Efficacy of Bacillus Thuringiensis Treatment on Aedes Population

Tropical Medicine and Infectious Disease

Article Eﬃcacy of Bacillus thuringiensis Treatment on Aedes Population Using Diﬀerent Applications at High-Rise Buildings

Zuhainy Ahmad Zaki 1, Nazri Che Dom 1,2,* and Ibrahim Ahmed Alhothily 2

1 Center of Environmental Health & Safety, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam 42300, Selangor, Malaysia; [email protected] 2 Integrated Mosquito Research Group (IMeRGe), Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam 42300, Selangor, Malaysia; [email protected] * Correspondence: [email protected]; Tel.: +60-3-32584447

Received: 20 March 2020; Accepted: 23 April 2020; Published: 1 May 2020

Abstract: Bacillus thuringiensis israelensis (Bti) is an effective biological insecticide for killing mosquito larvae. However, choosing the suitable application method for larviciding is critical in increasing its effectiveness. Therefore, this study aimed to determine the effectiveness of Bti (VectoBac®) WG using various applications at high-rise buildings. Three different applications of Bti treatment were applied at three high-rise buildings in Bandar Saujana Putra. The ULV machine is used for Pangsapuri Impian, a mist blower for Pangsapuri Seri Saujana and a pressured sprayer for BSP 21. BSP Skypark does not undergo treatment and acts as a control. The efficacy of Bti treatment was measured by analyzing the ovitrap surveillance data collected (POI and mLT) for pre and post-treatment. Post-treatment ovitrap surveillance indicates that the Aedes sp. mosquito density was lower than the density at the time of pre-treatment surveillance. Overall, the Aedes albopictus species in both an indoor and outdoor environment setting had shown a reduction. The highest Aedes sp. density reduction is seen through the use of mist blowers in outdoor settings for Aedes albopictus, (%POI reduction = 87.4%; %MLT reduction = 93.8%). The mist blower yielded results that is significantly higher compared to other larviciding applications; the order from greatest to the least was mist blower > pressured sprayer > ULV. It can be concluded that each application produces different degrees of effectiveness in reducing the Aedes sp. density in different environmental settings.

Keywords: Bacillus thuringiensis israelensis (Bti); high-rise buildings; larviciding applications

1. Introduction Dengue is endemic in Malaysia; it is found mainly in the urban and suburban areas [1]. The dramatic increase in dengue cases reported is due to several factors such as increased urbanization, housing designs, the mobilization and movement of the population, erratic water supplies and ineffective or unsustainable vector control [2,3]. Several strategies have been implemented in an effort to control the proliferation of dengue vectors, either at their immature aquatic stages or at their mature stages. Dengue cases have also been reported in high-rise buildings that offer places of residence in the form of flats, apartments or condominiums [2]. Human activities and poorly maintained sanitation in the surrounding area can trigger the breeding of mosquitoes [3]. High-rise buildings designed with rain gutters that make cleaning almost impossible offer the best breeding conditions for mosquitoes as regions with improper drainage and piping systems show high potential in becoming Aedes habitats [1]. The abundance of these species is influenced by the preference and inherent behaviors in oviposition of the female mosquitoes, as well as other biotic and abiotic factors [4,5]. Environmental factors such

Trop. Med. Infect. Dis. 2020, 5, 67; doi:10.3390/tropicalmed5020067 www.mdpi.com/journal/tropicalmed Trop. Med. Infect. Dis. 2020, 5, 67 2 of 12 as relative humidity, wind and temperature influence the occurrence and density of these species. Anthropogenic changes in the environment will also influence the abundance and distribution of these species [6]. There are a lot of potential breeding sites in high-rise buildings and it is difficult to control by just focusing on a singular control method. The increase in the number of breeding mosquitoes within the region is the main cause of dengue outbreaks [7]. In vector control, the application of insecticides is widely used with the aim to kill and/or reduce the population size of infected mosquitoes whenever cases of dengue are reported. The application of insecticides, especially pyrethroids, is carried out through space spraying in the form of cold or thermal fogging. The main purpose of space spraying is to kill the infected mosquitoes, thereby interrupting the cycle of transmission, and stopping the spread and occurrence of dengue [8]. Other control activity in order to reduce the density of Aedes sp. is larviciding. The larvicide agent, which can kill mosquito larvae, includes biological insecticides such as Bacillus thuringiensis israelensis (Bti), a microbial larvicide that is commonly used as a larvicidal agent [9]. Bacillus thuringiensis israelensis (Bti) is a Gram-positive bacterium that is used as a larvicide to cause death to Aedes larvae. Delta endotoxin from the Bti is ingested by larvae and binds to the cells of the epithelium in the mid gut, causing disruption to the osmotic balance within the mid gut’s epithelial cells by forming pores in the cell membrane and lysing the cells [10]. A lot of studies have been done in Malaysia to determine the effectiveness of each dengue control method [11,12]. Synthetic insecticides have been effectively used for the past several decades as a method of vector control. However, the effective use of chemical insecticides has been greatly impeded by several factors such as the development of physiological resistance within the target vector, the causing of environmental pollution resulting in the bio amplification of food chain contamination, and the development of harmful effects on other beneficial non-target animals [13]. Therefore, the need for more effective and environment-friendly control agents has become more urgent and as of the moment, biological agents seem to be the most promising and safest alternative. Bacillus thuringiensis israelensis (Bti), is an effective larviciding agent for Aedes sp., and this method is applicable in the mass treatment of dengue outbreak areas [14]. Larviciding using biological insecticides is effective in killing mosquito larvae and it can be conducted regularly to reduce the density of Aedes sp. within a region. Different treatment applications and different formulations are available. The Bti comes with different formulations, such as in the form of rice husks, wettable powder, water granules and water tablets. The larvicidal activity can be applied using an ultra-low volume (ULV) machine, a mist blower, a pressured sprayer, and by direct application. The process of choosing the application method of Bti treatments is critical in increasing its effectiveness in reducing the density of Aedes sp. Therefore, this study aims to understand the best application method of Bti treatments for high-rise buildings by comparing different methods of application and studying its impact on mosquito populations and species. This study is expected to contribute to the baseline knowledge of the possible effect of using different applications on Aedes sp. in Selangor, Malaysia.

