mosquitoes from (PKT) Pekalongan City and (PKB) Pekalongan Regency were spread in defferent coordinates, it indicated that there were many breeding places around the site of collection and in geographically showed the high genetic variation.