The Impact of Improved Lid of Underground Tanks “Tanka” On Breeding of An. Stephensi In Western Rajasthan, India

Himmat Singh (  hspawar@rediffmail.com ) National Institute of Malaria Research https://orcid.org/0000-0001-7682-651X Sanjeev Kumar Gupta ICMR-National Institute of Malaria Research: National Institute of Malaria Research Kumar Vikram ICMR: Indian Council of Medical Research Rekha Saxena ICMR-National Institute of Malaria Research: National Institute of Malaria Research Amit Sharma ICMR-National Institute of Malaria Research: National Institute of Malaria Research


Introduction
Arid and semi-arid regions of western India are prone to malaria outbreaks 1 . In western India's desert region, major malaria epidemics have occured 2 , while malaria incidences in semi-arid and arid regions are affected by rainfall 3 . After the establishment of the Indira Gandhi Canal in Western Rajasthan in 1970 4 , the ecological scenario in terms of cropland and vegetated areas increased by ~ 68% due to su cient water supply 5

. The breeding of malaria vectors in
Western Rajasthan is also aided by erratic rainfalls, migration and community water storage activities. The underground water tank (locally known as Tanka) is the primary water storage container in almost every house of Jodhpur, Jaisalmer, Barmer and Bikaner districts of Western Rajasthan. A Tanka is a rainwater collection system consists of covered, underground, impermeable cistern on shallow ground. Tankas collect rainwater from rooftops, courtyards, and arti cially constructed catchments 6 .
According to studies, the lid of Tanka became cracked or damaged due to severe temperature variation in western Rajasthan, and became potential breeding sites for mosquitoes, especially An. stephensi, the malaria vector in western Rajasthan 7,8 . During summer season (April-June), Anopheles larvae are found in limited number of Tankas with missing or damaged lids, which act as mother foci and cater mosquitos during the monsoon and post monsoon season 7 . The aim of this study was to prevent breeding of mosquitoes in Tankas by improvising design of the lid from traditional iron to polyvinyl plastic. This improved design was supposed to minimize An. stephensi breeding and density in the study village.

Materials And Methods
Study Site: Jaisalmer, located in the Thar desert between 26°.4' and 28°.23′ North Latitude and 69°.20′ and 72°.42′ East Longitude, is the largest district of Rajasthan and third largest in India. It shares international boundary in west with Pakistan and share district boundaries with Bikaner, Jodhpur and Barmer districts. Malaria has become a major problem in desert areas as a result of increased water availability for irrigation and drinking after introduction of Indira Gandhi Nahar Pariyojana 9 .
The district of Jaisalmer is divided into four blocks: Jaisalmer, Pokaran, Fatehgarh, and Bhaniyana. Pokaran has been the most affected by malaria compared to other blocks 10 . Ajasar village in Pokaran block, with population of around 1500 people, was chosen for the study due to higher API (> 5) and after consulting with the Medical O cer of CHC Pokhran during August 2017. Tota, the non-intervention village about 7 km from the study village and with similar geographical, ecological and malarious conditions was also selected for the research (Fig.1).
Entomological surveillance: Malaria transmission in the Thar desert of India is primarily caused by An. stephensi 11 , which breeds almost exclusively in the underground water reservoirs such as 'tanka' and 'beri' 7 .
To evaluate adult and larval mosquito breeding of An. stephensi, entomological surveillances were conducted during pre-intervention (May 2018), postintervention (June, 2018), and follow-up (May, 2019) Adult mosquitoes were collected using two methods: hand catch and total catch, as per standard WHO manual 12 .
Hand catch: Mosquitoes resting inside human dwellings, cattle sheds and mixed dwellings were collected by insect collectors using ash light and mouth aspirators during the early morning hours (6 to 8 AM). Standard keys were used to identify mosquitoes. Per Man Hour Density (PMHD) of each Anopheles species was calculated (PMHD= Total no. of mosquitoes collected / No. of person x Time spent in hours).
Total catch: Mosquitoes resting inside structure were sampled, also known as spray sheet collection. Indoor resting mosquitoes were collected on white cotton sheets using this approach after being knocked with pyrethrum extract. Cotton sheets were laid out on the oor of the room, and pyrethrum was sprayed to cover the area. After ten minutes, the mosquitoes killed by the spray were collected in petri dishes lined with wet cotton or lter paper from sheets spread on the oor and transported to the laboratory.
Larval collection: Breeding site surveys were conducted in all habitats around and within villages by standard WHO methods. Sampling was done with a dipper of white enamel bowl from Tanka, clay pot/bird pots, under construction Tanka, cement cattle tank, ground cement tanks and seepage water. Larvae were identi ed using standard identi cation keys after emergence at adult stage.
Intervention: A total of 200 Tanka's lids (18x18 inch) were replaced after receiving consent from house owners in Ajasar village. To prevent mosquito breeding in Tankas, improvisations in lid design were developed. The material of the lid was converted from traditional Iron or others materials to the polyvinyl plastic with rust resistant steel screws at the hinge joint of the lids (Fig. 2).
Information, Education and Communication (IEC) activities: During the pre-intervention phase, IEC activities included conducting a door-to-door survey, meeting with the Sarpanch, Block Development O cer (BDO) and community representatives to inform the community about the study's goal, bene ts, and obtain consent. Skilled manpower (known as Mistry") were also selected from the Ajasar village and trained on the entire process of replacing lids in the presence of the Sarpanch and other important community representatives ( Fig.3) Knowledge, Attitude and Practices (KAP) study: A KAP study was also conducted to assess community knowledge about malaria, mosquitoes, their breeding places, and the role of Tankas in malaria transmission before and after the intervention. A total of 120 household persons participated in the KAP study, both pre and post replacement of the lids.
Fitness of Tankas: Tankas with damaged or missing lids were labelled and geo-referenced using GPS for future replacement in Ajasar village only during preintervention phase. The tness of the lids, as well as their ability to prevent mosquito breeding was examined during the follow-up after one year.