2. Materials and Methods

2.1. Description of Study Site This study was conducted at Bandar Saujana Putra, in the Kuala Langat District of Selangor. Kuala Langat is situated in the southwestern part of Selangor and covers an area of 885 square kilometers, with a population of 260,261 people. In this study, four high-rise residential buildings; Pangsapuri Impian (PI), Pangsapuri Seri Saujana (PSS), BSP 21 (B21), and BSP Skypark (BSP) were selected as study sites. Figure1 shows the location of these high-rise buildings. These four localities of high-rise apartments had been selected due to a series of dengue outbreak incidences that had been reported by the Vector Control Unit of the Kuala Langat Health Oﬃce from 2014 to 2018. PI, PSS and B21 were chosen as the treatment locality, while BSP was selected as the control locality. Trop. Med. Infect. Dis. 2020, 5, x FOR PEER REVIEW 3 of 12

All localities were identified as high-rise buildings in sub-urban residential areas, where two of them are considered low-cost apartments (PI and PSS) and the other two are serviced apartments (B21 and BSP). PI and PSS are located side to side. PI is a five-story, low-cost apartment with eighteen blocks whereby each block consists of eighty houses and PSS is a low-cost apartment with five-story blocks consisting of ten blocks and each block has eighty units of houses. B21 consists of ten blocks of serviced apartments with eighteen and twenty-seven story buildings, while BSP is a serviced residence consisting of two blocks of apartments with six hundred and eighty-nine units of houses. Both PI and PSS have been in service for approximately eight years and the overall surroundings seem poorly maintained with unmanaged trash disposal, untidy communal yards and an overgrowth of vegetation. The surrounding area appears unclean with piles of garbage randomly scattered at almost every floor. In contrast, the surrounding environment of B21 and BSP is well kept with planted trees, with the area looking cleaner and well maintained in terms of sanitation with proper waste managementTrop. Med. systems Infect. Dis. in2020 place., 5, 67 However, a certain area in B21 was quite messy as the area3 of 12 was still under construction with works progressing in the area.

Figure 1.Figure Sampling 1. Sampling sites for sites four for four different different localities localities of high-risehigh-rise apartments apartments in Bandar in Bandar Saujana Saujana Putra; Putra; (PI) Pangsapuri(PI) Pangsapuri Impian Impian (orange), (orange), (PSS) (PSS) Pansgapuri Pansgapuri Seri Saujana Saujana (blue), (blue), (B21) (B21) BSP 21BSP (green) 21 (green) and (BSP) and (BSP) BSP Skypark (yellow). BSP Skypark (yellow). All localities were identified as high-rise buildings in sub-urban residential areas, where two of 2.2. Studythem Design are considered low-cost apartments (PI and PSS) and the other two are serviced apartments (B21 and BSP). PI and PSS are located side to side. PI is a five-story, low-cost apartment with eighteen Thisblocks study whereby applies each the block field consists research of eighty design houses and to PSS investigate is a low-cost the apartment density with of five-story dengue vector mosquitoesblocks within consisting selected of ten blocks high and-rise each build blockings has eighty in Kuala units of Langat, houses. B21 Selangor consists of after ten blocks applying of the biologicalserviced control apartments treatment with usingeighteen different and twenty-seven treatment story applications. buildings, while This BSP is study a serviced was residence conducted to identify whichconsisting treatment of two blocks application of apartments is more with sixeffective hundred to and be eighty-nineapplied in units high of-rise houses. building Both PI areas and (Table PSS have been in service for approximately eight years and the overall surroundings seem poorly 1). Positive ovitrap index (POI) and mean larvae per ovitrap (MLT) were used to assess the density maintained with unmanaged trash disposal, untidy communal yards and an overgrowth of vegetation. of AedesThe sp. surrounding with regard area to appears their distribution unclean with piles and of abundance. garbage randomly Then, scattered the treatments at almost everywere floor. carried out shortly afterIn contrast, the bas theeline surrounding study environmenthas been completed. of B21 and BSP This is wellwas kept conducted with planted for trees, about with twelve the area weeks in all the localities.looking cleaner Three and different well maintained types of in applications terms of sanitation were with applied proper using waste managementBti (VectoBac®) systems WG in at each locality place. over However, the twelve a certain-week area treatment in B21 was duration. quite messy The as the PI area locality was still was under treated construction by applying with Bti (VectoBac®)works WG progressing using a inn ultra the area.-low volume machine (ULV), PSS was treated using mist blowers (MB) and B21 2.2.were Study treated Design using a back-pack pressured sprayer (PS). BSP, as a control locality (C), had no

This study applies the field research design to investigate the density of dengue vector mosquitoes within selected high-rise buildings in Kuala Langat, Selangor after applying the biological control treatment using different treatment applications. This study was conducted to identify which treatment application is more effective to be applied in high-rise building areas (Table1). Positive ovitrap Trop. Med. Infect. Dis. 2020, 5, 67 4 of 12

