Species shifting composition of the Anopheles vector in Wongsorejo district - Banyuwangi, Indonesia

Malaria’s cases have been reported to occur annually in Wongsorejo district of Banyuwangi, East Java since 2002. However, there is a significant decrease of malaria cases during these last years, which might be related to the malaria vector species shifting composition. The objective of this research was to observe some important bionomic characteristics of malaria vector Anopheles in this area from 2015 until 2020 which include species identification, blood feeding behaviour and biting preference of vector. The data collection i.e. determination of Anopheles diversity and behaviour was conducted monthly for 3-6 months annually from 2015 – 2018. In 2019-2020 we sampled irregularly to see the trend. The results showed that there were species shifting of Anopheles vectors in this area. The proportion of Anopheles (An.) sundaicus and An. subpictus, which were previously reported until 2015 as the main Anopheles species in this area, significantly decreased in 2016 - 2018. An. indefinitus & An. vagus was becoming the majority of Anopheles species. During 2019-2020, An. vagus became the main species identified in the sampling area. The predominant species of An. vagus and An. indefinitus has exophagic and zoophilic preference behaviour. Furthermore, An. indefinitus has not previously been identified as a vector for malaria, compared to An. sundaius, and An. subpictus which are well known as an important primary malaria vector on Java Island, Indonesia.


1.
Introduction Malaria remains an important health threat in rural areas of Southeast Asia including Indonesia. Many attempts had been made to control, prevent, and even eliminate the disease. Studying diversity and behaviour of Anopheles (An.) mosquito, which is the vector of malaria, plays a crucial role in developing strategy for an effective and successful vector control program to combat malaria transmission [1]. The exploration and mapping of Anopheles species and its distribution are also important as Indonesia is an archipelago country with each of its islands or regions having very distinct ecological characteristics from one another.
Wongsorejo district, especially the Bangsring area is a coastal region in Banyuwangi, East Java Indonesia, which has been identified as a malaria-endemic area since 2002 [2]. The highest incidence of malaria was reported in 2011 with 127 cases as recorded at Wongsorejo Centre for Public Health (2013). Previous studies on bionomic of vectors and its diversity in Wongsorejo Banyuwangi showed that An. sundaicus was the main dominating species, however many other species vectors were also identified [2]. The objective of this research was to observe some important bionomic characteristics of malaria vector Anopheles in this area ( Figure 1) from 2015 until 2020, which include species identification and its structural composition, blood feeding behaviour and biting preference.  Figure 1. Sampling site location for Anopheles collection.

2.
Methods Female adult mosquitos were collected monthly using hand-held aspirators for six to seven months annually from 2015-2016 (November-April), 2016-2017 (November-April) and 2017-2018 (November-January). To observe further trends of species composition, additional irregular samplings (three times a year) were also conducted between in 2019 and 2020. Sampling was conducted every hour for 12 hours between 06.00 p.m. -06.00 a.m. Human landing and light-trap collections were made indoors and outdoors as well as around cattle shelters. The mosquitoes were then placed inside a labelled paper cup, identified and counted to species level by morphological characters using taxonomical book [3] and was also compared with the book of Anopheline Mosquitoes of Malaya & Borneo [4]. All data were descriptively analysed to see the diversity of the species as well as its biting behaviour.