Results
Impact of lid replacement on positivity of Tankas: In pre-intervention phase of the study, 22.1% (58/262) Tankas in the intervention village (Ajasar) and 27.1% (19/70) of Tankas in non-intervention village (Tota) found positive. Positivity was not statistically signi cant between intervention and non-intervention village (z=-0.8814, p=0.37886 > 0.05, not signi cant) indicates that there was no difference in positivity until the lids were replaced.
Impact of lid replacement on larval Collection: In addition to Tankas, mosquitoes were also collected from seepage water, pipeline leakage, bird pots, plastic tanks, coolers and cement tanks. In pre-assessment, An. stephensi was predominant in both intervention and non-intervention villages in Tankas while during both post-assessment and follow-up,An. stephensi emerged from Tankas in non-intervention village only .
Impact of lid replacement on Per Man Hour Density (PMHD) of Anopheles mosquitoes: An. stephensi had the highest PMHD (10.9 and 11.8 in intervention and non-intervention villages, respectively) in pre-assessment, followed by An. culicifacies (Fig.6). PMHD of An. stephensi in intervention village reduced signi cantly to 0.55 (94.95%) in post-intervention and to 0.22 (97.8%) in follow-up. The reduction of An. stephensi in non-intervention village was moderate, at 4.6 (61.1%) and 5.9 (50%) in post-intervention and follow-up phase, respectively.
Results of KAP study undertaken in intervention village: A KAP research was performed during a door-to-door survey conducted before and after the lids replacement, with questions about malaria breeding sites, mosquito larvae detection, and other topics. A total of 120 people from different households took part in the survey. There was a substantial increase in respondents' awareness levels. (Fig 7).
Pre-assessment result showed that 67% respondents were aware about the malaria disease and 37% knew that malaria spreads through the bite of female mosquitoes. Only 11% respondents were aware that larva is a part mosquito's life cycle whereas 20% of the respondents knew that mosquitoes breed in Tankas Since the community living in this zone were devoid of potable water, they used Tanka (underground tank) for year-round storage in nearly every house. As it is known, that An. stephensi adapted for breeding in Tankas (Underground tanks) and beris (Wells) throughout the year 7 . With the increase in water supply of Indira Gandhi Canal, the resident population increased in few decades and so more Tankas came into existence, fueling bigger mosquito breeding habitats and malaria transmission. Availability of water and improper lids allowed mosquito breeding in these Tankas.
Almost all houses in western Rajasthan use Tankas for their daily water needs and long term storage. Tanka is an important feature which improves selfreliance of the rural people, stabilizes rural employment skills, and cost-effective 6 . An. stephensi which is the major malaria vector of western Rajasthan predominantly breeds in these Tankas 7 . Our pre-awareness study (KAP) of the community showed that about 67% individuals were aware of malaria as disease spread by mosquitoes and about 44% knew it spreads due to infective bites of female Anopheles. This shows a fair awareness of the community about the diseases and its transmitting agent. However, community did not know much about larvae (13%) and 20% knew that breeding occurs in Tankas (Fig. 7). Similar results were observed in study conducted by Yadav et.al 15 , which also showed that about 20% of respondent have knowledge that Tanka is main source of mosquito breeding and its proper coverage is essential in prevention of mosquito breeding 16 . Our results showed that there was signi cant improvement in the knowledge of community (22-67%) about malaria, its transmission and breeding sites. The signi cant improvement may be due to active engagement of villagers in lid replacement process during the study. The knowledge gained by community will be helpful in understanding the importance proper lids and mosquito proo ng of Tankas.
Damage lids provide opportunity to An. stephensi to breed in these Tankas. During hot summer season, when outdoor temperature exceeds > 45 o C, these Tankas becomes resting place for the mosquitoes to avoid desiccation. During monsoon and post-monsoon season, An. stephensi breeds profusely and support malaria along with another vector species i.e. An. culicifacies. An culicifacies, is a seasonal malaria vector of the Western Rajasthan, which appears in post-monsoon and winter season only. Study by Joshi et al (2005) showed that An. stephensi is a persistent malaria causing vector of the region, which is supported by An. culicifacies in the post-monsoon season 17 Installation of improved polyvinyl lids has completely stopped breeding in these Tankas. As a result of which there was drastic reduction in density of An. stephensi (94.9% in post-intervention & 97.9% in follow-up after one year) in Ajasar the intervention village as compared to non-intervention Tota village. After one year of Installation of improved lids the tness was checked during May, 2019. A total of 98% (196/200) replaced lids were found t and were free from breeding. Furthermore, they had no rusting at hinge joints except for 4 lids, where 2 lids were found detached from cement binding, probably due to use of lids prior to cement settling, and 2 had breeding, possibly due to holding lids open for long periods of time.
According to the World Malaria Report 2019 18 , almost 85% of the global malaria burden is distributed in nineteen countries of Sub-Saharan Africa and India.  19 . However, reduction in parasite incidences also relate to good intervention. In case of Rajasthan there might be impact of reduced malaria in Odisha and other zones where inhabitants of western Rajasthan usually go for livelihood and labour migrate from these places to western Rajasthan for work also. Availability of vectors like An. stephensi during the immigration of such person coming to village either post-monsoon for crop purpose of visit of laborers for developmental activities might introduce parasite in the village system for transmission 17 .