Trop. Med. Infect. Dis. 2020, 5, x FOR PEER REVIEW 4 of 12 index (POI) and mean larvae per ovitrap (MLT) were used to assess the density of Aedes sp. with Trop. Med. Infect. Dis. 2020, 5, x FORtreatment PEER REVIEW applications applied to it. The treatments were conducted at 4 each of 12 locality for overall regard to their distribution and abundance. Then, the treatments were carried out shortly after the coverage. After each treatment application, post-treatment ovitrap surveillance was conducted to treatment applications baselineapplied to study it. The has beentreatments completed. were Thisconducted was conducted at each locality for about for twelve overall weeks in all the localities. evaluate post-treatment POI and MLT. The post-treatment ovitrap surveillance® was conducted in a coverage. After each treatmentThreesimilar di ffapplication, erentmanner types to the ofpost-t baseline applicationsreatment ovitrap wereovitrap surveillance applied surveillance and using it wasBti was(VectoBac done conducted shortly) WGafter to ateach each treatment locality was over the ® evaluate post-treatmenttwelve-week POIcompleted. and MLT. treatmentThe The results post-tre duration.wereatment used The as ovitrap a PImeasure locality surveillance to was compare treated was the byconducted effectiveness applying inBti ofa different(VectoBac treatment) WG using Trop. Med. Infect. Dis. 2020, 5, x FOR PEER REVIEW 4 of 12 similar manner to the baselineanapplications ultra-low ovitrap volume andsurveillance also machine to compare and (ULV), it the was results PSS done was of shortly the treated baseline after using ovitrapeach mist treatment surveillance blowers was (MB) with and the B21post wereovitrap treated completed. The results wereusingsurveillance used a back-pack as aafter measurepressured treatment. to compare sprayer (PS).the effectiveness BSP, as a control of different locality (C),treatment had no treatment applications treatment applications applied to it. The treatments were conducted at each locality for overall applied to it. The treatments were conducted at each locality for overall coverage. After each treatment applications and also to comparecoverage. the After results each of treatment the baseline application, ovitrap surveillancepost-treatment with ovitrap the post surveillance ovitrap was conducted to Table 1. Timeline of the study on the efficacy of Bti treatment on Aedes sp. using different applications surveillance after treatment.application,evaluate post-treatment post-treatment POI ovitrap and MLT. surveillance The post-tre wasatment conducted ovitrap to evaluatesurveillance post-treatment was conducted POI in and a mLT. at high-rise buildings. Thesimilar post-treatment manner to the ovitrap baseline surveillance ovitrap surveillance was conducted and it was in done a similar shortly manner after each to the treatment baseline was ovitrap Table 1. Timeline of thesurveillancecompleted. study on the The and efficacy results it was of Btiwere done treatment used shortly as onObservational a afterAedesmeasure each sp. tousing treatment compare Week different for the was Pretreatment applications effectiveness completed. (Grey of The different Color) results andtreatment were Post used as at high-rise buildings.a measureapplicationsLocality to and compare alsoApplications to the compare effectiveness the results of of di thefferent baseline treatmentTreatment ovitrap applications surveillance(Blue Color) with and the also post to ovitrap compare the resultssurveillance of the baselineafter treatment. ovitrap surveillance1 2 with3 the4 post 5 ovitrap6 surveillance7 8 9 after10 treatment.11 12 13 Observational Week for Pretreatment (Grey Color) and Post Pansapuri Ultra-low Locality Applications Treatment1st (Blue2nd Color) 3 rd    TableImpianTable 1. 1. (PITimeline Timeline) volume of the study study(ULV) on on the the efficacy efficacy of Bti of Btitreatmenttreatment on Aedes on Aedes sp. usingsp. using different diff erentapplications applications atPangsapuriat high-rise high-rise1 buildings. buildings.2 3 4 5 6 7 8 9 10 11 12 13 Pansapuri Ultra-low st nd rd Seri Saujana1st Mist2nd blower 3rd Observational 1  2Week  for Pretreatment 3  ★ (Grey Color)★ and★ Post Impian (PI) volume (ULV)(PSS) Observational Week for Pretreatment (Grey Color) and Post Treatment LocalityLocality ApplicationsApplications Treatment(Blue (Blue Color) Color) Pangsapuri Pressured BSP21 11 22 331 st 44 5 52 nd 66 7 3 rd 8 8 9✚ 9 10 1011✚ 1112 1213✚ 13 Seri Saujana Mist blower 1st sprayer 2nd 3rd ★ ★ ★ PansapuriPansapuri Ultra-lowUltra-low BSP Skypark Control 1st 11stst 2nd22ndnd 3rd33rdrd    (PSS) ImpianImpian (PI) (PI) volumevolume (ULV) (ULV) PressuredPangsapuri Pangsapuri st nd rd BSP21 Seri SaujanaNote: Baseline Mist study blower1 (pre-treatment) 2 1st based 3on ovitrap2nd surveillance✚ 3rd (grey✚ shading) F ✚ was conductedF for F sprayerSeri Saujana Mist blower 1st 2nd 3rd ★ ★ ★ (PSS)each locality. Each locality is applied with the Bti treatment using different applications (blue shading). BSP Skypark Control (PSS)1 st Pressured2nd 3rd    BSP21The ultra-low volume, ULV (), mist 1stblower (★), 2ndand pressured3rd sprayer (✚) are applied in sprayerPressured BSP21Pangsapuri Impian (PI), Pangsapu ti Seri Saujana1st (PSS) and2nd BSP 21 (B21),3rd respectively. ✚ BSP✚ Skypark ✚ Note: Baseline study (pre-treatment)BSP Skypark based Control onsprayer ovitrap surveillance1st (grey2nd shading)3rd was conducted for Note:was a control locality where no treatmentst hadnd been appliedrd during the study periods. Post-treatment each locality. Each localityBSP is applied SkyparkBaseline with study the ControlBti (pre-treatment) treatment using1 based differenton ovitrap2 applications surveillance 3 (grey(blue shading) shading). was  conducted  for each locality. Eachwas locality conducted is applied a week with after the compleBti treatmentting the using treatment different application. applications (blue shading). The ultra-low volume, The ultra-low volume, ULVULV ( ),), mistmist blower blower (F ),(★ and), and pressured pressured sprayer sprayer ( ) are applied(✚) are in applied Pangsapuri in Impian (PI), Pangsaputi Seri SaujanaNote: Baseline (PSS) and study BSP 21(pre-treatment) (B21), respectively. based BSPon ovitrap Skypark surveillance was a control (grey locality shading) where was no conducted treatment for had been Pangsapuri Impian (PI),2.3. Pangsapu Treatmentti Seri Application Saujana (PSS) and BSP 21 (B21), respectively. BSP Skypark appliedeach locality. during theEach study locality periods. is applied Post-treatment with the Bti was treatment conducted using a week different after completing applications the (blue treatment shading). application. was a control locality where no treatment had been applied during the study periods. Post-treatment TheA Btiultra-low formulation, volume, VectoBac®ULV (), mistWG blower(Lot No. (★), 114-114-3L)and pressured was sprayer used (in✚ ) thisare appliedstudy. Thein Bti was conducted a week after completing the treatment application. 