3.
Results & Discussion Total mosquitoes collected throughout the sampling period were 3723 which consisted of five species of Anopheles i.e: An. vagus, An. barbirostris, An. subpictus, An. sundaicus and An. indefinitus. Compared to the previously published paper from our research group [5], there was a decreasing tendency of the total number of mosquitoes from year to year (Table 1.) Previous results showed that An. sundaicus and An. subpictus were the main species in 2014 [5], however their numbers significantly decreased in the 2015-2020. An. vagus and An. indefinitus were thereafter becoming the majority of Anopheles species in this area.
In this study, we also elaborate the biting behaviour of collected Anopheles mosquitoes based on the number of mosquitoes which were resting on human and/or cattle/cattle shelter during sampling. All collected mosquitoes are predominately zoophilic, exophagic and exophilic species. Only one mosquito was found indoor at the position of human resting in 2014 [5], the remaining samples were outdoor and predominantly zoophilic. In 2016-2020, there were even no mosquitoes resting on human structure ( Table 2). From all of the observation period, the highest peak of mosquito's activities was from 18.00-19.00. It remained consistent until 23.00 but decreased starting from 24.00. The last Anopheles mosquito's activity was detected between 05.00 -6.00 a.m.  [6]. From the species composition, this study showed also that there was a decreasing number of An. sundaicus and An. subpictus which was accompanied by an increasing population of An. indefinitus and An. vagus. This change of species domination indicated a species shifting composition of Anopheles in this area. The species shifting composition in latitude or elevation could be in response to changing climate with a wide diversity of responses by individual species [7]. However, in addition to the abiotic factors, such as temperature and humidity, there was no significant change in those environmental conditions during the observation period. The lowest temperature and humidity recorded by our thermohygrometer were 23.6°C and 59.3(%RH), whereas the highest were 30.8°C and 91.2(%RH), respectively. Therefore, the changes in the species composition of vectors is very likely in response to human vector-control interventions around observation areas [8]. This commonly occurs when the vector species that are more sensitive to a specific vector-control measure become less common, leaving vector species that are less sensitive.
There are already many studies about An. sundaicus and An. subpictus being the main Anopheles species in coastal areas in Indonesia [9]. Among those 4 potential vectors of malaria in this area, An. sundaicus is mainly responsible for malaria transmission in the coastal area of Indonesia. This has been related with its slightly anthropophilic tendency while An. subpictus, An. barbirostris and An. vagus are predominately zoophilic, exophagic and exophilic species [6] which were relevant with this study. An. sundaicus has been also proven to be more often infected than any other species tested over a 30-year period across Sumatra (Nias, Sihepeng, Riau/Bintan Island, Lampung), Java (Pari Island, near Jakarta) and the Lesser Sunda Islands (Sumbawa, Flores and Adonara Islands) by CSP-ELISA analysis [10]. An. indefinitus was previously reported caught only in hilly-rice field areas in Java, associated with grassy pools, ponds and ditches [11]. We found only one report in Sikka, Flores mentioning about the presence of An. indefinitus in rice fields and lagoons but with limited numbers and low population density [12].
The high numbers and domination of An. indefinitus during three years of our study was unexpected and mainly observed in sampling areas around lagoons. It seems that the decrease of malaria cases is very likely due the change in vector species domination into An. indefinitus and An. vagus. Although only An. indefinitus is identified as a non-malaria vector in Indonesia, however the preference biting behaviour of An. vagus in this study were more into zoophilic and exophagic which are also relevant with the previous studies [13,14]. This was also supported by the fact that there are always domestic animals around the sampling site. Thus, it can be concluded that having a domestic animal around the settlement could attract this species, and hence, decrease the risk of malaria infection (cattle barrier) [15]. This study was also conducted on the peripheral area close to shrub and lagoon which are suitable breeding grounds for salt tolerant Anopheline mosquitoes. This will also provide a natural barrier of malaria transmission in coastal areas such as Wongsorejo.  Furthermore, as mentioned above, the shifting of species composition may be due to vector-control intervention, thus it could also influence the reduction number of endophilic biting vectors (indoors) but increased the proportion of exophilic biting vectors (outdoor) ( Table 2). Personal communication with people living around the observation area showed that use of insecticides are commonly applied indoors through spraying, burning or using electric mosquito repellent to avoid mosquito bites. Long-lasting insecticidal nets and indoor residual spraying programs have been also reported previously to be attributed to the change in vector's composition and behaviour [8].
To our knowledge, this is the latest detailed observation on bionomic of Anopheles vectors in this area. Although malaria outbreak was reported in Wongsorejo Banyuwangi with 127 cases in 2013 (Report of Dinas Kesehatan Banyuwangi 2010-2013), however since 2015, this city is declared as malaria free area (Official website of Banyuwangi city). Considering the geographical position of Banyuwangi as a junction border between Java and East Timur Indonesia (Bali and Nusa Tenggara), therefore migration of people between those areas also plays an important factor in the emergence of imported malaria [16]. However, apart from this fact, the species shifting composition of malaria vectors in this study should be also considered to give significant impact on malaria transmission.

Conclusion
There was a species shifting composition of Anopheles in the sampling site from 2015-2020. The previously reported main malaria vectors have been significantly decresed during the observation period. The predominant species of An. indefinitus and An. vagus has exophagic and zoophilic preference behaviour. This could be an important factor in the significant decrease of malaria cases in this area during these last years.