Conclusion
The current study has successfully shown that small interventions can e ciently solve an emerging problem. A signi cant reduction in density of An. stephensi was observed using mosquito proof lids, addressing the problem of persistent mosquito (An. stephensi) breeding in underground tanks. The modi ed polyvinyl plastic lids are cost-effective and have a life expectancy of 2 to 5 years if used properly. Lids not only keep mosquito away, but they also keep other opportunistic insects and reptiles like lizards, snakes, and rodents out of portable water.
Rajasthan is a low malaria transmission area, falling under Category 1 of the WHO's malaria elimination target for 2030. These novel measures could help to curb perennial malaria transmission by reducing the responsible vector, which may be responsible for year-round malaria transmission. Such simple long-term interventions are critical because they reduce routine intervention costs, reduce insecticide waste, and protect the environment. Map showing study areas of Jaisalmer district in Rajasthan, India Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 2
Polyvinyl mosquito proof lid of size 18X18 inches Figure 3 Training/demonstration of replacement of Tanka's lid Proportion of adult species collected by hand-catch method in intervention and non-intervention villages before and after replacement of lids.

Figure 5
Proportion of adult species collected by total-catch method in intervention and non-intervention villages before and after replacement of lids Figure 6 Per man hour density of Anopheles species in intervention and non-intervention village pre and post lid replacement Awareness among the community about malaria and its cause (n=120)