2.3.treatments TreatmentPangsapuri were Application Impian targeted (PI), towards Pangsapu allti Seriidentified Saujana actual (PSS) andand BSP potentia 21 (B21),l outdoor respectively. breeding BSP areas.Skypark Table 2 showswas all a controlthe equipment locality where used no in treatment this experimental had been applied field study.during theThe study ULV periods. sprayer Post-treatment can cover a wide A Bti formulation, VectoBac® WG (Lot No. 114-114-3L) was used in this study. The Bti treatments 2.3. Treatment Application areawas whilst conducted the mist a week blower after andcomple pressuredting the treatment sprayer application.provides a more targeted approach, which is were targeted towards all identified actual and potential outdoor breeding areas. Table2 shows all the A Bti formulation, VectoBac®contained to WG a specific (Lot area.No. The114-114-3L) application was is direct used in in the this sense study. that the The Bti Btiwere basically sprayed equipment2.3. Treatment used Application in this experimental field study. The ULV sprayer can cover a wide area whilst the treatments were targeted towardsdirectly intoall identified the potential actual breeding and potentiacontainers.l outdoor breeding areas. Table 2 mist blower and pressured sprayer provides a more targeted approach, which is contained to a specific shows all the equipment usedA in Bti this formulation, experimental VectoBac® field study. WG The(Lot ULVNo. 114-114-3L)sprayer can was cover used a wide in this study. The Bti area. The applicationTable is direct 2. The in specification the sense of that treatment the Bti applicationswere basically used sprayedin this study. directly into the potential area whilst the mist blowertreatments and pressured were targeted spraye towardsr provides all identified a more actual targeted and potentiaapproach,l outdoor which breeding is areas. Table 2 breeding containers. contained to a specific area.shows TreatmentThe allapplication the equipmentTypes is direct of used Area in in the this Recommendedsense experimental that the fieldBti were study.Equipment basically The ULV Capacity sprayed sprayer and can Capability cover a wide areaApplication whilst the mist blower and pressuredDilution spraye r provides a more targeted approach, which is directly into the potential2.4. breeding Pre and containers. Post-Treatment Ovitrap Surveillance containedULV to a specificGutter, area. dump The application750 g/12 is directL in theFull sense tank that 35 the L can Bti coverwere basicallyup to 300 sprayed m². directly into the potential breeding containers. Table 2. TheThe specification profile of of Aedessite, treatment gardens density applications was measured used in throughthis study.Discharge the data rate collected 300 mL/min. from conventionalThere are two ovitrap surveillance. Ovitrapping was done to obtain baseline datanozzle of sizes; the infestation minimum at profile 1 hour before able to treatment Table 2. The specification of treatment applications used in this study. Treatment Types of(pre-treatment) Area Recommended and after treatment Equipment (post-treatment) Capacity usingdisperse and the Capability 18 same L. Vehicle method. speed The is baseline5–8 km/h. profile of Application AedesTreatmentMistsp. blower density DilutionTypes Drainage, was measuredof Area drain throughRecommended50 g/12 a conventionalL FullEquipment ovitraptank of 12Capacity surveillance L can cover and Capability thatup to was50 m². conducted ULV Gutter,from dumpApplication 3 December 750 g/12 2018 L to 4 March Full 2019Dilution tank (14 35 weeks). L can Thiscover was upDischarge to done 300 at m². allrate: four 120 localities ml/min to obtain the Gutter, dump 125 g/12 L site, gardensbaselineULV information Gutter, of Aedes dump ovipositions.Discharge750 g/12 rate After L 300 that, mL/min. a Full series tank There of 35 three areL can two treatments cover up to were 300 conductedm². for site, gardens There are 6 speeds. Speed level 1 has all localities. The firstsite, gardens treatment nozzle was initiated sizes; minimum on 25 MarchDischarge at 1 2019,hour rate twoable 300 weeksmL/min.to after There the are pre-treatment two dispersion distance of 2 m and maximum surveillance. The second treatmentdisperse was conducted 18 L. Vehicle onnozzle 3speed April sizes;is 2019 5–8 minimum andkm/h. the thirdat 1 hour treatment able to on 8 May Water reservoir, 250 g/L speed level 6 can disperse to a range of Mist blower Drainage, drain 50 g/12 L Full tank of 12 L candisperse cover up 18 to L. 50 Vehicle m². speed is 5–8 km/h. 2019. A post-treatmentpond ovitrap surveillance was conducted directlybetween after each 12–15 treatment. m In this study, theMist ovitraps blower were Drainage, placed atdrain places deemed50 Dischargeg/12 asL “semi rate: indoors” Full120 tankml/min and of 12 “outdoors”. L can cover up Semi to 50 indoors m². would Discharge rate: 120 ml/min Gutter,refer dump to areas inside125 g/12 the L building itself, namely areas covered by the roof of the building whilst outdoors Gutter, dump 125 g/12 L site, gardens There are 6 speeds. Speed level 1 has site, gardens There are 6 speeds. Speed level 1 has dispersion distance of 2 m and maximum dispersion distance of 2 m and maximum Water reservoir, 250 g/L speed level 6 can disperse to a range of Water reservoir, 250 g/L speed level 6 can disperse to a range of pond pond between 12–15 m between 12–15 m

Trop. Med. Infect. Dis. 2020, 5, 67 5 of 12 refers to areas outside the building area, including the surrounding environment [5]. The ovitraps were placed randomly near potential breeding spots in order to capture more accurate results. Other considerations taken is for them to be placed in areas with less physical and environmental interference in order to reduce the risk of misplaced or malfunctioning ovitraps. The ovitraps were recovered after five days from their designated areas and were brought back to the laboratory for larvae species identification. At every locality, ovitrapping was done during three independent visits. For each visit, a total of a hundred ovitraps were placed. Fifty ovitraps were placed accordingly in semi indoors and outdoors settings and they were distributed randomly up to level five of the buildings.

Table 2. The speciﬁcation of treatment applications used in this study.

Treatment Recommended Equipment Capacity and Types of Area Application Dilution Capability ULV Gutter, dump site, 750 g/12 L Full tank 35 L can cover up to gardens 300 m2. Discharge rate 300 mL/min. There are two nozzle sizes; minimum at 1 hour able to disperse 18 L. Vehicle speed is 5–8 km/h. Mist blower Drainage, drain 50 g/12 L Full tank of 12 L can cover up to 50 m2. Discharge rate: 120 mL/min Gutter, dump site, 125 g/12 L gardens There are 6 speeds. Speed level 1 has dispersion distance of 2 m and Water reservoir, pond 250 g/L maximum speed level 6 can disperse to a range of between 12–15 m Pressured sprayer Drainage, drain 50 g/12 L Full tank of 12 L can cover up to 50 m2. Maximum discharge rate: Gutter, dump site, 125 g/12L 120 mL/min depends on the gardens pressure given. Light-weight, easy to be carried everywhere. Uses manual hand pump to control the dispersion distance of sprayer.

2.5. Measurement of Positive Ovitrap Index (POI) and Mean Larvae per Trap The density of the Aedes sp. mosquito of pre- and post-treatment was measured by calculating the POI and mLT for each different species at different environmental settings. Both recovered and missing ovitraps were recorded. Positive ovitraps with positive mosquito eggs were identified. A comparison of the POI in each environmental setting for each of the four localities was calculated to determine the Aedes profile of the area. The POI was determined by dividing the number of positive ovitraps with the number of recovered ovitraps during collection, multiplied by 100 to obtain a percentage [7]. Mosquito eggs were brought to the laboratory for the hatching process. The number of developed larvae was subsequently counted and these were observed at the third instar to identify the Aedes sp. The number of Aedes larvae was calculated and recorded for each positive ovitrap. The identification process is conducted with supervision from entomologists for more accurate results. The identification of larvae was carried out by looking at the comb teeth and siphon characteristics. The mLT was determined by dividing the total number of larvae with the number of recovered ovitraps. The POI and mLT for both semi indoor and outdoor settings were then categorized to reflect information based on the two separate species; Aedes aegypti and Aedes albopictus.

2.6. Statistical Analysis The results from ovitrap surveillance between 3 December 2018 to 4 March 2019 were gathered for analysis. Positive ovitrap index (POI) and mean larvae per ovitrap (MLT) were calculated to assess the density of Aedes sp. with regard to their distribution and abundance. The post study was conducted in a similar manner to the baseline study shortly after the treatments were completed in order to identify Trop. Med. Infect. Dis. 2020, 5, 67 6 of 12 the Aedes sp. density based on POI and mLT results at all localities after the treatment. The result was used as a measure to compare the effectiveness of different treatment applications and also to compare the results of the baseline ovitrap surveillance with the post ovitrap surveillance after treatment. All data obtained from this study were analyzed using the Statistical Package for the Social Sciences (SPSS) software version 23. The significant difference was determined by using the Paired sample t-test, Analysis of Variance (ANOVA) and hierarchical regression.

3. Results The summary data of the efficacy of the Bti (VectoBac®) WG treatment based on POI and mLT were described in Table3. Table3A shows the percentage of reduction in POI between pre-treatment and post-treatment surveillance. For semi-indoor settings, the data shows that the Aedes albopictus species is clearly affected by the Bti treatment for all application methods, whilst the treatment has no effect on the density of Aedes aegypti species. In contrast, the data for the outdoor settings show that both species are affected by the Bti treatment, regardless of the application method. Table3B shows the percentage of reduction in mLT between pre-treatment and post-treatment surveillance. In both settings, Aedes albopictus shows a reduction in terms of numbers of larvae, while for the semi indoor setting, Aedes aegypti only show a reduction in mLT for the treatment application using a pressured sprayer. Generally, the POI and mLT decreased dramatically at all localities, two weeks after initiating Bti (VectoBac®) WG treatments, especially in the outdoor environmental setting, showing that this kind of control treatment activity is suitable for outdoor environments.

3.1. Distribution of Pre- and Post-Treatment for All Applications Based on the Positive Ovitrap Index (POI) of Dengue Vectors in Different Environmental Settings Figure2 shows the results of pre- and post-treatment for all applications based on the POI of the Aedes aegypti and Aedes albopictus species in different environmental settings. The results of a paired-sample t-test show that there is no significant difference for both species on the reduction of POI using Bti treatments for all applications in semi-indoor settings (Figure2A,B). In contrast, the results of the paired t-test for outdoor settings show a significant difference between pre- and post-treatment. The application method of the pressure sprayer recorded a significant reduction of POI with POIpre = 5.4% and POIpost = 2.0%; (p = 0.02) for the Aedes aegypti species, while the mist blower and ULV applications both show a reduction of POI with POIpre = 31.7% and POIpost = 4.0%; (p = 0.01) and POIpre = 36.3% and POIpost = 7.9%; (p = 0.02) respectively, for the Aedes albopictus species in the outdoor environment for high-rise buildings (Figure2C,D). Therefore, it can be concluded that there is a reduction of POI for both species in outdoor settings after treatment with Bti (VectoBac®) WG by using the mist blower and the ULV. This application is suitable for Bti treatment in controlling larvae in outdoor environmental settings seeing as the Aedes albopictus species recorded the highest reduction during pre- and post-treatment in both environmental settings, especially in the outdoor setting. Trop. Med. Infect. Dis. 2020, 5, 67 7 of 12

Table 3. Percentage reduction of positive ovitrap index (POI) and mean larvae per trap (MLT) based on species between pre- and post-treatment according to semi indoors and outdoors setting.

A. Positive Ovitrap Index (POI) Semi Indoor (POI) Outdoor (POI) Treatment Aedes aegypti Aedes albopictus Aedes aegypti Aedes albopictus Pre Post % Pre Post % Pre Post % Pre Post % ULV 3.4 14.4 +76.4 16.4 9 45.1 5.1 3.1 39.2 36.3 7.9 78.2 − − − Mist blower 6.1 12.1 +49.6 9.5 8.1 14.7 6.8 2.3 66.2 31.7 4.0 87.4 − − − Pressured sprayer 5.7 6.4 +10.9 7.0 3.7 47.1 5.4 2.0 63 11.4 3.0 73.7 − − − Control 3.7 10.5 +64.8 5.7 5.8 +1.7 4.4 2.1 52.3 13.1 3.4 74 − − B. Mean Larvae per Trap (MLT) Semi Indoor (POI) Outdoor (POI) Treatment Aedes aegypti Aedes albopictus Aedes aegypti Aedes albopictus Pre Post % Pre Post % Pre Post % Pre Post % ULV 0.5 3.6 86.0 2.7 1.7 37.0 2.1 0.7 66.7 14.8 1.1 92.6 − − − Mist blower 1.0 2.2 54.5 1.5 1.3 13.3 2.7 0.3 88.9 11.2 0.7 93.8 − − − Pressured sprayer 2.0 1.5 25.0 2.3 0.9 60.9 1.8 0.5 72.2 3.9 0.6 84.6 − − − Control 1.6 2.4 33.3 2.5 1.2 52.0 1.4 0.6 57.1 4.2 0.9 78.6 − − − Note: Symbol of ( ) shows there is an eﬀect on the treatment while (+) shows there is no eﬀect of the treatment on the density of dengue vectors. − Trop. Med. Infect. Dis. 2020, 5, 67 8 of 12

Aedes aegypti Aedes albopictus

Pre Post B Pre Post A 20 20 ** ns ns ns ns ns ns ns

15 15

10 10 POI

5 5

Semi indoor settingSemi indoor

0 0 ULV MB PS C ULV MB PS C

Pre Post Pre Post C15 D50 ** ** ns ns ns ** ns **

30 POI 20 5

Outdoor setting

0 0 ULV MB PS C ULV MB PS C

Figure 2. Distribution of pre- and post-treatment based on the positive ovitrap index (POI) of dengue Figure 2. Distribution of pre- and post-treatment based on the positive ovitrap index (POI) of dengue vectors (A & C: Aedes aegypti; B & D: Aedes albopictus) in different environment settings. Note: treatment vectors (A & C: Aedes aegypti; B & D: Aedes albopictus) in different environment settings. application of Bti (VectoBac®) WG are coding with ULV, MB and PS to represent ultra-low volume Note: treatment application of Bti (VectoBac® ) WG are coding with ULV, MB and PS to represent sprayer, mist blower, pressured sprayer, respectively, and C as the control without any treatment. Mean ultra-low volume sprayer, mist blower, pressured sprayer, respectively, and C as the control without numbers and standard errors are shown. Significant differences between pre and post-treatments are any treatment. Mean numbers and standard errors are shown. Significant differences between pre noted (ns: not significant, ** p-value < 0.05). and post-treatments are noted (ns: not significant, ** p-value < 0.05). 3.2. Abundance of Pre- and Post-Treatment for All Applications Based on Mean Larvae per Trap (MLT) of Dengue3.2. Abundance Vectors in of DiPrefferent- and EnvironmentalPost-Treatment Settingsfor All Applications Based on Mean Larvae per Trap (MLT) of Dengue Vectors in Different Environmental Settings The paired sample t-test was also used to examine the effect of Bti (VectoBac®) WG treatment on the densityThe paired of the sample mosquitoes t-test throughwas also variousused to treatmentexamine the applications. effect of Bti The(VectoBac® results show ) WG thattreatment there is noon significant the density di offference the mosquitoes in terms through of mean various larvae pertreatment trap pre- applications. and post-treatment The results in show both that species, there for allisapplication no significant methods difference in semi-indoor in terms of mean settings. larvae Nonetheless, per trap pre the- and pressured post-treat sprayment method in both recordedspecies, a for all application methods in semi-indoor settings. Nonetheless, the pressured spray method significant reduction in mLT for Aedes albopictus in semi-indoor settings with mLTpre = 2.3 larvae/trap recorded a significant reduction in MLT for Aedes albopictus in semi-indoor settings with MLTpre = 2.3 and mLTpost = 0.9 larvae/trap; (p = 0.03) (Figure3A,B). The results show a significant di fference in the larvae/trap and MLTpost = 0.9 larvae/trap; (p = 0.03) (Figure 3A,B). The results show a significant reduction of density for both species pre- and post-treatment in the outdoor setting. Larviciding using difference in the reduction of density for both species pre- and post-treatment in the outdoor setting. the ULV and the pressured spray were found to create a significant reduction in mLT for Aedes aegypti Larviciding using the ULV and the pressured spray were found to create a significant reduction in (ULV: mLTpre = 2.1 larvae/trap and mLTpost = 0.7 larvae/trap; (p = 0.01); pressured spray: mLTpre = 1.8 MLT for Aedes aegypti (ULV: MLTpre = 2.1 larvae/trap and MLTpost = 0.7 larvae/trap; (p = 0.01); pressured larvae/trap and mLTpost = 0.5 larvae/trap; (p = 0.01)). The results obtained showed that larviciding spray: MLTpre = 1.8 larvae/trap and MLTpost = 0.5 larvae/trap; (p = 0.01)). The results obtained showed ® usingthat larvBti (VectoBaciciding using) WGBti (VectoBac® treatments ) canWG reducetreatments the can density reduce of Aedesthe density albopictus of Aedesusing albopictus all application using methods (ULV: mLT = 14.8 larvae/trap and mLT = 1.1 larvae/trap; (p = 0.01); mist blower: mLT all application methodspre (ULV: MLTpre = 14.8 larvae/trappost and MLTpost = 1.1 larvae/trap; (p = 0.01); mist pre p =blower:11.2 larvae MLT/trappre = and11.2 mLTlarvae/trappost = 0.7 and larvae MLT/trap;post = 0.7 ( = larvae/trap;0.01) and pressured (p = 0.01) spray:and pressured mLTpre = spray:3.9 larvae MLT/pretrap and= 3.9 mLT larvae/trappost = 0.6 and larvae MLT/trap;post = (p0.6= larvae/trap;0.02) (Figure (p3 =C,D). 0.02) (Figure 3C,D).

Trop. Med. Infect. Dis. 2020, 5, x; doi: FOR PEER REVIEW www.mdpi.com/journal/tropicalmed Trop. Med. Infect. Dis. 2020, 5, 67 9 of 12

Trop. Med. Infect. Dis. 2020, 5, x FOR PEER REVIEW 2 of 12

Ae. aegypti Ae. albopictus A Pre Post B Pre Post 6 6

ns ns ** ** 5 ** ns ns ns 5

4 4

3 3

MLT MLT

2 2

1 1

Semi indoor settingSemi indoor

0 0 ULV MB PS C ULV MB PS C

C Pre Post D 6 20 Pre Post ** ns ** ns ** ** ** ** 5 15 4

MLT 10 MLT

2 5

Outdoor setting 1

0 0 ULV MB PS C ULV MB PS C

FigureFigure 3. 3. AbundanceAbundance of pre-pre- and post-treatmentpost-treatment based based on on mean mean larvae larvae per per trap trap (MLT) (MLT) of of dengue dengue vectors (A & C: Aedes aegypti; B & D: Aedes albopictus) in different environment settings. Note: vectors (A & C: Aedes aegypti; B & D: Aedes albopictus) in different environment settings. Note: treatment treatment application of Bti (VectoBac® ) WG are coding with ULV, MB and PS to represent an ultra- application of Bti (VectoBac®) WG are coding with ULV, MB and PS to represent an ultra-low volume low volume sprayer, mist blower, pressured sprayer, respectively, and C as control without any sprayer, mist blower, pressured sprayer, respectively, and C as control without any treatment. Mean treatment. Mean numbers and standard errors are shown. Significant differences between pre- and numbers and standard errors are shown. Significant differences between pre- and post-treatments are post-treatments are noted (ns: not significant, ** p-value < 0.05). noted (ns: not significant, ** p-value < 0.05).

3.3.3.3. E Efficacyfficacy of of BTI BTI (Vectobac(Vectobac®)®) WGWG TreatmentTreatment onon DiDifferentfferent ApplicationApplication M Methodsethods at at H High-Riseigh-Rise B Buildingsuildings TheThe results results show show thatthat eacheach applicationapplication method has has a a different different level level of of effectiveness effectiveness in in reducing reducing thethe density density ofof Aedes sp.sp. Therefore, Therefore, a ahierarchical hierarchical multiple multiple regression regression test testwaswas used used to determine to determine the thebest best treatment treatment application application that thatis suitable is suitable and effective and effective in reducing in reducing Aedes Aedessp. densitysp. density within the within study the studyarea. area.For this For study, this study, control control locality locality was used was usedas the as controll the controlleded variable. variable. To determine To determine whether whether the thecontrolling controlling effects effects are are significant, significant, a threea three-step-step hierarc hierarchicalhical regression regression was was conducted. conducted. Firstly, Firstly, the the effeffecectt of of independent independent variablesvariables forfor larvicidinglarviciding application, application, namely namely ULV, ULV, mist mist blower blower and and pressured pressured sprayer, was estimated. Then, the controlling variable (control) was included to measure whether the sprayer, was estimated. Then, the controlling variable (control) was included to measure whether the controlling variables have a significant and direct impact on the dependent variables (POI and MLT). controlling variables have a significant and direct impact on the dependent variables (POI and mLT). Lastly, the interaction terms between independent variables and moderating variables were entered Lastly, the interaction terms between independent variables and moderating variables were entered to to show the additional variance. The result was tabulated in Table 4. In summary, the mist blower show the additional variance. The result was tabulated in Table4. In summary, the mist blower yielded yielded results that were significantly higher when compared to other larviciding applications; the results that were significantly higher when compared to other larviciding applications; the order from order from greatest to the least was mist blower > pressured sprayer > ULV. This reflects the greatest to the least was mist blower > pressured sprayer > ULV. This reflects the effectiveness of these effectiveness of these applications for larviciding using Bti (VectoBac® ) WG treatments in outdoor applications for larviciding using Bti (VectoBac®) WG treatments in outdoor environments. environments.

Trop. Med. Infect. Dis. 2020, 5, 67 10 of 12

Table 4. Coeﬃcients result for controlling factor for (A) positive ovitrap index (POI) and (B) mean larvae per trap (MLT) using hierarchical multiple regressions.

A. Coefficients Result for Controlling Factor for Positive Ovitrap Index (POI) Standardized Unstandardized Coefficients t Significance Model Coefficients B Standard Error Beta Control 0.035 0.849 0.013 0.041 0.968 − − − ULV 2.378 1.034 0.885 2.298 0.051 − − − Mist blower 2.885 0.358 1.302 8.052 0.000 − − − Pressured sprayer 9.459 2.143 1.807 4.413 0.002 B. Coefficients Result for Controlling Factor for Mean Larvae per Trap (MLT) Standardized Unstandardized Coefficients t Significance Model Coefficients B Standard Error Beta Control 0.081 0.131 0.191 0.616 0.552 − − − ULV 0.270 0.243 0.638 1.113 0.298 − − − Mist blower 0.419 0.084 1.199 4.985 0.001 − − − Pressured sprayer 1.065 0.503 1.290 2.118 0.067

4. Discussion The control of dengue vectors is already discussed by several authors [8,10,14,15]. The results obtained in this study recommended that the Bti (VectoBac®) WG treatment can be conducted using various application methods to reduce the density of mosquito larvae in high-rise buildings, especially for outdoor environments. Many methods are available for the control of dengue vectors, for example, environmental control, chemical control, biological control, genetic control, human-behavioral control and others [16]. The results obtained show the implication of using different applications of Bti (VectoBac®) WG. None of the studies prior have looked into the efficacy of different application methods; it is important in choosing the right application to be used for control activities. Post-treatment ovitrap surveillance indicates that the Aedes sp. mosquito density was lower than the density at the time of pre-treatment surveillance. Overall, the Aedes albopictus species in both indoor and outdoor environment settings had shown a reduction in numbers after being treated with Bti (VectoBac®) WG. The highest Aedes sp. density reduction is seen through the use of mist blowers as the treatment application method in outdoor settings for Aedes albopictus, where both POI and mLT shows a higher degree of effect after treatment (%POI reduction = 87.4%; %MLT reduction = 93.8%). These results show that the mist blower is the most suitable treatment application for larviciding in high-rise building areas. The dispersal range of a mist blower allows for a focus on targeted areas, making it much better when compared to treatment using ULV. These biological insecticides are proven to be effective in killing Aedes sp. larvae [17–20]. The results show overall effectiveness in reducing Aedes sp. density in the study area through larviciding activities done by using this biological treatment. Larviciding using the mist blower is found to be the best treatment application for outdoor environmental settings whilst for semi indoor settings, the pressured sprayer is found to be a more suitable treatment application in killing Aedes albopictus larvae under field conditions. The effectiveness of Bti (VectoBac®) WG in controlling dengue vectors had been extensively demonstrated worldwide. Indeed, choosing the right application method will increase the effectiveness of control activities. The efficacy of Bti treatment using mist blower directly to the breeding site is higher than ULV spraying. The study showed a significant difference in the POI value between the control and the treated high-rise buildings, where the percentage of reduction in POI in the treated high-rise building was higher than the controlled locality. This showed that Bti (VectoBac®) is proven to be effective in killing Aedes sp. larvae and in controlling the Aedes sp. population in high-rise Trop. Med. Infect. Dis. 2020, 5, 67 11 of 12 buildings. Under field conditions, larviciding using a mist blower was found to be the best application method for outdoor environmental settings whilst for semi indoor settings, the pressured sprayer was found to be more suitable in killing Aedes albopictus larvae. In a nutshell, the most effective treatment application was found to be by using the mist blower (p = 0.004). This form of application method is found to be effective in controlling Aedes albopictus in an outdoor environment due to the ability to produced small droplets that can remain airborne for a sufficiently long time in the treated area to give a knock-down effect to the targeted flying insects. This is an important factor in protecting the environment, especially in terms of avoiding any adverse impact on insect pollinators. Additionally, the mist blower machine could be carried up by personnel and the dispersal is focused toward targeted areas. This application method is said to be able to overcome the problem faced by local authorities and health department officers in doing full-coverage of vector control activities. However, each application method produces different degrees of effectiveness in reducing both species of Aedes sp. in different environmental settings. Thus, the right application method based on housing type and environmental setting is necessary for the effective reduction of the Aedes sp. population.

5. Conclusions The application of Bti (VectoBac®) WG in vector control management is relevant and serves as an effective tool to control the Aedes sp. population. It is recommended to be conducted regularly. Nevertheless, it is important to choose the right application method in order to make sure the effectiveness of control activities. On the contrary, the long-term solution to the dengue scourge is proper maintenance and cleanliness. The reduction of larval breeding sites is preferred as one of the most effective strategies. This method combined with other control methods may present a strong strategy in reducing the Aedes sp. population in the long run. The findings from this study will help to identify which application method is most effective for Bti treatment in high-rise buildings. Hence, it is recommended that the Bti (VectoBac®) WG treatment be conducted using various application methods to reduce the density of mosquito larvae in high-rise buildings, especially for outdoor environments. Therefore, hopefully, this study can act as a guideline for future control activities in high-rise buildings.

Author Contributions: Z.A.Z. and N.C.D. was involved in all phases of the study, including study design, data collection, data analysis and write up of the manuscript; Z.A.Z. and N.C.D. supervised the study, and revised the manuscript; I.A.A. and N.C.D. were involved in the statistical analysis of data. N.C.D. is the guarantor of the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors sincerely thank the Ministry of Health, Malaysia for providing ground data on the ovitrap surveillance data for this research work. The University of Technology MARA and Ministry of Higher Education (MOHE) Malaysia are also duly acknowledged. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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